Keywords: DNA replication, molecular combing, DNA barcoding, single-molecule, nanochannel, epifluorescence

In eukaryotes, DNA replication starts at multiple origins that are activated following a specific program. Population methods allow genome-wide analysis of DNA replication. However, single-molecule methods are required to monitor cell-to-cell variability, detect rare events and measure individual replication fork speeds. With the existing techniques, newly-synthesized DNA is labelled with thymidine analogs and revealed with fluorescent antibodies. Fibres containing a locus of interest can be identified by fluorescent in situ hybridization. These steps are complex and the throughput is low. This work proposes novel, antibody-free tools to detect replication tracts and identify the locus of origin of all DNA molecules at much higher throughput. DNA replicated in the presence of a fluorescent dUTP was purified and specifically barcoded by using a nicking endonuclease, followed by limited nick-translation with another fluorescent dUTP. This allowed alignment to a reference genome map. DNA was then stained with the fluorescent DNA intercalator YOYO-1. Direct epifluorescence revealed the DNA molecules, their replication tracts and their barcodes in three distinct colours. Replicated segments showed a stronger YOYO-1 fluorescence, demonstrating that replication bubbles can be directly detected without metabolic labelling. Finally, these tools were coupled to a nanofluidic device: DNA was driven into 13,000 parallel nanochannels and automatically imaged, massively increasing the throughput. Altogether, these results provide a starting point for genome-wide, single-molecule mapping of DNA replication in eukaryotic organisms.

I would like to thank the members of the jury: Bénédicte Michel and Aloys Schepers, to accept to revise my thesis in the middle of August. Thanks to Gilles Fischer, to chair the committee, and to Torsten Krude, to fight the Brexit and to come from Cambridge. Thanks to Arach Goldar, to be always present all along my PhD, and to amaze me with a mixture of biophysics and magic (yes, I'm still trying to understand why, theoretically, the YOYO-1 signal shouldn't be "double"). Thanks to the others that followed me during the PhD: to Gaël Millot, I would have never been able to include the nanochannel results in this thesis without your "scoop" on the existence of this technology, and to Jean-François Allemand to give me the access to the white-room at the physic department and to be always available to discuss.

Many thanks to the "captain of the ship", Olivier Hyrien, to accept me at the IBENS, and to give me the full responsibility of this project («You are responsible for all your successes and all your failures»). Thank you to always force me to think with my head (every time I enter in your office with a question, I exit with a reply, and with two or three novel questions!), and to never limit my "brilliant" ideas (which were immediately summarised with: «You are probably the most expensive M2 student of all the times». Ok, it was true, but check the price of the AlexaFluor-dUTP!). Thank you for the good wine (oups…time) spent together, and to always remember me why we do research. Thanks to keep doing science for science (and not for the glory from the scientific community). Many thanks to Benoit: you brought a breath of fresh air in the lab, and a lot of good science. I would have never finished the manuscript without your help and support. I'm looking forward to start new experiments with your criticisms and your competences. Thanks to "wonder-woman" Mag: biologist, bioinformatic and mother (super Pablo!): how can you do that? I wish to be half as efficient as you are. Thank you, Xia, to share your thesis preoccupations with me, and to work so hard for three years: we have never given up for one second even in the most difficult periods, downstairs. Thank you, Nikita, to be "brave" and accept to start the huge bioinformatics effort necessary to analyse my data. I hope your thesis will be full of joy (I also hope you give me some results tomorrow. No, really. Where are my results?). Thanks to the old lab members. To Nat: I wish you were here with your family. I'll never forget your positive energy that is still supporting me. Thank you Malik! To stay with me to distribute 12 mL of Xenopus egg extracts in 5 µL aliquots at 2 am, and to teach me how to seduce a frog. Our frozen extracts still partially works 2 years after…that's not magic: it's the touch of the sun from the south. Thanks to the last arrived: Florence. I would have liked an engineer before the end of my thesis (I'd have slept a bit more…), but I'm very happy to wait if this engineer is you! Thanks to the doctoral school "Complexité du Vivant", for the PhD grant. And to some other French institutions: thank you to consider me "Italian" on French-reserved grants, and to consider me "French" on foreign-reserved grants. That's kind.

Thanks to the collaborators. To the ENS in Lyon (Alain Arneodo, Benjamin Audit, Françoise Argoul and Stéphane Roux) to make me waking up at 4 am, but also to enchant my soul with wavelet transforms and some other mathematical things that I promise…I would if I could (understand). Thanks to Auguste Genovesio (IBENS): your platform is the most important tool in this building for us, poor "wet-lab" researchers! Thank you, Elton Rexhepaj, cause this **** computer doesn't want to understand my language (but, apparently, your is ok). Thanks to Valérie Barbe (Genoscope): in a scientific world where "time is money" you believed in our ideas, and you still continue to do it. I am grateful for your excitement in this nanochannel project and for your patience. Thank you, Wahiba, to wonderfully finalise my samples and to run the Irys beast.

Thanks to the functional genomic (S2) section. I'm sorry for shouting in the corridor, for the angry emails about the microscope, for the chairs flying in the radioactive room, for my daily, "non-diplomatic" (euphemism) way to resolve problems. Thank you Hervé, to be the one understanding that I simply like when "things work" and to respect my constant nervousness. Thank to your team. To Isa, you've been a great colleague and a dear friend since the beginning. You still are. Emélie (yes, you're still here for me): we miss you, but the world needs even more passionate biology teachers. Thanks to Joanne, Virginia, Rémi, Rahul, Raphael, Jerome for the time spent together. Thanks to OB to bring tons of "useful" material on my bench, to Lydia to constantly animate the section, and to Floriane, Anne-Catherine and Xiao. Thank you, Alice, to be always available for me and to have brought to the section such a positive energy and your amazing lab. Thank you Laure, to make me smile every single time I meet you, even without talk (and to not make me pay 20 cents for every bad word I've said). I'm actually depressed for your departure. Thank you Vinko. You were also here with me quite all the Saturdays and the Sundays until 10 pm. I would have never been able to finish this year, without your friendship. Thanks for the 5,000 Italian coffees, for the pizzas/sushi, and for the drinks, despite the unacceptably expensive price of beers in France. Thanks to Caro: I'm keeping all your post-it on my desk wall, and in my heart. I bring you with me, always. Thank you, Chloé: I've no idea how you do, but every time I talk to you, you say something that I don't expect and I laugh till I can't breathe…do not stop doing that, please! Thanks to Benoit, to remember me that your flat is less expensive and bigger (but outside Paris, gne gne). Thanks to the EM team, especially to Guillaume, to remember me that I should stay calm, and to Vero, to incite my daily rebellions and to always say what she thinks. Thanks to the bioinformaticians of the section, especially to Yves, to be able to link all of us together, and to Alexandra, to always smile (yeah, you're miss sunshine of the section). Thanks to the past members of the S2, especially the Darzacq lab, to introduce me to the fluorescent microscopy.

Thanks to Brigitte. You would deserve a page, all for you. I don't have words to say how much we need you. Thank you to be my "mother" at IBENS. Thank you, Abdoul, to make her nervous. You are a cool couple. Many thanks to Martine (laverie) and to all the informatics service to make our job here possible, and to be so incredibly efficient.

Thanks to the S2 microscopy room. To "steal" me 4 months of my thesis, and a ridiculous amount of time per week. Thanks to Xavier (and Astou!), to teach me everything about the Nikon Ti. Thanks to help me in putting the furniture together, and to mount/test/maintain that "monster". Thank you to injure yourself (twice) for our section. That black room wouldn't exist without you (my images, as well). Thank you Benjamin, to help me whenever you could, and to Nikon, to make a software as expensive as its number of bugs.

Thanks to all the friends from the M2 at the Magistère Européen de Génétique. Fra, Lara, Ale, Madda, Giulia, Teo (Deb, Cri, …): every time we meet we have great fun. It's like the time never pass and I always feel you by my side.

Thanks to my family here: Jack, Frarro, Andre. You have made me feel at home, every day, for 3 years. Thanks for the inniorantia time, for the Appa Alti Livelli, for the food and the good wine, for the water leakages every 2 weeks, for the door blocked and the nights outside the flat, for the hole in the gas pipe to hang a lantern, for the 1500 euros-extortion of the boiler and the weeks without light in the toilet. Don't ask me HOW, but all this **** was a fantastic experience, with you. Thanks to support me despite my many limits.

Thanks to Marco (God only knows how much I miss you), Stefania (to never let me go, to keep forgiving me for my absence) and Marta (I will never forget that 14 th July, before your departure). You, famiglia cuore, you taught me the value of friendship. Thanks to Nat and our group (especially to Turo and Chiara), to ask me, constantly, what we are doing here. Thanks to the Italian friends in Paris, especially Robi, I miss the time of our disputes.

Thanks to Paolo, Frenchy, David, Tati, Nic, Beppe, Margio, Claude. Some friends are always close in your heart, it doesn't matter the distance. You are as the good ageing wine.

Dulcis in fundo, thanks to my family. To my mother, to show me that believe in Something is the most rational way to reply to our desire for happiness (yes, science can't). Thanks for your constant support and for the sacrifices done for our family. To my father, to show me that miracles do exist and that in life nothing is lost, when you want to restart. To my sisters Marta and Miriam, 'cause sometimes you need to leave to realise what you've left. I miss you, I miss big-Stefano on one side, and Ale, Japo, Michele and Niccoló on the other side. Thanks for your help, to keep me company during all these years, and to make my very shorts Italian trips gorgeous. Thanks to my grandma Rosetta, example of extraordinary faith and of untiring joy of life, and to Elio, Giannina and Gelmino, to keep an eye on me from up there.

Thank you, Laurianne. Thank you for believing in me. Thank you, to constantly show me that science is not sufficient be happy. Thank you to accept my limits and to love our differences. Thank you, to make me always smile with your simplicity and to comfort me. Thank you, to stick with me despite my periodical nervousness and the too many moments of absence along this year together. This thesis and my stay here in Paris during difficult times wouldn't have been possible, without you. Thanks to Sylvie, Michel (Funny), Matthieu, Marie-Laure et les mamies! For the short time spent together, and to make me feel part of your family.

For the people that, arrived at this line, did not found (unjustly) their name: I'm sorry. Thanks to have contributed, in a way or another, to the realisation of this manuscript. Please to forgive my omission and to understand my emotion at the time of writing.

« L'unica gioia al mondo è cominciare. E' bello vivere perché vivere è cominciare, sempre ad ogni istante. » « The only joy in the world is to begin. It is good to be alive because living is beginning, always, at every moment. »

Cesare Pavese

« One day the machines will be able to solve all the problems, but none of them can deliver us one. »

Albert Einstein

Résumé

Vers l'analyse en molécule unique de la réplication de l'ADN eucaryote à l'échelle du génome entier.

Chez les eucaryotes, la réplication de l'ADN démarre au niveau de multiples origines activées suivant un programme précis, qui peut être analysé à l'échelle du génome sur des populations cellulaires. Cependant, l'étude de la variabilité intercellulaire, la détection d'évènements rares et la mesure de la vitesse des fourches de réplication nécessitent des analyses en molécule unique. Avec les techniques actuelles, l'ADN néosynthétisé est marqué avec des analogues de la thymidine et révélé par des anticorps fluorescents. Les molécules d'intérêt sont identifiées par hybridation fluorescente in situ. Ces étapes sont complexes et le débit est faible. Cette thèse développe de nouvelles méthodes de détection et d'identification des molécules d'ADN réplicatives sans anticorps et à haut débit. L'ADN est répliqué en présence d'un dUTP fluorescent, purifié puis marqué en code-barre spécifique permettant l'alignement sur le génome de référence par coupure avec une endonucléase simple brin et incorporation d'un autre dUTP fluorescent. L'ADN est ensuite coloré avec un intercalant fluorescent, le YOYO-1. Les molécules d'ADN, leurs segments néorépliqués et leurs code-barres sont observés en trois couleurs différentes par épifluorescence directe. Les segments répliqués ont une fluorescence YOYO-1 plus intense, ce qui permet de détecter les bulles de réplication sans marquage métabolique. Ces outils ont été couplés à un dispositif nanofluidique dans lequel l'ADN est conduit dans des milliers de nanocanaux et imagé automatiquement, ce qui augmente massivement le débit. L'ensemble de ces résultats ouvre la voie à la cartographie pangénomique de la réplication de l'ADN en molécule unique.

Mots clés : réplication de l'ADN; peignage moléculaire; code-barre fluorescent; molécule unique; nanocanaux; épifluorescence.

Table of contents

I. Introduction I.1 Prerequisites on DNA replication

The "genetic research" era that led to the DNA replication studies, opened with the identification of the molecule responsible for the genetic inheritance. Thomas H. Morgan's fruit flies experiments demonstrated that genes, the physical and functional units of heredity, are located on chromosomes. This notion, termed "Chromosomal Theory of Heredity" [START_REF] Morgan | Sex Limited Inheritance in Drosophila[END_REF]), however, could not identify the element responsible for the genetic inheritance. Both deoxyribonucleic acid (DNA) and proteins were plausible candidates. It took almost two decades to discover that the genetic information is transferrable [START_REF] Griffith | The Significance of Pneumococcal Types[END_REF], and other sixteen years to prove that the "transforming principle" is the DNA [START_REF] Avery | Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types : Induction of Transformation by a Desoxyribonucleic Acid Fraction Isolated from Pneumococcus Type Iii[END_REF]. Residual sceptical scientists, arguing that genes of bacteria must have very different compositions compared to those of complex organisms, finally accepted DNA as the genetic material after the bacteriophage T2 infection experiments conducted by Alfred D. Hershey and Martha C.

Chase [START_REF] Hershey | Independent functions of viral protein and nucleic acid in growth of bacteriophage[END_REF]. Meanwhile Erwin Chargaff had shown that the pyrimidine and purine bases are present in equal proportion in the DNA, and that the composition of the DNA varies between organisms [START_REF] Vischer | The separation and quantitative estimation of purines and pyrimidines in minute amounts[END_REF]. The rigorous validation of the Chargaff's rules was finally provided by James D. Watson and Francis H. C. Crick with the elucidation of the DNA structure [START_REF] Watson | Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid[END_REF]. This paper, a cornerstone of the modern research, not just confirmed the Chargaff's rules and that the DNA is the chemical substance defining the genes, but inspired an entire era with its famous citation: "It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material." 1 Eventually, this intuition of the semi-conservative replication model was demonstrated only four years later [START_REF] Meselson | The Replication of DNA in Escherichia Coli[END_REF][START_REF] Taylor | The Organization and Duplication of Chromosomes as Revealed by Autoradiographic Studies Using Tritium-Labeled Thymidinee[END_REF]. Since then, many discoveries were done, setting the stage for DNA replication studies. Today, we know that despite the huge variability in known eukaryotic genome sizes (i.e. spanning from the 2.25 million base pairs of the parasite Encephalitozoon intestinalis to the stunning 133 billion base pairs of the marbled 1 [START_REF] Watson | Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid[END_REF]. Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid. Nature 171: 737-8 lungfish Protopterus aethiopicus), the DNA is highly organised and its duplication is tightly regulated within a dedicated conserved time-slot, the S-phase.

It is in this frame that the DNA replication takes places: this very delicate process of life responsible for the genetic inheritance is meticulously orchestrated, together with all the other necessary operations, at any moment, within the "concert" of the cell survival.

I.2 The DNA replication background: early studies I.2.1

The first visualisation of replication "bubbles": a fibre autoradiography story

In 1963, Cairns coupled3 H-thymidine metabolic labelling of replicating DNA in Escherichia coli cells, with fibre spreading and autoradiography. For the very first time DNA replication could be physically visualised. Replication appeared to start at a single site, and to be bidirectional [START_REF] Cairns | The bacterial chromosome and its manner of replication as seen by autoradiography[END_REF]. At the meantime genetic studies conducted on the same organism by Jacob, led to the formulation of the "Replicon Theory": a cell contains one or several autonomous units of DNA replication, termed "replicons", that each require a cis-acting element, named "replicator", and a trans-acting element, the "initiator", for their replication [START_REF] Jacob | On the regulation of DNA synthesis in bacteria: the hypothesis of the replicon][END_REF] (Figure I-1 A). The interaction between these two elements results in an autonomous replication unit called "replicon". It took almost ten years to isolate the replicator, oriC [START_REF] Yasuda | Cloning and mapping of the replication origin of Escherichia coli[END_REF], and another five to determine the initiator, DnaA [START_REF] Chakraborty | Purification of the E. coli dnaA gene product[END_REF], but finally the theory was proved by in vitro reconstitution of the initiation process [START_REF] Bramhill | Duplex opening by dnaA protein at novel sequences in initiation of replication at the origin of the E. coli chromosome[END_REF]. It is the initiator itself that is in charge of binding and unwinding the replicator in order to recruit the helicase (DnaB) directly on singlestranded DNA. Finally, the recruitment of additional factors culminate with the assembly of two divergent replication forks (reviewed in [START_REF] Bell | Helicase loading at chromosomal origins of replication[END_REF]). Almost a decade after Cairns, his discoveries were confirmed by [START_REF] Prescott | Bidirectional replication of the chromosome in Escherichia coli[END_REF], but the position of the replication start site was still unknown. Then, in 1968, Huberman and Riggs successfully adapted the DNA fibre autoradiography analysis to the study of mammalian chromosomes.

Many key points could be outlined. DNA replication is bidirectional also in eukaryotic cells, but starts at multiple sites [START_REF] Huberman | On the mechanism of DNA replication in mammalian chromosomes[END_REF])(Figure I-1 B). These sites were therefore called "replication origins" (ORIs), and appeared to be coordinated in clusters. Clusters of five to ten replicons2 complete replication synchronously in about one hour, implying sequential activation of different clusters along the S-phase (8 to 10 hours, typically). The DNA fibre autoradiography also allowed measurements of fork velocities (2-3 kb/min) 3 and quantification of the distances between replicons (20-400 kb) [START_REF] Huberman | On the mechanism of DNA replication in mammalian chromosomes[END_REF]. This was doable because the structure of the DNA was already known [START_REF] Watson | Molecular structure of nucleic acids; a structure for deoxyribose nucleic acid[END_REF]: each residue in the double helix occupies 3.4 Å, therefore, it is possible to establish a correspondence between the µm distance (measured by autoradiography) and its number of bases. Later studies revealed no agreement comparing the number of replication units within different clusters (R. Hand observed very few replication units within the cluster [START_REF] Hand | DNA replication in mammalian cells. Altered patterns of initiation during inhibition of protein synthesis[END_REF], while Willard and Latt reported several dozens of replicons [START_REF] Willard | Analysis of deoxyribonucleic acid replication in human X chromosomes by fluorescence microscopy[END_REF]). Moreover, [START_REF] Yurov | The units of DNA replication in the mammalian chromosomes: evidence for a large size of replication units[END_REF] proved that mammalian replicons could be much longer than previously thought, up to 2 Mb4 . It was clear from all these experiments that, although the fork velocities were almost constant among different cell types and studies, the intervals between ORIs and the synchrony of their activation were highly variable. The reasons accounting for this "flexibility" in ORI usage were unknown, but scientists reasoned that it could be a way to fit specific needs of the cell. The cell needs change the replication program according to its developmental and differentiation stage (reviewed in [START_REF] Berezney | Heterogeneity of eukaryotic replicons, replicon clusters, and replication foci[END_REF]). The concept of flexibility of ORIs usage was further stimulated when Taylor showed that holding the cells at the beginning of the S-phase increases the replicon number by reducing ORIs spacing [START_REF] Taylor | Increase in DNA replication sites in cells held at the beginning of S phase[END_REF]. Therefore, replication stress can modify the "normal" DNA replication pattern, which is not anymore to be considered immutable. A| The replicon model as proposed by [START_REF] Jacob | On the regulation of DNA synthesis in bacteria: the hypothesis of the replicon][END_REF]: the replicon is a circular structure harbouring two genetic determinants. A trans-acting element, the initiator protein, coded by a "structural gene" (SG1, blue), recognises a specific cis-acting element, the replicator (orange). This interaction allows the beginning of DNA replication. B| DNA fiber autoradiography of mammalian chromosomes (Chinese hamster cells, CH). CH cells were pulse labelled with 3 H-thymidine for 30 min, and chased by non-radioactive thymidine for another 45 min. Cells were collected and lysed. The DNA trapped on dialysis filters was subjected to autoradiography. Replication occurs at several starting sites (i.e. replication origins, indicated by orange arrows) and proceed in a bidirectional fashion from each of these. Adapted from [START_REF] Huberman | On the mechanism of DNA replication in mammalian chromosomes[END_REF].

I.2.2 The discovery of eukaryotic replicator: ARS in plasmids as a replication assay

Despite the fast progression of knowledge, it was still unclear whether replication ORIs correspond or not to specific genomic sequences in eukaryotes. The replicon model inspired novel essays to study DNA replication: selectable markers coded in specific plasmids could be maintained independently of chromosomal replication only if the plasmid contained a replication ORI (Figure I-2). Thanks to this idea, eukaryotic replicators were first isolated from the budding yeast Saccharomyces cerevisiae [START_REF] Stinchcomb | Isolation and characterisation of a yeast chromosomal replicator[END_REF]. Specific sequences smaller than 100-150 bp conferred to other DNA molecules the ability to autonomously replicate when transfected into host cells. Such sequences were therefore called "autonomously replicating sequences" (ARSs). Yeast ARSs share little sequence homology (<10%) with each other. However, an 11-bp degenerated T-rich consensus sequence, called "ARS consensus sequence" (ACS), is necessaryalthough not sufficientto establish an ARS. Later studies showed that a second non-consensus element, located at the 3' of the ACS, is also required [START_REF] Newlon | The structure and function of yeast ARS elements[END_REF]. Please notice that, a priori, the genetic replicator may not necessarily coincide with the replication origin (i.e. the site where replication actually begins). Replicators isolated in bacteria and viral genomes did act as replication origins, strongly supporting this idea, but the definitive proof in yeast required the invention of a more powerful DNA replication assay: the 2D agarose gel electrophoretic analysis. ARS assay allows to identify sequences that behave as replicators (ORIs). A| The genome is fragmented in random pieces that are cloned in a plasmid vector that contains a specific selectable marker. B| The library is transfected to the host cells, which contain all the replication machinery (initiator and additional markers). C| If the cell is able to grow in a selecting media it means that it incorporated a plasmid with the ability to autonomously replicate within the cell. The colony can be isolated and expanded for further ARS characterisation. Alternatively, the cell can gain the ability to grow by integrating the selectable marker into one of its chromosomes by integrative recombination, but this is a rare event.

I.2.3 Origins have different efficiencies: the advent of 2D agarose gel electrophoresis

In 1987, Brewer and Fangman and Huberman et al independently invented 2D agarose gel electrophoretic techniques to map replication origins [START_REF] Brewer | The localization of replication origins on ARS plasmids in S. cerevisiae[END_REF][START_REF] Huberman | The in vivo replication origin of the yeast 2 microns plasmid[END_REF]. The technique used by Brewer and Fangman was in fact first developed by Bell and Byers to identify X-shaped recombination intermediates [START_REF] Bell | Separation of branched from linear DNA by two-dimensional gel electrophoresis[END_REF].

In the Huberman et al "neutral/alkaline" 2D gel technique, total genomic DNA was digested with a restriction enzyme and subjected to a first, neutral gel electrophoresis that separated restriction fragments according to mass. The replication intermediates were separated from the corresponding unreplicated fragments because of their increased mass and lower electrophoretic mobility. The second migration (i.e. second dimension, 2D) was performed at a 90° angle, under alkaline conditions that denatured the DNA and separated the parental and nascent strands. After blotting and revealing DNA with specific probes covering different regions of the target fragment, it was possible to detect DNA ORIs based on the electrophoretic pattern of the separated nascent strands [START_REF] Huberman | The in vivo replication origin of the yeast 2 microns plasmid[END_REF]. This method, however, was very sensitive to the nicks introduced in the parental DNA during isolation and manipulation.

In the "neutral/neutral" 2D gel technique of Brewer and Fangman, the first migration is similar to the neutral/alkaline technique but the second migration is performed in neutral conditions, at both high voltage and high agarose concentration [START_REF] Brewer | The localization of replication origins on ARS plasmids in S. cerevisiae[END_REF])(Figure I-3). These conditions permit to separate restriction fragments based on the branch topology: branched structures have lower electrophoretic mobility compared to unbranched molecules of the same mass and fragments of similar mass that carry a single fork or two convergent or divergent forks migrate differently (for a review about 2D gels, I suggest the original one written by [START_REF] Fangman | Activation of replication origins within yeast chromosomes[END_REF]). Later on, their neutral/neutral 2D electrophoresis analysis was widely employed, resulting in an explosion of new concepts. We initially learnt that, on ARS plasmids, replication starts always and only at ARS sequences [START_REF] Brewer | The localization of replication origins on ARS plasmids in S. cerevisiae[END_REF]. 2D gels also showed that in their chromosomal context, the ARS sequences were activated (i.e. ORIs "fired") in very different fractions of the cell cycles (from 0 to 100%), introducing the concept of ORI "efficiency". This efficiency turned out to correlate with the time at which the ORI was activated: efficient ORIs tend to fire earlier in the S-phase than inefficient ones. Moreover, 2D gels discriminate whether a given locus is replicated from an internal initiation or passively replicated by forks emanating from outside origins.

I.2.4 Origins are redundant: novel insights from electron microscopy studies

Cloning experiments on the yeast genome allowed an estimation of the density of ARS elements. At the beginning of the 90s, moieties of the chromosomes 3, 4 and 5 had been systematically tested for the presence of ARSs, revealing about one ARS element every 20 kb.

Meanwhile, electron microscopy (EM) of replicating DNA had confirmed the replicon sizes observed in yeast by fibre autoradiography [START_REF] Petes | Yeast chromosomal DNA: size, structure, and replication[END_REF]. With EM it was no longer necessary to expose the fibres to the photographic emulsion for several months in order to reveal the radiolabeled tracts. However, ARS elements seemed to be in excess over the real ORIs number: while cloning experiments estimated a density of one ARS every ~20 kb, EM observations detected one active ORI every ~40 kb. In a 2D gel electrophoresis study of the replication intermediates performed by [START_REF] Linskens | Organization of replication of ribosomal DNA in Saccharomyces cerevisiae[END_REF], it was shown for the first time that only a small proportion of the ARSs are actually fired within a given genomic region [START_REF] Linskens | Organization of replication of ribosomal DNA in Saccharomyces cerevisiae[END_REF]. This is consistent with the observation described above that ORI efficiencies are broadly distributed (from 0% to 100%). Thus, chromosomes contain more ARSs than the number of activated ORIs in a single S-phase. Experiments elegantly narrated in [START_REF] Fangman | Activation of replication origins within yeast chromosomes[END_REF] suggested that ARS could be silenced, e.g. by the proximity of the telomere. ARSs that do not fire under normal circumstances, could be activated by perturbing chromatin structure, inactivating checkpoints, or delaying their passive replication by perturbing fork progression, and were called "dormant ORIs".

I.2.5

The eukaryotic initiator (ORC) and the pre-replicative complex (pre-RC)

The eukaryotic initiator was first isolated in yeast, as a heterohexameric complex called the origin recognition complex (ORC) that binds the yeast replicator in vitro [START_REF] Bell | ATP-dependent recognition of eukaryotic origins of DNA replication by a multiprotein complex[END_REF]. Mutations of the ORC genes, indeed, resulted in defects in ORI firing. It was shown that ORC also interacts with ARSs in vivo throughout the cell cycle [START_REF] Diffley | Protein-DNA interactions at a yeast replication origin[END_REF][START_REF] Diffley | Two steps in the assembly of complexes at yeast replication origins in vivo[END_REF]. However, ORC binding is not sufficient to start DNA replication. Additional proteins are recruited by ORC in G1-phase, to form a pre-replicative complex (pre-RC, [START_REF] Diffley | Protein-DNA interactions at a yeast replication origin[END_REF]). ORC, together with replication factors Cdt1 and Cdc6, load the minichromosome maintenance proteins 2-7 (MCM2-7), which form the core motor of the replicative helicase, in an inactive form around double-stranded DNA. This event is commonly described as origin "licensing". Please note that, in contrast to the bacterial initiator, ORC is unable to unwind the replicator alone. During S-phase, S-phase protein kinases Dbf4-dependent (DDK) or Cyclin-dependent (CDK) and many other factors switch the inactive MCM2-7 into an active conformation leading to the assembly of two diverging replisomes (for a detailed review about the proteins involved in the ORIs licensing and firing, see [START_REF] Fragkos | DNA replication origin activation in space and time[END_REF]).

Although a larger number of MCM proteins than ORC can be loaded on the DNA during G1phase, only a small fraction actually fire in the subsequent S-phase. In higher eukaryotes, unfired MCMs proved to be a responsive backup system for both helping to complete the normal S-phase replication [START_REF] Lucas | Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos[END_REF], as well as rescuing artificially stalled forks [START_REF] Woodward | Excess Mcm2-7 license dormant origins of replication that can be used under conditions of replicative stress[END_REF].

I.3 Insights from early replication studies

From the replication studies presented above (spanning from the early 60s to the early 90s), it was clear that the replication process was much more complex and dynamic than initially thought. In 30 years of DNA replication studies, we came across unexpected concepts like the "flexibility" in ORI usage (both in space and time), the "efficiency" of a given ORI and finally the notion of ORIs "redundancy".

For these reasons, the search for DNA replication ORIs became the major topic since the beginning, immediately after the Cairns' experiments in 1963 [START_REF] Hyrien | Peaks cloaked in the mist: The landscape of mammalian replication origins[END_REF], Urban et al 2015). Understanding the nature of the DNA synthesis initiation sites is clearly the keystone in understanding eukaryotic cell proliferation.

Hereafter I highlight the key, unsolved questions, which I believe animated the replication debate then as now:

1. What determines where the potential replication ORIs are located?

2. What decides which, among all the potential ORIs, will be activated?

3. What defines when in the S-phase, a given ORI will be activated?

I.4 Pre-"omics" studies I.4.1 Random vs. site-specific initiation in metazoans

Eukaryotic DNA contains many DNA replication start sites. By regulating when and where DNA replication begins, the cell ensures that the entire genome is duplicated within a reproducible time. If ORIs selection was completely random, segments of the genome may fail to replicate at each cell cycle, which would be catastrophic for genome stability. This is because random ORIs positioning would imply a geometric distribution of inter origin distances (IODs), resulting in a large number of unreplicated segments at the end of the S-phase (i.e. a problem known as the "random completion problem", described by [START_REF] Laskey | Chromosome replication in early Xenopus embryos[END_REF] and revised by [START_REF] Hyrien | Paradoxes of eukaryotic DNA replication: MCM proteins and the random completion problem[END_REF]). Strikingly, exogenous DNA from any source could be replicated when microinjected in Xenopus laevis eggs or incubated into a Xenopus egg extract, a system able to recapitulate the steps occurring during eukaryotic DNA replication [START_REF] Harland | Regulated replication of DNA microinjected into eggs of Xenopus laevis[END_REF][START_REF] Mechali | Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast[END_REF]. For plasmids, the replication efficiency was independent of their sequence, but dependent of their size (i.e. the bigger the plasmid, the more efficient the replication). A similar experiment performed in human cells gave comparable results [START_REF] Krysan | Isolation of human sequences that replicate autonomously in human cells[END_REF]. Interestingly, autonomous replication assays generally failed to isolate metazoan DNA replication ORIs, but useful information could be gathered by 2D gel electrophoresis: in Xenopus (either in vitro and ex vivo) as well as in human cells, 2D gels of replicating plasmids were consistent with random initiation events [START_REF] Hyrien | Plasmid replication in Xenopus eggs and egg extracts: a 2D gel electrophoretic analysis[END_REF][START_REF] Krysan | Replication initiates at multiple locations on an autonomously replicating plasmid in human cells[END_REF].

More importantly, non-transcribed chromosomal ribosomal DNA5 (rDNA) and tandem repeats of histone genes were also found to replicate randomly in early (pre mid-blastula transition, pre-MBT6 ) embryos of Xenopus and Drosophila, respectively [START_REF] Hyrien | Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos[END_REF][START_REF] Shinomiya | Analysis of chromosomal replicons in early embryos of Drosophila melanogaster by two-dimensional gel electrophoresis[END_REF]. These experiments showed that random initiation is not a peculiar feature of exogenous DNA, but is compatible with the physiological nuclear replication. It was later demonstrated that spatially random initiation is compatible with a reproducible replication ending time because 1) potential origins are redundant and 2) the rate of origin activation increases as S-phase progresses, which speeds up replication of the occasional large genomic segments that failed to activate origins in early S-phase [START_REF] Herrick | Replication fork density increases during DNA synthesis in X. laevis egg extracts[END_REF][START_REF] Hyrien | Paradoxes of eukaryotic DNA replication: MCM proteins and the random completion problem[END_REF][START_REF] Lucas | Mechanisms ensuring rapid and complete DNA replication despite random initiation in Xenopus early embryos[END_REF].

In contrast to these results, many EM and 2D gel studies in Xenopus, Drosophila and sea urchins showed that transcriptionally active rDNA is preferentially replicated starting from the intergenic spacer between the rDNA's repeats (reviewed in [START_REF] Hernandez | Conserved features in the mode of replication of eukaryotic ribosomal RNA genes[END_REF]). The obvious question is: when is the transition between random and site-specific initiation taking place? The transition appeared at the MBT stage both in Xenopus and in Drosophila [START_REF] Hyrien | Transition in specification of embryonic metazoan DNA replication origins[END_REF][START_REF] Sasaki | Specification of regions of DNA replication initiation during embryogenesis in the 65-kilobase DNApolalpha-dE2F locus of Drosophila melanogaster[END_REF]. Thus, the establishment of the transcription activity interestingly coincides with the spatial specification of replication initiation, suggesting an epigenetic control of DNA replication. It is interesting to note that the absence of "classic" replicators in higher eukaryotes does not mean that the position of ORIs cannot be specified by other means than DNA sequence. Several potential epigenetic mechanisms have indeed been proposed for replication origin specification in metazoans (see [START_REF] Hyrien | Peaks cloaked in the mist: The landscape of mammalian replication origins[END_REF]). This is in stark contrast with the DNA-sequence based origin specification observed in budding yeast.

I.4.2 The DHFR locus: a closer look at replication initiation reveals controversies

Many insights about mammalian DNA replication came from the study of the Chinese hamster dihydrofolate reductase (DHFR) gene. This locus was initially studied in a methotrexate-resistant Chinese hamster ovary (CHO) cell line (i.e. CHOC400), harbouring ~1000 copies of a 240 kb region containing the DHFR gene. By labelling synchronised cells with 3 H-thymidine at the onset of the S-phase and visualizing the earliest labelled amplified restriction fragments, it was shown that replication initiates at specific sites within the repeated units [START_REF] Heintz | An amplified chromosomal sequence that includes the gene for dihydrofolate reductase initiates replication within specific restriction fragments[END_REF]. All these early-labelled fragments mapped within a 55 kb "spacer" located between the DHFR and the 2BE2121 genes [START_REF] Looney | Isolation of the amplified dihydrofolate reductase domain from methotrexate-resistant Chinese hamster ovary cells[END_REF]).

Interestingly, the two genes were transcriptionally active, reminiscent of the Xenopus and Drosophila discovery presented above (see paragraph I.4.1), and transcribed in a convergent direction. These results suggested that it might be possible to precisely map the replicators within the spacer and gave the general hope that DNA replication initiation in the mammalian chromosomes could be sequence specific. Early studies revealed the presence of at least two preferential initiation regions (ori-β and ori-γ) within the intergenic spacer. However, 2D gels studies brought evidence that initiation could happen basically everywhere inside the 55 kblong spacer (reviewed in [START_REF] Hamlin | A winding road to origin discovery. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology[END_REF]). Thus, these authors proposed the term of "initiation zone". The dispersive and inefficient nature of initiation events within the DHFR initiation zone was later confirmed by mapping the location of in vivo-labelled short nascent strands (SNS) or the strandedness of in vivo-labelled Okazaki fragments using hybridization to cloned DNA probes [START_REF] Hamlin | A winding road to origin discovery. Chromosome research : an international journal on the molecular, supramolecular and evolutionary aspects of chromosome biology[END_REF] and at a single-molecule level by DNA combing [START_REF] Lubelsky | Pre-replication complex proteins assemble at regions of low nucleosome occupancy within the Chinese hamster dihydrofolate reductase initiation zone[END_REF]. Altogether these studies highlighted somewhat favoured initiation sites (ori-β, ori-β' and ori-γ), but these only accounted for a low fraction of initiation events through the zone.

I.4.3 Narrow and efficient origins vs broad, initiation zones

Before the "omics7 " era, and especially before the advent of the "next generation sequencing8 " (NGS) techniques, ORIs had to be mapped locus-by-locus. Because of the difficulty in isolating the rare and fragile initiation events, very few ORIs were mapped in the early 2000s [START_REF] Hyrien | Peaks cloaked in the mist: The landscape of mammalian replication origins[END_REF], Urban et al 2015). In terms of ORI location, the most studied loci were the MYC and the β-globin genes (together with the DHFR gene above described, see paragraph I.4.2). For MYC, both strand-polarity and nascent strand abundance assays (Figure 1990[START_REF] Vassilev | An initiation zone of chromosomal DNA replication located upstream of the c-myc gene in proliferating HeLa cells[END_REF]. Experiments detected ORI activity after ectopic relocation [START_REF] Malott | Activity of the c-myc replicator at an ectopic chromosomal location[END_REF]. All these experiments suggested that MYC gene replication is under the control of a narrow and efficient initiation zone located just upstream of the gene. Analogous results were obtained for the β-globin gene: a thin initiation zone was mapped between the δ-globin and the β-globin genes and this ORI was suppressed by deleting a 5 kb DNA fragment straddling it [START_REF] Kitsberg | Replication structure of the human beta-globin gene domain[END_REF]. Moreover, ectopic relocation experiments gave similar results to the ones obtained at the MYC locus [START_REF] Aladjem | Genetic dissection of a mammalian replicator in the human beta-globin locus[END_REF]. Thus, as for MYC, a narrow and efficient ORI seemed the most appropriate description of the mechanism ensuring replication of the β-globin locus. However, more recent nascent strands experiments revealed dispersed initiation events both at the MYC [START_REF] Waltz | DNA replication initiates non-randomly at multiple sites near the c-myc gene in HeLa cells[END_REF] as well as the β-globin [START_REF] Kamath | Multiple sites of replication initiation in the human beta-globin gene locus[END_REF] genes.

I-4 A-D) identified a localised ORI 5' to the MYC gene (McWhinney & Leffak
Single-molecule DNA combing experiments (see paragraph I.7.2 for an explanation of the technique) at fragile site FRA6E detected broad and scattered initiations over ~800 kb covering the PARK2 gene with a slight preference for its upstream region (~200 kb, [START_REF] Palumbo | Replication dynamics at common fragile site FRA6E[END_REF]). Thus, despite the early mapping studies targeting specific loci suggested highly efficient and narrow initiation sites, later analysis converged towards the definition of broad initiation zones. In such zones, initiation appears to be widely dispersed. Many other studies supported the idea of broad initiation zones by single-molecule observations: a region covering 1.5 Mb on the chromosome 14q11.2 showed large initiation zones that preferentially mapped in intergenic regions [START_REF] Lebofsky | DNA replication origin interference increases the spacing between initiation events in human cells[END_REF], but also the mouse Igh, the human POU5F1, NANOG and FRM1 loci [START_REF] Gerhardt | The DNA replication program is altered at the FMR1 locus in fragile X embryonic stem cells[END_REF][START_REF] Schultz | Single-molecule analysis reveals changes in the DNA replication program for the POU5F1 locus upon human embryonic stem cell differentiation[END_REF], as well as the human telomeres [START_REF] Drosopoulos | Human telomeres replicate using chromosome-specific, rather than universal, replication programs[END_REF], showed broad initiation zones. To conclude, from the early 2D gels analysis in metazoans (where dispersed initiations where proposed to account for the inability to detect efficient ORIs), to the more recent discoveries by single-molecule techniques, broad initiation zones appears to be a general feature of mammalian cells.

I.4.4 Temporal control of origin activation: early studies

In eukaryotic cells, different genomic regions replicate at different times during the Sphase. This may be explained if unequally spaced ORIs were all activated at the start of Sphase, so that the most distant regions from an origin would be the latest to replicate. However, late replicating regions are large enough to contain multiple ORIs [START_REF] Drouin | Analysis of DNA replication during S-phase by means of dynamic chromosome banding at high resolution[END_REF].

Furthermore, the rate of eukaryotic chromosome replication is rather constant during the Sphase, whereas it should continuously decrease if no new ORIs were activated after the start of S-phase (Fangman & Brewer 1991). In addition, early autoradiography experiments had shown that ORIs are organised in clusters that are activated at different times in S-phase. Analysis of the right telomere of yeast chromosome V [START_REF] Ferguson | A yeast origin of replication is activated late in S phase[END_REF] showed that the telomere is replicated late in S-phase from the closely located ARS 501, whose activity is only detected late in S-phase by 2D gels. Ectopic relocation experiments shuffling ARS501 (late) within the ARS1 (early) chromosomal context (and vice versa) proved that the activation time of these ARS elements depended on their position rather than on the relocated DNA sequences [START_REF] Ferguson | A position effect on the time of replication origin activation in yeast[END_REF]. This highlighted a novel level of complexity in the control of eukaryotic DNA replication initiation: the determinants for ORI function are separable from the determinants of ORI firing time. In the case of ARS 501 it is the telomere that creates a signal for late ORI activation [START_REF] Wellinger | Lack of positional requirements for autonomously replicating sequence elements on artificial yeast chromosomes[END_REF]. The mechanisms were unclear.

Replication timing needed to be studied genome-wide in order to infer the replication program.

I.5 The advent of genome-wide analysis of DNA replication I.5.1 Technologies for rapid pangenomic mapping of the ORIs

Before the "omics" era, the 2D gels analysis (explained in paragraph I.2.3), as well as the purification of replication bubbles9 , short nascent strands10 (SNS) and Okazaki fragments11

were only applied to small portions of the genome (see [START_REF] Depamphilis | The search for origins of DNA replication[END_REF] 13 RIP mapping consists in mapping the transition from continuous to discontinuous replication by phophorylating the 5' end of all DNA fragments, followed by primer removal in order to expose the 5'-hydroxyl group (previously end labelled with 32 P). Cells can be lysed directly inside the agarose well to reduce further manipulation. 2) SNS can be purified by recovering BrdU-positive SSS using anti-BrdU antibodies after size selection excluding Okazaki fragments on alkaline gel, neutral sucrose gradient, or isopycnic centrifugation. 3) Finally, since NS are synthetized elongating an RNA primer, SNS can be enriched thanks by a λ-exonuclease digestion removing all the SSS that are not protected by an RNA. F| Okazaki fragments can also be isolated using different approaches. 1) Cells are radio-labelled and selected on alkaline gel prior to elution of 50-300 nt-long SSS. 2) Alternatively, they were isolated by size selection on ligase-deficient yeast cells. 3) Recently, OK were isolated by size-selection on denatured, EdU-labelled DNA. Biotin was coupled to EdU thanks to "click"-chemistry and EdU-positive SSS were captured on streptavidine beads. G| Leading strand bias can also be used to find replication ORIs by isolating BrdU-NS with an isopycnic separation on cells treated with emetine to block lagging strand synthesis. See text for references. Modified from [START_REF] Hyrien | Peaks cloaked in the mist: The landscape of mammalian replication origins[END_REF]. [START_REF] Rhind | DNA replication timing[END_REF]. In vertebrates, early-replicating regions are usually rich in active genes, with a solid positive correlation between transcription and early replication timing detected in higher eukaryotes (in metazoans, at least 3/4 of the coding genome is replicated in the first half of the S-phase) [START_REF] Hiratani | Global reorganization of replication domains during embryonic stem cell differentiation[END_REF], for a review see [START_REF] Rhind | DNA replication timing[END_REF]). However, these features are not sufficient to specify the replication timing (Rivera-Mulia & Gilbert 2016). Later studies have proposed that chromatin accessibility is the most reliable predictor for replication timing [START_REF] Gindin | A chromatin structure-based model accurately predicts DNA replication timing in human cells[END_REF]. Replication timing is also clearly related to the development of an organism, under the control of epigenetic factors. In mammals, more than 50% of the genome is subject to extensive changes in replication timing during development [START_REF] Hiratani | Global reorganization of replication domains during embryonic stem cell differentiation[END_REF][START_REF] Ryba | Evolutionarily conserved replication timing profiles predict long-range chromatin interactions and distinguish closely related cell types[END_REF].

I.5.2.2 Spatiotemporal replication program

A strong connection has long been observed between replication timing and spatial nuclear organisation. In mammals, early-replication occurs within the inner part of the nucleus while late-replication happens at the periphery (see [START_REF] Rhind | DNA replication timing[END_REF] for a review).

Recent genome-wide analyses by chromatin conformation capture (Hi-C14 ) have revealed a strong correlation between replication timing and the 3D chromatin organisation. In fact, this correlation is one of the strongest ever observed in genomics [START_REF] Rhind | DNA replication timing[END_REF]).

Chromatin appears to be organised in two compartments that are spatially separated and that What defines origin avidity for the limiting factor(s) is unclear, although it has been shown that the chromatin context established in G1, affects the probability of a given ORI to compete for limiting factors in S-phase [START_REF] Kim | Early-replicating heterochromatin[END_REF]. It is also possible that the diffusion rate of the activators differs between early-and late-replicating nuclear domains [START_REF] Gauthier | Control of DNA replication by anomalous reaction-diffusion kinetics[END_REF].

It is still debated whether establishing a specific spatiotemporal DNA replication program improves the fitness, or if replication timing simply reflects the chromosomal organisation of the cell, without any biological benefit [START_REF] Rhind | DNA replication timing[END_REF]. Interestingly, a reproducible DNA replication timing pattern can be recognised in a cell-population although single-molecule studies have shown that each cell fires different cohorts of ORIs to replicate its genome [START_REF] Czajkowsky | DNA combing reveals intrinsic temporal disorder in the replication of yeast chromosome VI[END_REF][START_REF] Patel | DNA replication origins fire stochastically in fission yeast[END_REF]. Indeed, if individual ORIs are activated in a stochastic manner (reflecting their characteristic efficiencies), they will fire, on average, at reproducible times, with the most efficient ORIs activated earlier, on average, than the least efficient ones [START_REF] Bechhoefer | Replication timing and its emergence from stochastic processes[END_REF].

I.5.2.4

Sequence-specific metazoan origins? "G-rich" evidences from λ-SNS studies

As already explained (see paragraph I.4.1), pre-omics studies failed to identify specific sequence motifs in metazoan ORIs, as opposed to S. cerevisiae. Later studies showed that metazoan ORCs can bind any DNA sequence [START_REF] Remus | DNA topology, not DNA sequence, is a critical determinant for Drosophila ORC-DNA binding[END_REF][START_REF] Vashee | Sequence-independent DNA binding and replication initiation by the human origin recognition complex[END_REF], although with a slight preference for AT-rich sequences on dsDNA and for G-quadruplexes16 (G4s) on ssDNA and RNA [START_REF] Hoshina | Human origin recognition complex binds preferentially to G-quadruplex-preferable RNA and single-stranded DNA[END_REF]. These and other experiments suggested that specific DNA sequence motifs might not be the determinant element for ORC binding. However, several genome-wide origin mapping studies reported an enrichment of λ-SNS in guanine-rich sequences. λ-SNS peaks were found to be enriched in G4 motifs [START_REF] Besnard | Unraveling cell type-specific and reprogrammable human replication origin signatures associated with G-quadruplex consensus motifs[END_REF], GC-rich sequences [START_REF] Cayrou | New insights into replication origin characteristics in metazoans[END_REF] and CpG islands17 (CGI) [START_REF] Cayrou | Genome-scale analysis of metazoan replication origins reveals their organization in specific but flexible sites defined by conserved features[END_REF]. Moreover, such structures also correlated with ORI efficiencies (i.e. the more efficient ORIs are G4-or CGIassociated) and ORI timing (G-rich ORIs are generally early ORIs). Contrary to these studies, [START_REF] Karnani | Genomic study of replication initiation in human chromosomes reveals the influence of transcription regulation and chromatin structure on origin selection[END_REF] found enrichment in AT sequences at ORI sites and, more recently, Foulk et al (2015a) demonstrated that ORIs can be found both at AT-and GC-rich regions after correcting for λ-exo biases (discussed in paragraph I. 5.3.2). Strikingly, bubble-seq experiments disagreed with λ-SNS studies on the necessity of G4s at replication ORIs [START_REF] Mesner | Bubble-seq analysis of the human genome reveals distinct chromatin-mediated mechanisms for regulating early-and late-firing origins[END_REF].

These experiments do not exclude that some ORIs actually coincide with G4s and CGIs, but they simply show that G-rich sequences might not be the main feature determining ORIs' position. Taking in account all these results, it is most likely that at least 1/3 rd of the total replication ORIs localise at or near G4s, and that 1/10 th to 1/3 rd are found at CGIs [START_REF] Urban | The hunt for origins of DNA replication in multicellular eukaryotes[END_REF], many of the latter being also G4-associated.

I.5.2.5 Whole-genome nucleotide compositional skew & replication fork directionality analysis

In the second half of the 90s, Lobry showed that bacteria have different nucleotide composition on the complementary strands: the leading strand possesses more G and T than C and A [START_REF] Lobry | Asymmetric substitution patterns in the two DNA strands of bacteria[END_REF]. This is because the two strands are subjected to different substitution rates during evolution. It is therefore possible to define the GC and TA skews as SGC= (G-C)/(G+C)

and STA=(T-A)/(T+A), respectively. SGC plot were afterward used to detect replication ORI and termini in bacteria as abrupt upward and downward shifts, respectively, in the skew profile [START_REF] Grigoriev | Analyzing genomes with cumulative skew diagrams[END_REF]. Similarly, skew profiles allowed to predict ORIs location in mammals [START_REF] Touchon | Replicationassociated strand asymmetries in mammalian genomes: toward detection of replication origins[END_REF]. Interestingly, such predicted ORIs fall within intergenic regions and open chromatin regions [START_REF] Audit | Open chromatin encoded in DNA sequence is the signature of 'master' replication origins in human cells[END_REF]. The upward shifts in the skew (i.e. "S-jumps") delimit "domains" as the region in between two successive shifts. Within the domain the skew profile decreases linearly, forming, together with its borders, an N-shaped profile [START_REF] Huvet | Human gene organization driven by the coordination of replication and transcription[END_REF].

Thus, skew domains are called "N-domains". N-domains turned out to match very well with Ushaped domains of the timing profiles (Figure I-5 A-B, E-F): S-jumps in the skew profiles correspond to early replicating peaks (borders of the U-domains), while the centre of the Ndomains correspond to late-replicating zones (valleys of the U-domains) [START_REF] Hyrien | From simple bacterial and archaeal replicons to replication N/U-domains[END_REF].

The replication time gradient was afterwards shown to depend on both the replication fork speed and the replication fork directionality in the relationship dt/dx = (R-L)/v 18 [START_REF] Baker | Replication fork polarity gradients revealed by megabase-sized U-shaped replication timing domains in human cell lines[END_REF][START_REF] Guilbaud | Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome[END_REF]. In other words, both the N-and U-shape of skew and timing domains, imply a linear inversion of the replication fork directionality across their length.

The linear inversion of the average direction of replication forks may simply emerge as the population average of forks emanating from border origins and terminating at different sites.

However, this imposes a broad dispersion of ORI firing time such that a fork emanating from a border ORI can sometimes traverse the entire domain before the other ORI fires, and vice versa.

To account for megabase-sized N/U-domains, this dispersion would need to be larger than the actual duration of S-phase. This strongly argues that additional ORIs fire inside the domain in a specific temporal sequence that results in the linear inversion of the average fork direction.

indeed, an ORI firing "cascade" model, in which forks emanating from highly efficient ORIs at the U-borders stimulate other ORIs while moving toward the U-valley, could quantitatively 18 The derivative of the replication timing (dt/dx), depends on fork speed (v) and on the proportion of rightward (R) and leftward (L) moving forks in the cell population. Initiation zones were enriched in ORC binding sites, specific chromatin marks (DNAse I hypersensitive sites, H3K4me1 and H2A.Z histone variant) and frequently colocalised with TAD borders. Termination also happens dispersedly over large zones at positions determined by the location and firing time of adjacent ORIs, and often overlapping active genes [START_REF] Petryk | Replication landscape of the human genome[END_REF]. In contrast to λ-SNS studies (see paragraph I.5.2.4), OK-seq showed that most of the initiation sites do not associate with G4s or CGIs, although an enrichment of CGIs was observed at the initiation zones borders. Overall, OK-seq better agreed with bubble-trap techniques and showed broad initiation zones, probably supporting one stochastic initiation event within the zone. The initiation sites were coordinated to the transcriptional activity (i.e. the replication fork progression was co-oriented with the transcription) and appeared epigenetically regulated in order to fit the cell-type-specific replication programs.

I.5.2.7 General features of mammalian genome replication

Many genome-wide studies agreed that there is an interplay between transcription and replication. Often ORIs were found at the 5' end of actively transcribed genes. This is especially true in the case of early-replicating ORIs. Late-replicating regions, on the other side, were The first high-throughput study of DNA replication mapped short single-strand (SSS)

DNA, thought to represent newly synthetized DNA, in lymphoblastoid cell lines using microarray chips [START_REF] Lucas | High-throughput mapping of origins of replication in human cells[END_REF]. ORIs at various well-studied loci including β-globin, FMR1, LMNB2 and myc could be mapped, together with an unknown 1 Mb region on chromosome 22. Some new ORIs could be detected, however, the amount of SSS DNA recovered was ~1000 fold higher than the expected amount, suggesting huge contaminant DNA in the library [START_REF] Hyrien | Peaks cloaked in the mist: The landscape of mammalian replication origins[END_REF]. Such DNA was most likely coming from pre-existing nicks present in parental DNA. λ-exo digestion was therefore introduced to eliminate SSS while preserving the RNA primed molecules19 [START_REF] Cadoret | Genome-wide studies highlight indirect links between human replication origins and gene regulation[END_REF]. λ-SNS-chip was adopted to map ORIs on the 1% of the genome of HeLa cells studied by the ENCODE20 project. In the first experiments λ-exo treatment was mandatory in order to detect replication ORIs (in the absence of treatment, up to ~30 fold less peaks were detected) [START_REF] Cadoret | Genome-wide studies highlight indirect links between human replication origins and gene regulation[END_REF]. However, in a later study, a comparable number of ORIs peaks was obtained with or without λ-exo treatment [START_REF] Valenzuela | Preferential localization of human origins of DNA replication at the 5'-ends of expressed genes and at evolutionarily conserved DNA sequences[END_REF]. On the other hand, when comparing SNS and BrdU-SNS, no difference was revealed by single-locus PCR analysis [START_REF] Kumar | Utilization of the same DNA replication origin by human cells of different derivation[END_REF]. These results raised a debate on whether it is possible or not to detect real SNS without prior λ-exo digestion, considering that the vast majority of "SSS" are in fact broken strands. Interestingly, it was afterwards proposed that the strength ORIs might have been overestimated about tenfold considering that an accumulation of small (~200 bp), duplex DNA molecules (proposed to represent abortive initiation intermediates), was detected in total genomic DNA 21 [START_REF] Gomez | Overreplication of short DNA regions during S phase in human cells[END_REF].

Thus, the SNS peak strengths depended on whether the SNS signal was normalized against total -Only 10-14% of the bubble-chip ORIs overlapped 26-35% of λ-SNS-chip;

-As low as 2.2-12.8% of BrdU-SNS-chip overlapped 6.4-33.4% of λ-SNS-chip;

-Feeble concordance was observed in between λ-SNS-chips: 5-6%.

This very poor concordance between studies was initially attributed to their presumed incomplete sets of DNA replication ORIs (i.e. "non-saturation" condition). Although recently confirmed, the lack of saturation is not sufficient to explain the large variability, and the concordance remains incomplete even at saturation [START_REF] Besnard | Unraveling cell type-specific and reprogrammable human replication origin signatures associated with G-quadruplex consensus motifs[END_REF]: of the λ-SNS-seq peaks, 50-65% agree with bubble-chip and 9-20% with BrdU-SNS-chip. Finally, according to the latest study [START_REF] Picard | The spatiotemporal program of DNA replication is associated with specific combinations of chromatin marks in human cells[END_REF], 45-46% of the λ-SNS-seq peaks overlapped the 36-37% of the bubble-seq, and 56.5% of the BrdU-SNS-seq peaks accorded with 50.2% of these λ-SNSseq peaks.

ORC1-ChIP-seq was also used in human cells and identified 13,600 Orc1 binding sites, which do not reveal any sequence consensus (Dellino et al 2013). Only 11-30% of these peaks overlapped λ-SNS peaks, and 47% overlapped bubbles. Note that ORC has many functions other than DNA replication, so that ORC-ChIP may correspond to other functional genomic elements than ORIs.

21 Such molecules possess a 5' RNA primer and therefore are not digested upon λ-exo treatment.

Summarising, bubble-based methods still disagree with SNS-based ones. Bubble traps identified large initiation zones that are variable between cell lines, while SNS called sharp initiation peaks that are more conserved between cell lines. One important consideration is that bubble traps were also validated by 2D gel electrophoresis, which confirmed a purity in replication bubbles of >80% [START_REF] Mesner | Bubble-chip analysis of human origin distributions demonstrates on a genomic scale significant clustering into zones and significant association with transcription[END_REF]. Moreover, the recent OK-seq analysis performed on human DNA [START_REF] Petryk | Replication landscape of the human genome[END_REF], which agrees with both the Repli-seq and the compositional skew profiles (see paragraph I.5.2.6), better concords with bubbles than λ-SNS.

Altogether these data highlight the necessity to use different techniques to map DNA replication (and different peak calling algorithms, possibly) to cross-validate the discoveries made.

I.5.3.2 SNS, the λ-exo concern: about the reliability of current genome-wide methods

The low concordance among λ-SNS studies and between the λ-SNS and the other available techniques, suggested that non-ORI peaks could also be detected. Both singlemolecule data [START_REF] Conroy | Lambda exonuclease digestion of CGG trinucleotide repeats[END_REF][START_REF] Perkins | Sequence-dependent pausing of single lambda exonuclease molecules[END_REF][START_REF] Van Oijen | Singlemolecule kinetics of lambda exonuclease reveal base dependence and dynamic disorder[END_REF] as well as ad hoc λexo studies performed on G0-phase DNA (Foulk et al 2015b), demonstrated that λ-exo does not digest with equal efficiency all the DNA sequences: AT-rich sequences are more efficiently digested than GC-rich ones. It is likely that G4s-and GC-protected sequences are enriched in the λ-SNS analysis, even though they do not correspond to nascent strands. Another possible contamination could come from occasional insertion of ribonucleotides during genome duplication, or extra-chromosomal circular DNAs present in the cells. λ-exo would not digest RNA protected sequences, nor circular DNA, and these sequences would also be enriched in the final library [START_REF] Urban | The hunt for origins of DNA replication in multicellular eukaryotes[END_REF]. Consistent with all these possible sources of bias, many peaks of the λ-SNS studies were shown to overlap peaks obtained on λ-SNS libraries performed on G0 cells (Foulk et al 2015b). Thus, the λ-exo libraries used until today most likely carry both real SNS and false positives (non nascent strands). Various solutions were tried in order to control for these false positives, but the most promising one is to use the λ-"SNS" sequences performed on G0 cells as a control to normalise for non-replicating DNA. This, together with the strand-specific sequencing and well-defined conditions to reduce G4s formations during λexo digestion, could help obtaining pure SNS [START_REF] Urban | The hunt for origins of DNA replication in multicellular eukaryotes[END_REF].

I.6 Interplays between transcription and replication

Both replication and transcription are very processive 22 reactions operating on the same substrate, the DNA, and they may therefore interfere with each other. In eukaryotes, where genome duplication is restricted to a particular moment of the cell-cycle, one possibility is to separate the moment of DNA replication from the transcription step. Although transcription does occur during the S-phase, it has been shown by fluorescent microscopy that transcription generally takes place in spatially distinct domains than those under active replication [START_REF] Wei | Segregation of transcription and replication sites into higher order domains[END_REF]. In the case when replication and transcription simultaneously occur on the same DNA molecule, two types of collision are conceivable: frontal collisions and co-directional collisions.

DNA replication is preferentially co-oriented with transcription in bacteria [START_REF] Kunst | The complete genome sequence of the gram-positive bacterium Bacillus subtilis[END_REF] as well as in metazoans [START_REF] Huvet | Human gene organization driven by the coordination of replication and transcription[END_REF][START_REF] Petryk | Replication landscape of the human genome[END_REF], where ORIs are preferentially found at the 5' end of actively transcribed genes (described in paragraph I.4.1), which reduces the possibility of frontal collisions. Early EM experiments on replicating rDNA in yeast showed that replication and transcription can also simultaneously occur on the same DNA molecule [START_REF] Saffer | Electron microscopic study of Saccharomyces cerevisiae rDNA chromatin replication[END_REF]. The two processes could coexist because of the presence of specific elements, called replication fork barriers (RFBs), which are located 3' to each rDNA unit. RFBs can block replication forks travelling in the opposite direction than transcription and assure the co-directional replication and transcription of the rDNA [START_REF] Brewer | A replication fork barrier at the 3' end of yeast ribosomal RNA genes[END_REF], for reviews see [START_REF] Lambert | Impediments to replication fork movement: stabilisation, reactivation and genome instability[END_REF], [START_REF] Michel | Rescue of arrested replication forks by homologous recombination[END_REF], [START_REF] Hyrien | Mechanisms and consequences of replication fork arrest[END_REF]). Replication and transcription machineries travel at comparable speeds in eukaryotes [START_REF] Darzacq | In vivo dynamics of RNA polymerase II transcription[END_REF], thus minimising the probability of co-directional collisions. However, co-directional collisions remain possible in bacteria as replication forks travel ~12-30 fold faster than transcription machineries (for review see [START_REF] Merrikh | Co-directional replicationtranscription conflicts lead to replication restart[END_REF]). In addition, in higher eukaryotes, the timing of transcription and replication programs are coordinated: transcribed genes are generally early replicated during S-phase, while non-transcribed genes are replicated later [START_REF] Fraser | Cell-cycle regulated transcription associates with DNA replication timing in yeast and human[END_REF][START_REF] Goldman | Replication timing of genes and middle repetitive sequences[END_REF]. This might give the cell more time to deal with potential interference problems and make sure that replication is completed (reviewed in [START_REF] Evertts | Back to the origin: reconsidering replication, transcription, epigenetics, and cell cycle control[END_REF]).

Despite all these mechanisms, collision events can occur and block replication forks. In eukaryotes, this activates the S-phase checkpoint that will stabilize and restart the stalled forks (reviewed in [START_REF] Zeman | Causes and consequences of replication stress[END_REF]). Failure to do so is deleterious : transcription is now 22 The processivity of an enzyme is its ability to catalyse multiple reactions without releasing its substrate.

regarded as a prevalent source of replication stress and genetic instability (see Santos-Pereira and Aguilera (2015) for a review).

I.7 Towards single-molecule, genome-wide ORI mapping I.7.1 Advantages of single-molecule techniques

As discussed above (see paragraph I.5.2), despite their far-from-perfect agreement, population-based techniques have massively increased our knowledge of the DNA replication process. However, these techniques reveal an average behaviour in a cell population, without accounting for cell-to-cell variability. Conversely, if molecules could be observed one-by-one, all the complexities and peculiarities that are "smoothed" in the population analysis could be detected. We have seen that there is a large flexibility in ORI usage, but this does not prevent phase [START_REF] Jackson | Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells[END_REF]. The extent of replicated DNA and the velocity of fork progression could be quantified at a SM level. However, the variability in the stretching factor (~10%), the poor fibre alignment and the presence of many bundles of fibres complicated the fibre analysis making reliable measurements difficult to perform (see [START_REF] Parra | High resolution visual mapping of stretched DNA by fluorescent hybridization[END_REF], [START_REF] Jackson | Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells[END_REF]; reviewed in Techer et al ( 2013)).

Later on, a technique capable of physically stretching and aligning DNA molecules on hydrophobic glass coverslips, named "DNA (or molecular) combing24 " was invented [START_REF] Bensimon | Alignment and sensitive detection of DNA by a moving interface[END_REF][START_REF] Bensimon | Stretching DNA with a Receding Meniscus: Experiments and Models[END_REF]. DNA molecules could be anchored to a hydrophobic surface thanks to their high binding specificity for the DNA ends and were stretched by a receding air-water interface generated by capillary force between two glass coverslips (one silanated25 and one untreated). The binding of DNA ends to the silanated coverslip is thought to happen because at a specific pH26 the DNA ends (that are exposing polar groups and are more negative than their mid-segment) can specifically bind to the ionisable groups coating the hydrophobic surface [START_REF] Allemand | pH-dependent specific binding and combing of DNA[END_REF]. The rest of the DNA cannot form these interactions, and it is left linearized and dry behind the receding meniscus. Between combed DNA molecules there is a low variability in the stretching factor (~2.3% according to the original article [START_REF] Bensimon | Alignment and sensitive detection of DNA by a moving interface[END_REF], see paragraph III.2.1.3 in the Results section for a detailed quantification) and directly measuring the micrometric length (i.e. in µm) equals measuring a well-defined genomic distance (i.e. in kb) without the need of an internal reference [START_REF] Michalet | Dynamic molecular combing: stretching the whole human genome for high-resolution studies[END_REF]. By incubating the hydrophobic glass coverslip inside the buffered DNA solution and by lifting it at a constant speed (typically 300 µm/sec), the stretching could be further controlled (i.e. "dynamic" molecular combing, [START_REF] Michalet | Dynamic molecular combing: stretching the whole human genome for high-resolution studies[END_REF]. See Figure I-6).

The DNA combing technique was rapidly adapted to DNA replication studies. To do so, a prior pulse-labelling of the newly-synthetized DNA with digoxigenin-dUTP and biotin-dUTP was introduced in order to reveal early-and late-replicated DNA in Xenopus egg extracts [START_REF] Herrick | Replication fork density increases during DNA synthesis in X. laevis egg extracts[END_REF]. Xenopus egg extracts is a powerful cell-free system able to support the synchronous assembly of working nuclei that undergo efficient semi-conservative replication [START_REF] Blow | Initiation of DNA replication in nuclei and purified DNA by a cell-free extract of Xenopus eggs[END_REF]). Thymidine nucleotide analogs added to the extracts are efficiently incorporated in the newly-synthetized DNA. The two metabolic labels could be afterwards revealed with fluorescent anti-digoxigenin and anti-streptavidin antibodies, respectively. DNA combing allows to visualise ORI firing events, measure inter-ORIs distances (IOD) but also to monitor the replication fork progression on the stretched (i.e. "combed") DNA molecules and to compute the rate of initiation per length of unreplicated DNA (for reviews, see [START_REF] Lebofsky | Single DNA molecule analysis: applications of molecular combing[END_REF] and [START_REF] Techer | Replication Dynamics: Biases and Robustness of DNA Fiber Analysis[END_REF]). Thanks to all these novel measurable parameters, DNA combing rapidly became the most used SM method to study DNA replication. Combing and IdU can freely permeate the cytoplasmic membrane in mammalian cells [START_REF] Jackson | Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells[END_REF]. Expression of several copies of the Herpes simplex thymidine kinase (TK) gene under the control of a strong constitutive promoter allowed incorporation of exogenous BrdU and DNA combing analysis also in TK+ budding yeast cells 27 [START_REF] Lengronne | Monitoring S phase progression globally and locally using BrdU incorporation in TK(+) yeast strains[END_REF]. Moreover, performing FISH on combed molecules permit their genomic identification [START_REF] Pasero | Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus[END_REF].

Thus, with DNA combing it is either possible to anonymously analyse all the DNA molecules present on the coverslips and gather general information about the replication mechanism in a given genome, or to probe a specific target locus thanks to FISH (which identify the locus-oforigins of the "FISHed" molecules).

Summarising, DNA combing studies allow the quantification of a variety of SM parameters (for reviews, see [START_REF] Lebofsky | Single DNA molecule analysis: applications of molecular combing[END_REF] and [START_REF] Techer | Replication Dynamics: Biases and Robustness of DNA Fiber Analysis[END_REF]):

-Fraction of replicated DNA; -Replication bubble size;

-Inter-bubble distances and inter-ORI distances (IODs);

-Fork density and velocity. 27 The task would otherwise be impossible because of the lacking of TK in yeast. There are other bottlenecks linked to the FISH probing: 1) FISH is further complicating the experiments introducing hybridisation/washing steps harsher than the ones already required by the detection of incorporated labels at replication forks; and 2) it imposes to concentrate on a small genomic region for a single combing experiment. To give an idea of the tight restriction imposed by FISH-probing, one must think that to study a region of ~1-2 Mb, corresponding to only ~0.05% of the human genome, it is required to design and prepare a collection of ~25 probes29 in order to unambiguously align the DNA molecules on the reference genome. This is extremely time-consuming and tedious. On human cells, obtaining a fibre coverage of ~200x

(corresponding to a replicative coverage of ~5-10x) on a 1 Mb locus requires ~6 months. The analysis is manually performed and requires careful blind evaluation from multiple researchers in order to discriminate false positives (e.g. bundles of fibres). Moreover, when targeting a specific locus, ~99.95% of the molecules that were carefully extracted, combed and revealed, are actually anonymous and therefore non-informative.

The last bottleneck to the dissemination of molecular combing studies is the difficulty to develop software to automate the analysis. The high background noise, together with the crenelated signals, makes this a daunting task. To my knowledge, only 3 attempts have been done in almost 20 years 30 , but none of them was made available to the scientific community for further development. The future of the SM analysis of DNA replication will strongly reside also in the efforts put by the scientific community to develop and share reliable and accurate automated analysis tools.

Due to these barriers, ORIs have been historically mapped locus-by-locus and only a few dozens of them could be identified during the last decades, some of which are still debated [START_REF] Hyrien | Peaks cloaked in the mist: The landscape of mammalian replication origins[END_REF]. SM techniques able to automatically identify at a high throughput the locus-oforigin and the DNA replication signal of every single fibre are required to provide the scientific community with the whole-genome, SM data necessary to correctly understand the eukaryotic DNA replication program.

I.7.4 Possible achievement of genome-wide, single-molecule techniques

Following what was presented in the genome-wide studies section (I.5.2), it is pretty clear that ensemble-(i.e. population-) and SM-based methods provide very different results.

Both approaches are necessary and non-redundant. SM data are required to clarify some unresolved issues. First of all, the quantification of ORI efficiencies need to be tackled with high-throughput SM methods. Most genome-wide approaches do not allow precise quantification of origin efficiency. λ-SNS tend to highlight narrowly localized, highly efficient origins, whereas bubble trap, OK-seq and SM studies (the latter being so far limited to a few loci) suggest a predominance of broad initiation zones consisting of multiple inefficient sites.

Second, SM observations of multiple active replicons on the same molecule allow to analyse the spatial and temporal correlations between neighbouring ORIs and neighbouring fork speeds.

For example, since it is possible to infer both the firing time and the location of multiple origins on the same fibre, it would be possible to discover ordered ORI firing patterns (i.e. spotting preferential positions with respect to the existing forks). Third, SM techniques visualize equally well site-specific and dispersed initiation events, whereas genome-wide mapping techniques that search peaks of abundance of initiation intermediates can only reveal the most efficient origins. Fourth, SM studies can reveal the full dispersion of replication fork speeds at a locus; SNS-seq and bubble-seq do not allow fork speed measurements, and replication timing and OK-seq only allow indirect and low resolution measurements of mean fork speed.

SM studies might be useful to fully explore ORI flexibility in the yeast genome. Both pre-and post-"omics" studies detected ORIs at specific, well-described ARSs. However, Gros

I.7.5 Inspiring tools for developing novel, high-throughput, single-molecule techniques

The first important bottleneck of the current SM techniques is the obligation to use antibodies to reveal the replicated tracts (discussed in paragraph I.7.3). However, thanks to the advances in fluorescent imaging (e.g. total internal reflection fluorescent microscopy, TIRFM)

and the increasing interplay between biology and physics (i.e. micro-and nano-fluidic applied to biology), this bottleneck can be resolved. In 2010, J.C. Walter's and A.M. van Oijen's laboratory was able to visualise the replication status of bacteriophage λ-DNA replicated in a microfluidic flow cell (opportunely anchored by its ends) in the presence of Xenopus egg extract and dig-dUTP [START_REF] Yardimci | Uncoupling of sister replisomes during eukaryotic DNA replication[END_REF]. By flushing a detergent buffer and staining the DNA with SYTOX31 and with anti-dig-fluorescein antibodies, the replication process was monitored.

Interestingly, under TIRFM, DNA stained with SYTOX showed a remarkable doubling of fluorescence intensity corresponding to the anti-digoxigenin fluorescent tracts. Thus, fluorescent DNA intercalators are, a priori, sufficient to reliably detect replication bubbles and estimate ORI positions as their mid-points. However, bubble detection alone does not allow to measure replication fork velocities. In order to do so, a double-labelling scheme is needed.

Luckily, several orthogonal methods for antibody-free, metabolic labelling of DNA have been recently reported. The thymidine analog 5-ethynyl-2'-deoxyuridine (EdU) is small and can polymerase [START_REF] Xiao | Rapid DNA mapping by fluorescent single molecule detection[END_REF]. In a similar experiment, several AlexaFluor647-dUTPs, rather than just one ddNTP, were incorporated at the target nicking site [START_REF] Chan | Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity[END_REF]. In the first case TIRFM was required to detect the single fluorophores, while in the latter study the labels where detected by fluorescence-resonance energy transfer (FRET) collected from the AlexaFluor. Very recently, genomic mapping was revolutionised when the power of fluorescent DNA barcoding was combined with the high-throughput of a nanofluidic device able to automatically imaging hundreds of thousands of single DNA molecules. Molecules are electrophoretically driven in an array of 13,000 parallel nanochannels, stopped and imaged, for multiple cycles. The result was astonishing: ~900.000 DNA molecules longer than 150 kb could be aligned thanks to their fluorescent barcoding, resulting in an outrageous ~70-fold coverage of 93% of the human genome [START_REF] Cao | Rapid detection of structural variation in a human genome using nanochannel-based genome mapping technology[END_REF]. To emphasise the power brought by these techniques, the term "next generation mapping" (NGM) was coined (reminiscent of the "NGS effect" on the sequencing field).

I.7.6 Goal and achievements of the thesis

The goal of this thesis was to develop novel, fast and efficient tools to map DNA replication at the single-molecule level, genome-wide.

Here we show that:

1. By staining DNA with YOYO-1, a fluorescent DNA intercalator, is possible to directly distinguish the replication bubbles from the unreplicated tracts thanks to their double fluorescence intensity.

I.7.7 Strategies

In order to achieve this goal, we decided to solve the two major problems discussed above (paragraph I.7.

II.1 Preparation of polystyrene coated coverslips

II.1.1 Glass coverslips cleaning

Glass coverslips (22×22 mm, Esco) were placed in teflon mini-racks in a well separated and vertical position ensuring the maximum contact with solutions during washing steps.

Coverslips where rinsed in Milli-Q water and soaked for 5 minutes in isopropanol in an ultrasonic bath (Branson 3800, Emerson) to remove organic impurities. Then, they were rinsed again in Milli-Q water to remove traces of isopropanol prior to a final soaking in chloroform (both in ultrasonic bath). Coverslips were let completely dry and exposed for 2 min to plasma treatment (Expanded Plasma Cleaner, Harrick Plasma). The clean coverslips were then baked for >1 h at 110°C in a dry oven to completely dehydrate them immediately before the hydrophobic coating. All chemicals were purchased from VWR.

II.1.2 Polystyrene coating

Polystyrene (MW 280,000; Sigma Aldrich) was dissolved in toluene (Sigma Aldrich) at a concentration of 50 mg/mL. The solution was deposed on a glass coverslip cleaned as in II.1.1, and spin-coated for 30 s at 3000 rpm. Polystyrene-coated coverslips were conserved at room temperature repaired from humidity and used within 2 months. Although only ten nicking endonucleases (NEs) are commercially available at the time of writing (New England Biolabs), even short viral genomes (like λ-DNA) contain some locations where two nicking sites are sufficiently close to each other to allow a selective oligo displacement (see Table II Adapted from [START_REF] Kuhn | Labeling of unique sequences in double-stranded DNA at sites of vicinal nicks generated by nicking endonucleases[END_REF].

II.2 Alkaline and neutral agarose gel electrophoresis to monitor fluorescent dUTP incorporation

We reasoned that we could design a specific probe that 1) is able to substitute the selected target oligonucleotide (i.e. the region flanked by two NE recognition sites) in a displacing reaction and 2) that is also complementary to the cohesive extremity (cos) of λ-DNA

II.5.2 Fluorescent barcoding, DNA staining and sample run

The nicking at Nt.BspQI sites, the YOYO-1 DNA staining and the sample run was performed according to the manufacturer specifications for the Irys system (BioNano Genomics).

III. Results

III.1 Premise

The Results chapter first describes the advances obtained by developing the DNA combing technique, which was the original goal of my thesis (section III.2). A second part concerns the use of a nanochannel device to stretch and image a larger number of DNA molecules (section III.3). Preliminary results are also illustrated, but their exploitation and analysis are still ongoing tasks.

The molecular combing section is constituted of five paragraphs. The last one (paragraph III. Finally, the nanochannel part reports preliminary results and set up future goals, and is a starting point to discuss the future of the single-molecule studies of DNA replication.

III.2 Molecular DNA combing III.2.1 Surface testing, definition of optimum DNA combing conditions

Testing the quality of the hydrophobic surfaces prior to DNA combing of biological samples is a prerequisite to such experiments. A good molecular combing experiment depends equally on the quality of the biological sample and the quality of the substrate chosen to stretch the DNA.

Scrupulous attention is required for the preparation and the characterisation of the hydrophobic coverslips, as their quality can vary widely between similarly prepared batches.

Their quality can be controlled by stretching simple, pure, double-stranded DNA specimens as bacteriophage λ-DNA.

Among the various parameters defining a "good" hydrophobic substrate for molecular combing, we can at least identify: Here I detail the results obtained with both silane-and polystyrene-coated surfaces, for each of the above-mentioned parameters.

Silane-coated surfaces are considered as the gold standard for single molecule analysis of DNA replication by DNA combing [START_REF] Allemand | pH-dependent specific binding and combing of DNA[END_REF][START_REF] Bensimon | Alignment and sensitive detection of DNA by a moving interface[END_REF][START_REF] Bensimon | Stretching DNA with a Receding Meniscus: Experiments and Models[END_REF][START_REF] Michalet | Dynamic molecular combing: stretching the whole human genome for high-resolution studies[END_REF]. This is in part because DNA stretched on such surfaces does not easily detach from the substrate during the harsh denaturation and hybridization steps required for FISH, in contrast to other types of surfaces. However, since FISH was not required in our experiments, there was no need for such harsh treatments, and we were not constrained to exclusively use silane-coating. We therefore also tested polystyrene-coated coverslips, which are easier and cheaper to produce.

III.2.1.1 Density of adsorbed DNA molecules

The number of molecules adsorbed on a coverslip is a critical parameter. Ideally, one would like to have the whole field-of-view completely covered by DNA fibres, without overlapping molecules. pH, salt and buffer concentration influence the density of molecules [START_REF] Benke | pH-and salt-dependent molecular combing of DNA: experiments and phenomenological model[END_REF].

During my Master internship, I confirmed that pH 6.0 is the correct choice when When combing at 250 mM this value further raised to 8.17 folds. This huge difference most probably depends on the different chemical characteristics of the surface. Based on these data, I concluded that our surfaces are performing very well in adsorbing DNA at concentrations similar to the published data [START_REF] Allemand | pH-dependent specific binding and combing of DNA[END_REF][START_REF] Michalet | Dynamic molecular combing: stretching the whole human genome for high-resolution studies[END_REF].

Silane-coated coverslips seems to perform better, but the polystyrene ones also permit to obtain an adequate density of combed molecules.

III.2.1.2 Percentage of exploitable DNA fibres

Only linear DNA molecules can be analysed in a combing experiment. We say that a given DNA molecule is "specifically" adsorbed on the coverslip when it binds the surface through its DNA ends. This is thought to happen only within a very narrow pH window. On the contrary, globular molecules, which arise from "unspecific" DNA binding, should never be observed after having defined reliable combing conditions. I never observed globular DNA structures when testing the surfaces using control λ-DNA, however, I could repeatedly observe The most important parameter to evaluate is the stretching factor, which is essential to establish a correlation between the micrometric and the genomic distance (i.e. from μm to kb).

This factor might slightly change between different batches of surfaces and needs to be constantly monitored after their production.

After An interesting observation is that is possible to recognise a "harmony" in the distribution of molecule lengths: a second cluster, roughly located at half of the size corresponding to fulllength λ molecules, can be observed. In addition, when combing on polystyrene surfaces (Figure III-8 C-E), a third peak is present at short lengths (i.e. around 3 μm). This might derive from pre-existing breakage in the preparation of λ-DNA used to comb, but I have no precise explanation on this point.

Notice that, although is still possible to comb on polystyrene coverslips at 250 mM MES, no evident full-length peak can be identified. Thus, this concentration is too high to obtain the characteristic profile that enables to evaluate the stretching factor. and at 100 mM (N=460) MES. More than 35% (50 mM) and more than 20% (100 mM) of molecules are specifically localised in a 1 µm bin corresponding to the full-length λ-DNA molecules. In all cases, the same preparation of λ-DNA (62.5 pg/µL) at pH 6.0 was used. B| Polystyrene surfaces. Unlike silane, a tri-phasic distribution can be recognised at 50 mM (N=422) and at 100 mM MES (N=622). The longersize peak, again, correspond to full-length molecules and contains ~20% (50 mM) or >25% of the molecules in a small 1 µm bin. At 250 mM (N=705), no evident full-length peak can be recognised. In all cases, the same preparation of λ-DNA (500 pg/µL) at pH 5.5 was used.

In order to evaluate the precise stretching factor in the different combing conditions I opted for a non-linear regression of the size frequencies. The bell-shaped, full-length peaks were fit with Gaussian distributions, and the means of the Gaussians were taken as "real" value, l, for the evaluation of the stretching factor (Figure III-9). The ratio l/l0, were l0 corresponds to the crystallographic length of non-stretched λ-DNA (l0 = 16.2 μm), is defined as stretching factor (s). For silane, s was 1.537 in the case of 50 mM MES and 1.373 for 100 mM MES. For polystyrene, the correspondent s values were 1.664 (50 mM) and 1.530 (100 mM). At 250 mM, the calculated value of 1.10 is certainly under-estimated, due to the impossibility to discern a full-length peak. Therefore, polystyrene coverslips result in a higher stretching capacity (up to 66.4% overstretching) compared to silane (up to 53.7%). This could also result in an increased fraction of broken molecules, explaining the above-described higher percentage of short molecules (i.e. the peak at 3 μm). In conclusion, with both silane and polystyrene coverslips: 1) a precise evaluation of the stretching factor is possible, 2) this factor decreases by increasing the MES concentration, and 3) it is not possible to evaluate the stretching factor when exceeding 100 mM MES.

Knowing the size of the λ-DNA genome (48,502 bp) and knowing its contour length upon stretching (l), dividing the genome size (kb) per l (µm) is possible link the genomic to the micrometric distance. For silane, this is resulting in the correspondence: 1 μm = 1.95 kb (at 50 mM MES). This value is very close to the well-known "1 μm = 2 kb" correspondence which is usually described as "gold-standard" in literature [START_REF] Michalet | Dynamic molecular combing: stretching the whole human genome for high-resolution studies[END_REF]. For polystyrene, however, at the same buffer concentration, the correspondence was 1 μm = 1.80 kb. This value is also in agreement with previous studies on hydrophobic surfaces [START_REF] Allemand | pH-dependent specific binding and combing of DNA[END_REF]. Thus, a slightly higher spatial resolution can be achieved with polystyrene coverslips compared to silane ones.

III.2.1.4 Alignment of DNA fibres

The density of DNA molecules on the coverslip is only a partial indication of its quality.

The degree to which molecules are aligned in the same direction must also be evaluated. Molecules shorter than 10 kb where arbitrarily excluded from the analysis. Too short molecules are difficult to measure because of the error in defining end positions. U-shaped and Y-shaped molecules were also excluded from the analysis. I concluded that, on average, the surfaces produced "in-house" showed a very reproducible alignment of the stretched molecules, helping the univocal identification of DNA fibres within the molecule array. For some unclear reasons, the misalignment of DNA fibres seems to slightly increase by increasing the MES concentration on silane, but not on polystyrene surfaces.

III.2.1.5 Uniformity of the fibres' fluorescent signal

Before developing methods capable to detect replication bubbles by direct DNA fluorescent intensity, I needed to choose the better performing substrate in signal detection.

In order to evaluate the fluctuations of the YOYO-1 signal on unreplicated DNA, I measured the mean molecule intensity (i.e. the average intensity value within the molecule32 )

and the standard deviation of the molecule intensity of 354 (50 mM) and 460 (100 mM) λ-DNA fibres for silane, and 422 (50 mM), 622 (100 mM) and 705 (250 mM) fibres for polystyrene.

Molecules of all sizes were taken in account for this analysis. Y-shaped molecules were excluded. In all cases, λ-DNA was stained at a 1:10 YOYO-1 to DNA base pairs ratio, and the same microscope setup was used. Interestingly, molecules combed on polystyrene surfaces showed higher mean intensities than on silane, and their distributions were broader at all MES The same consideration is valid at 100 mM MES (CVPolystyrene100 = 0.246 vs CVSilane100 = 0.206).

At 250 mM MES (CVPolystyrene250 = 0.253), the comparison is not possible. In other words, these results show that when comparing the mean intensities of different molecules (i.e. intermolecule variability of the mean fluorescent intensities) there is less variability on silane surfaces than on polystyrene ones. Thus, silane surfaces, especially at 50 mM MES, seem to be more suitable to our purpose to detect DNA replication bubble by direct fluorescence alone.

33 Also known as variance coefficient. It measures the relative variability in one sample. Summary:

1. When looking at the variability in the mean fluorescent signal between different molecules (inter-molecule variability), less variability is observed with silane than with polystyrene;

2. When looking at the variability in the fluorescent intensity within molecules (intramolecule variability), less variability is also observed with silane than with polystyrene; I concluded that silane was the best choice to develop the detection of DNA replication bubbles by their higher fluorescence intensity than unreplicated DNA segments.

III.2.2 Fluorescent labelling of specific nicking endonucleases sites

The possibility to label DNA molecules at specific sequences using fluorescent nucleotide analogs is a relatively recent advance in molecular biology. When I started the experiments, few protocols for fluorescent DNA barcoding had been reported [START_REF] Xiao | Rapid DNA mapping by fluorescent single molecule detection[END_REF], [START_REF] Chan | Natural and engineered nicking endonucleases--from cleavage mechanism to engineering of strand-specificity[END_REF], see paragraph I.7.5 for details).

The first data we obtained using Cy3-dUTP were unsatisfactory: some molecules showed the expected fluorescent labels, but the background noise was too high to detect specific fluorescent patterns (data not shown). As I was not able to understand what was causing the high background noise, I decided to break down the problem into parts and first investigate whether fluorescent nucleotide precursors can be incorporated at the expected nicked sites.

III.2.2.1 Early essays

After bioinformatic analysis, we reasoned that we could take advantage of the existing commercial endonuclease Nb.BbvCI. The enzyme, which recognizes a specific doublestranded DNA sequence (5'-CCTCAGC-3') but cleaves only one strand, has 7 target motifs along the short λ-phage genome (48,502 base-pairs). In particular, two sites (30.9 kb and 31. After incubation with Nb.BbvCI, genomic λ-DNA was nick-translated with DNA polymerase I or Klenow Fragment (5' and 3' exo-) in the presence of Cy5-dUTP. Every 15 minutes of a 90 minutes course, aliquots were taken and immediately stopped by EDTA addition. Samples were electrophoresed through a 0.7% agarose gel and the gel was stained with ethidium bromide. A| Ethidium bromide detection (Ex. source: 532 nm; Em. filter: 575 nm long-pass (LP)). After 15 minutes, both in DNA polymerase I-and in Klenow-mediated reactions, but not in negative controls, a 17.5 kb band (red arrows) was detected, as expected from the crossing of vicinal nicks. The other expected band (31 kb) was not resolvable from the full-length genomic λ-DNA (48.5 kb). See text. B| Cy5 detection (Ex. source: 635 nm; Em. filter: 665 nm LP). The signal detection was specific as demonstrated by the absence of signal from the markers and negative controls. The generation of double-strand breaks was due to nick-translation reaction with incorporation of Cy5-dUMPs. The intense smear at the bottom of the gel reflects unincorporated Cy5-dUTPs.

We therefore expected that the Klenow reaction would produce the expected labeling pattern of double-stranded DNA molecules whereas the DNA polymerase I reaction would generate partially single-stranded labelled molecules. We introduced a brief purification step in G-50 microspin columns, stained with YOYO-1 and combed the remaining material. After incubation with Nb.BbvCI, λ-DNA was nick-translated with DNA polymerase I in presence of Cy5-dUTP (red) resulting in complete breakage between vicinal nicks (see explanation in the text). The DNA was purified in G-50 microspin columns, stained with YOYO-1 (1 to 20 bp ratio, green), combed on silicone-coated surfaces and immediately visualized by epifluorescence microscopy without mounting. Small fragments were generated, and not all NE sites were 100% labelled, but it was generally possible to assign the labelled fragments to a particular region of the full-length molecule based on its labelling pattern and length. Scale bar: 5 μm (~10 kb). Similar results were obtained with both silane and polystyrene coverslips. In these cases the YOYO-1 signal was more constant along the length of the molecule but the dotty background presumably due to unincorporated Cy5-dUTP was stronger. After incubation with Nb.BbvCI or Nt.BbvCI, genomic λ-DNA was nick-translated with DNA polymerase I in presence of AF647-dUTPs. Every minute (for total 5 minutes) aliquots were taken and their reaction was immediately stopped with EDTA. Aliquots and controls were then loaded into a 0.7% agarose gel and stained with ethidium bromide. A| Ethidium bromide detection (Ex. source: 532 nm; Em. filter: 575 nm longpass, LP). After only 1 minute, all the full-length λ-DNA was already converted into the expected bands: 31 kb and 17.5 kb for Nb.BbvCI, and 30 kb and 18.5 kb for its isoschizomer, Nt.BbvCI (cutting on the opposite strand then Nb.BbvCI). These bands were further converted in additional products reflecting the progression of the labelling reaction. Note that the time required to add EDTA to stop the aliquots at 0 minutes was enough to break an important fraction of molecules; B| AF647-dUMP detection (Ex. source: 635 nm; Em. filter: 665 nm LP). The AF647-dUMP signal shows that the breakage was indeed due to the progression of the labelling reaction. At 0 minutes, a weak amount of fluorescence is already incorporated in the DNA in both labelling reactions (Nb.BbvCI and Nt.BbvCI) but not in the control. The weak fluorescence detected for the control reaction at 5 minutes is attributed to the filling of the cohesive extremities of λ-DNA.

In order to reduce the breakage at vicinal nicks, I tried to slow down the polymerases by labelling at lower temperatures than used here (37°C). This only slightly retarded the accumulation of the broken fractions of λ-DNA, but strongly reduced the labelling efficiency After incubation with Nt.BbvCI, genomic λ-DNA was nick-translated at different temperatures with DNA polymerase I in presence of AF647-dUTP. At 0, 1, 2 and 5 minutes aliquots were taken and their reaction was immediately stopped with EDTA. Aliquots and controls were then loaded into a 0.7% agarose gel and stained with ethidium bromide. A| Ethidium bromide detection. At 37°C, after only 2 minutes the full-length λ-DNA was already converted in 2 fragments (30 kb and 18.5 kb). At 24°C and 4°C, a time >2 minutes but <5 was required. Note that the experimental time needed to add EDTA and its diffusion to chelate magnesium to block the reaction at time-point 0 minute, was sufficient to start the labelling reaction. At 5 minutes, a smear extending below 18.5 kb is appearing, indicating successive fragmentation at other nicking sites. B| AF647-dUTP signal detection. Lowering the working temperature for DNA polymerase I has a massive impact in reducing the incorporation of fluorescent nucleotide precursors. At 37°C much fluorescence is incorporated all along the smear, indicating the successive breakage of the molecules due to the labelling reaction. At 24°C, the polymerase had enough time to generate the double-strand break only at the described site, and only the 31 kb and 17.5 kb fragment are labelled. At 4°C, the amount of fluorescence do not increase with the time. The fragmentation might be due to the exonuclease activities of DNA polymerase I, but the mechanism is unclear.

(
Considering the results obtained with DNA polymerase I, I decided to spend some more Then, I discovered that the nicking procedure itself slightly contributes to break the DNA prior to the labelling. A small amount of DNA is broken at the adjacent nicking sites (i.e.

"fragility hotspots") just after the nicking procedure . Although this fraction of broken molecules was initially negligible, it became very important after inactivating the nicking enzyme at 80°C for 20 minutes, as was done in all the above-shown experiments. This temperature is apparently sufficient to melt the double helix between vicinal nicks resulting in double-strand breaks. However, I found that incubating the DNA for 10 minutes at 70°C

immediately after the heat-inactivation of the enzyme restored a high proportion of full-length molecules. This temperature was low enough to permit reannealing of the ~500 base-pairs between the two nicking sites, yet too high to promote the reannealing of the 12-base-pairslong cohesive ends of λ-DNA. These results led me to introduce a supplementary nick-repair step after the DNA barcoding. In summary, each step of the barcoding protocol, presented challenges to resolve:

1. DNA fragmentation occurred during the heat-inactivation of the nicking-endonuclease; A| Agarose (0.7%) gel electrophoresis (ethidium bromide): a very small breakage of full-length molecules was observed when digesting λ-DNA with Nt.BspQI (left gel). Increased fragmentation was observed after purification of the nicked DNA in G-50 microspin columns: the 34.5 kb and 14 kb fragments are consistent with breakage at the predicted fragility hotspot (see B|). No fragmentation was introduced when purifying control DNA. A higher proportion of broken molecules was observed after heat-inactivating the nicking endonuclease at 80°C for 20 minutes (34.5 kb and 14 kb fragments, orange arrows). The proportion of full-length molecules was restored to the control level when incubating the DNA at 70°C for 10 minutes immediately after the heat-inactivation (right gel, red arrow). Decreasing gradually (1°C per minute) the temperature to 16°C, did not significantly restore full-length molecules. B| Predicted fragments generated on neutral gel electrophoresis of Nt.BspQI digested λ-DNA: the expected double-stranded fragments and their lengths are indicated. C| Alkaline agarose (0.7%) gel electrophoresis (stained with SYBR Gold): the fragmentation of λ-DNA at the fragility hotspots after G-50 purification was not due to pre-existing nicks, since undigested λ-DNA was not broken by G-50 column spin (left gel). The migration pattern of nicked sample was not perturbed by the incubation at 70°C, as expected (right gel). D| Predicted fragments generated on alkaline gel electrophoresis of Nt.BspQI digested λ-DNA: the expected single-stranded fragments and their lengths are indicated both for the top and the bottom strand.

To solve the labelling problem, I switched to Taq DNA polymerase, which possesses a strand displacement activity together with an efficient 5'→3' flap endonuclease action that degrades the displaced strand, eliminating the need to digest with RecJf. I firstly showed that

Taq polymerase was able to incorporate fluorescent dUMPs at the expected nicked sites (Figure

III-27 B).

Then, I introduced a repair step using the PreCR Repair Mix. The mix consists of a cocktail of various enzymes and is very efficient in sealing nicks. Indeed, after repairing the DNA, a strong 48.5 kb band was restored on alkaline gel conditions. This indicates that, at least for a considerable proportion of molecules, nick-translated sites were correctly sealed (Figure In conclusion, I solved the above-described problems and defined a protocol that is able to fluorescently barcode DNA molecules at specific and predictable sequence motifs while maintaining full-length molecules.

III-27 A).

III.2.3 Fluorescent replication signal detection

III.2.3.1 Fluorescent dUTP incorporation: nuclei reconstitution experiments

The first thing to monitor when using the Xenopus egg extracts system for DNA replication studies is the assembly of functional nuclei. Replication of demembranated sperm nuclei introduced into Xenopus egg extracts only occurs after chromatin decondensation and nuclear membrane assembly steps. Indeed, sperm chromatin was rapidly decondensed after its incubation in the extract and the replication-competent nuclei were formed within 40 minutes.

At 60 minutes, the vast majority of the nuclei had undergone active DNA replication (Figure Long unlabelled stretches as in b) and c) likely correspond to unreplicated segments or unreplicated molecules. Indeed, the TCA assay precipitation assay in presence of AF647-dUTP showed that only the 74% of the DNA was replicated in this sample (see paragraph III.2.5, Figure 1A in De Carli et al (2016)). However, it is difficult to explain the short gaps found in all the labelled molecules: if even the smallest gaps represented unreplicated DNA, their frequent occurrence would imply a very high rate of fork stalling and origin activation to account for the high density of red tracts. Another possibility is that the incorporated AF647-dUMP might have been partly removed by DNA repair mechanisms. To our knowledge, no evidence for such activity has been reported. Finally, the signal may be quenched by interaction with the combing surface or by photobleaching. These hypotheses are discussed in [START_REF] Carli | Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules[END_REF] (see paragraph III.2.5). In this article, we came to the conclusion that the latter hypothesis, a mechanism of signal quenching by the interaction with the surface or by photobleaching, was the most probable. Here I will present results obtained in trying to characterize this phenomenon. 2) a combination of all dNTPs + AF647-dUTP, and (3) a combination of dATP+dCTP+dGTP (dACG) + AF647-dUTP. In all cases, both the 500 bp and 10 kb fragments incorporated AF647-dUMP in the newly synthetized strand when it was present in the dNTP cocktail. The 10 kb products obtained with Klenow fragment were more smeared than the ones obtained with Taq or LongAmp DNA polymerases. This might reflect the incapacity of the polymerase to extend all the 10 kb stretch, resulting in partial singlestranded portions, but it could also be due to the formation of the single-strand flaps derived from the strand-displacement activities of Klenow (exo-). However, in all the cases, except for with Taq polymerase, the amount of fluorescent signal was higher when the dNTP cocktail contained unlabelled dTTP in addition to the fluorescent dUTP.

III-29).

In the case of the 500 bp amplicon with Taq polymerase, the ethidium bromide signal (representing total DNA, whatever its labeling extent), was comparable in the presence or absence of dTTP, but the fluorescent signal was stronger in its absence. This suggested that the reaction was approximately equally efficient in the presence or absence of dTTP but the presence of dTTP competitively reduced the amount of incorporated fluorescent dUTP, as expected. This was not the case for the 10 kb amplicon with Taq polymerase, where both the ethidium bromide and the AF647 signal were lower in the absence of dTTP. This suggests that the lack of dTTP became limiting for synthesis of large but not small amplicons by Taq polymerase. A similar stimulatory effect of dTTP was observed for both small and large amplicons with the other two polymerases. DNA polymerases might prefer to incorporate the canonical dNTP versus the modified ones. They might also slow or arrest due to the incorporation of the modified dNTP. Eventually, the presence of unmodified dTTP might help the stalled polymerase to restart. The mechanism generating high molecular weight amplification products of the 500 bp template in the presence of Klenow (exo-) polymerase is unclear. PCR fragments of 500 bp or 10 kb were purified and hybridised with a primer. The primer was extended with 3 different polymerases (Taq pol, LongAmp or Klenow exo-) and in 3 different conditions: 1) in the presence of all the canonical nucleoside triphosphate (dNTPs), or 2) a combination of all dNTPs + AF647-dUTP, or 3) a combination of dATP+dCTP+dGTP (dACG) + AF647-dUTP. The gel was stained with Ethidium Bromide and the two channels were acquired at the fluor-imager. A| Ethidium bromide: all the products are of the expected lengths. B| AF647-dUMP: smears appeared in the Klenow reactions probably reflecting uncompleted primer extension and presence of single-stranded flaps (see text). For Taq pol and LongAmp Taq pol, discrete bands can be observed at the expected lengths.

In order to gain information about the single-strands, the same reaction products were also electrophoresed in an alkaline gel (Figure III-34). The migration patterns confirmed the correct labelling of 10 kb-long fragments for all the polymerases in the presence, but not in the absence, of unmodified dTTP. However, a smear appeared on the 10 kb lanes with Klenow (exo-) reaction. This confirms that Klenow cannot easily extend the primer over long distances, at least in the presence of AF647-dUTP. LongAmp DNA polymerase turned out to be the best polymerase to obtain strongly labelled 10 kb-long fragments. None of the three polymerases was able to produce a detectable amount of labelled 10 kb bands in the absence of canonical dTTP. Intriguingly, in the case the 500 bp amplicon with Taq polymerase, a fluorescent 500 bp band was detected either in the absence or presence of dTTP, but the signal was stronger in the presence of dTTP, in apparent contradiction with the neutral gel analysis. We have no explanation for this discrepancy. In conclusion, the gel experiments suggest that we were able to create 10 kb-long, double-stranded DNA molecules that have incorporated AF647-dUMP all along their length, but the density of the incorporated labels is difficult to evaluate due to the required presence of dTTP for their efficient production. It is possible that the density of AF647 label is too weak to allow efficient detection by DNA combing. However, this seems difficult to reconcile with the large heterogeneity of fluorescent stretch lengths and intensities observed on the combed DNA molecules. In any case, the labelling of such molecules appeared much weaker than that produced by replication of sperm nuclei in egg extracts supplemented with AF647-dUTP. We therefore decided to focus on the analysis of molecules replicated in egg extracts and did not further attempt to artificially produce strongly fluorescent DNA molecules in more controlled conditions.

III.2.3.3 Observation of doubling of DNA fluorescent intensity along a combed Y-shaped

DNA substrate

To prove that replication bubbles can be detected based on their expected double YOYO-1 intensity, we constructed a lariat DNA structure from λ-DNA, in which a single-

III.2.4.2 Definition of a working protocol

After spending much time in trying to adjust the combing conditions without success, I decided to systematically test the effect of each step of the barcoding procedure on the ability of the DNA to comb correctly. I thus tested the impact of all the buffers and the proteins necessary to nick and to label the DNA. I discovered that the addition of buffers' volumes equivalent to the ones used in the barcoding reactions (few µL of 10x buffers) to the combing reservoir (1.5 mL), strongly affected DNA combing (Figure III-39). This is true both for the addition of NEBuffer 3.1, used in the nicking of the DNA, as well as for ThermoPol buffer, adopted in the labeling and in the repair reactions. A control λ-DNA solution (250 pg/µL) was used to test for the quality of DNA combing on silane surfaces. Different amounts of various enzymes were sequentially added to the control reservoir A| to mimic the amounts used in the fluorescent barcoding protocol B-C|. Supplementary proteins were also added to further investigate the effect of exceeding amounts E-F|. All the images correspond to 4 fieldsof-view with a 100X objective and a classic 512x512 captor. Scale bar: 10 µm (20 kb).

With these results in mind, I tried different purification methods, but the only one that allowed complete protein removal and buffer wash out, while minimizing DNA breakage, was to re-capture the DNA in agarose plugs. To do this, we exploited the fact that inactivation of the nicking endonuclease at 80°C also melt the agarose plug, allowing to perform the nicktranslation with Taq polymerase in molten agarose at 72°C prior to re-solidification of the agarose plug at 4°C. This allowed all the subsequent buffer changes required for the nick repair

III.3 High throughput single-molecule analysis of DNA replication using nanochannel arrays

In order to test the application of the tools for detecting and mapping replication bubbles using the nanochannel array technology, we decided to create a fluorescent replication map of λ-DNA as we did with DNA combing (see paragraph III.2.5, De Carli et al (2016)) and to compare both results.

III.3.1 Description of the nanochannel system

The nanofluidic device that we used (Irys Instrument, BioNano Genomics) has been designed to automatically generate whole-genome physical maps. To do so, it takes advantage of a fluorescent DNA barcoding method based on the very same principle that we proposed (see paragraphs I.7.7, point 2). DNA molecules are nicked with specific nicking endonucleases, the nick is extended using a DNA polymerase and fluorescent nucleotide analog and repaired.

While generating single-molecule physical maps, the device is especially useful for large-scale genome variant discovery, as well as for rapid de novo assembly of whole genomes and automated genome finishing (e.g. hybrid scaffolding).

However, although the device was designed for research fields different from the DNA replication one, it has specific features that are convenient for our goal to automate the detection and the mapping of DNA replication bubbles. The core of the Irys system is a disposable chip (IrysChip, BioNano Genomics) that contains 2 flowcells, thus enabling the analysis of 2 different samples for each run. Each flowcell is composed of a massive array of parallel nanochannels (13,000), each of which has a 45 nm diameter. DNA in solution is untangled in the chip applying an electrophoretic force, resulting in a uniform stretching of DNA molecules

(Figure III-42).
Thanks to an autofocus system, 3 laser lines (coupled to high speed filter wheels), a motorised XY stage and an ultra-low noise EM-CCD camera, the machine enables automatic high-resolution and high-throughput multicolour images of long DNA molecules.

Such DNA molecules are automatically detected, based on the YOYO-1 molecule intensity, and aligned on the reference genome, thanks to the fluorescent barcoding. Thus the Irys system is, a priori, the perfect device for our needs. Each run is composed of 30 cycles of loading, untangling and imaging of DNA molecules (for each of the 2 flowcells). In order to cover all the nanochannel chip, in each cycle 1140 images (512 x 512 pixels) are taken for each channels. In a complete run, 34,200 images are acquired for each colour channel, resulting in an impressive throughput (50-100 Gbp/chip).

In a recent analysis of human genome structural variation, use of a single nick-labelling enzyme

IV. Discussion

The work presented in this thesis consists in developing novel tools to study DNA replication at single-molecule (SM) level. The value of this work resides in improving the capacity that the scientific community has to reply to the three queries that I posed in the introduction:

1. What determines where the potential replication ORIs are located?

2. What decides which, among all the potential ORIs, will be activated?

3. What defines when in the S-phase, a given ORI will be activated?

In the case of the mammalian genomes, despite more than a decade of genome-wide studies, these three questions remain unsolved. First, population-averaged techniques either show an insufficient resolution to map individual origins (Repli-seq), or show only limited concordance between independent studies (e.g. SNS-seq vs. bubble-seq vs. OK-seq). SM mapping of individual replication bubbles remains the most convincing evidence for active replication origins, but the throughput has remained too low to help resolve controversies elsewhere than at very few loci. Second, population-averaged techniques cannot trace an accurate landscape of eukaryotic DNA replication without the analysis of cell-to-cell variability that is only attainable by SM studies. We report here significant advances that promise to bring SM techniques the required throughput for genome-wide studies of DNA replication.

IV.1 Challenges encountered in developing novel single-molecule tools

IV.1.1 Challenges in finding reliable combing conditions

One of the major difficulties that I found in developing replication tools based on the DNA combing technique is related to the technique itself. Many parameters substantially influence the DNA combing quality (see section III.2.1), and it is often difficult or impossible to have all of them under control. For example, hydrophobic surfaces are usually produced "inhouse" (because of their exaggerated cost on the market) and must be immediately checked at the time of the production. However, they should also be periodically controlled (e.g. every two weeks or monthly) because of the variability during storage. The room temperature, the humidity and the quality of the chemicals used to prepare the surfaces are only some of the parameters playing a determinant role in their ageing. Even when taking the utmost care, variability in combing quality is observed between different coverslips and within different zones of the same coverslip. Evaluation of surface quality (see section III.2.1) cannot be routinely performed in the absence of an automated software for analysis of the combed DNA.

Thus, the quality of the stretched fibres strongly depends on the experience of the experimenter and on his/her ability to discern acceptable and inacceptable conditions at the time of data acquisition. For this reason, further developing DNA combing might sound a daunting task even for laboratories that routinely use this technique. I tried to simplify the molecular combing process by using alternative coatings that are easier and cheaper to produce than the "classic" silanisation protocol. In particular, cleaning the coverslips with plasma-treatment followed by spin-coating with a polystyrene solution resulted in very interesting substrates for DNA combing. Although not recommended in replication analysis because of the variability in the YOYO-1 signal (see paragraph III.2.1.5), polystyrene surfaces can be reliably used to test nicklabelling protocols and for genomic mapping studies of simple organisms (as the signal/noise ratio is very high and nicking sites are efficiently detected).

IV.1.2 Challenges in developing a fluorescent DNA barcoding

Concerning the development of the fluorescent DNA barcoding, the task turned out to be much more difficult than expected. It took almost two years to develop a protocol able to gently nick-label the DNA molecules without breaking the vast majority of them. This required systematic testing of various DNA polymerases. In doing so we noticed that the products obtained using diverse polymerases were very different and showed different combing suitability. For example, we discovered that single-stranded DNA (ssDNA) flaps of short lengths (<1 kb) generated by strand-displacement are sufficient to impair DNA combing. Most likely, DNA could not stretch when containing flaps because ssDNA portions are attaching very efficiently on the hydrophobic coverslip, preventing its elongation under the force of the receding meniscus. Thus, when stretching λ-DNA for surface testing, the ability to specifically adsorb the DNA ends derives not only from the "correct" pH choice, but also from the presence of the single-stranded cohesive extremities of λ-DNA. Other substrates without cohesive ends, closer to actual DNA samples, would be a better choice for these testing steps.

The major problem, however, was to reach a compromise between the fluorescent nickextension and the occurrence of double-strand breaks (DSBs). We expected that extending too much the nicks would result in a DSB when the nicks are relatively close (<1 kb) and the forks travel in a convergent direction (in fact, we used this prediction to observe the occurred nicklabelling reaction at the expected NE sites, see paragraph III.2.2.1). However, we did not expect that adjacent nicks behave per se as fragility hotspots (see paragraph III.2.2.2). Molecules presenting nearby nicks (~300-500 bp apart) repeatedly broke at these fragility hotspots during subsequent DNA manipulation. Purifying (either in columns, by phenol-chloroform or by resin)

or heating (~80°C) the nicked DNA for further molecular biology reactions was enough to break a substantial proportion of molecules. Therefore, we decided to repair the DNA prior to molecular combing, which resulted in a working protocol. Importantly, the determinant parameter for mapping ability is the density of nicks (e.g. 1 every ~10 kb, [START_REF] Cao | Rapid detection of structural variation in a human genome using nanochannel-based genome mapping technology[END_REF]).

Thus, the nicking enzyme is chosen accordingly with this parameter (once this is set, the density of fragile sites automatically ensues) and cannot be chosen with the principal aim to minimise the fragility hotspots. The protocol developed here should allow a reliable nick-labelling of DNA from various sources. In the 60 minutes of the nick extension, Taq DNA polymerase could have, a priori, incorporated as many nucleotides as to entirely hemi-substitute the 48.5

kb-long λ-DNA. However, independently of the large incubation time, DNA polymerases stop incorporating fluorescent dUTPs after some dozens of bp, for sure less than ~250 bp 42 (see paragraph III.2.2.3). The reason explaining this upper limit is unclear to me. The most probable explanation is a polymerase stalling mechanism occurring in the absence of canonical dTTPs in the reaction mix. Taq DNA polymerase probably has some trouble incorporating AlexaFluor647-dUTPs for stretches of several dozens of nucleotide, resulting in its blockage. 42 Otherwise the λ-DNA would break with a DSB, considering that it was nicked with Nt.BspQI which generates two nicks ~500 bp apart on opposite strands.

This speculation requires further investigation, but whatever the mechanism this fact certainly helps the experimenter to perform an efficient nick-labelling without extending too-much the fluorescent stretch (see section III.2.2 to have an idea of the difficulty controlling the extension rate with other DNA polymerases).

IV.1.3 Challenges in developing a direct fluorescent bubble detection

The most straightforward task was the visualisation of replicated tracts by direct fluorescence without the need for antibody signal detection. This was possible thanks to the Xenopus egg extracts system, were fluorescent nucleotide precursors can be directly added to the reaction mix, contrary to cell systems. To my knowledge, this was the first time that replication tracts could be directly detected using fluorescent dUTPs, without antibody signal amplification. Fluorescent dUTPs were used for this task considering that the concentration of endogenous dTTPs in the egg extracts is estimated to be ~7 µM, while all the other dNTPs are In this thesis we also presented a replication bubble detection based on a simple staining of the replication intermediates with the fluorescent intercalator YOYO-1. This tool has a different potential compared to the incorporation of fluorescent dUTPs, because it is applicable to DNA extracted from any source. This was definitely the more difficult task and this is still not completed. For almost two years I tried to define combing conditions that always and reproducibly allow the bubble detection based on the YOYO-1 fluorescent profile. However, this was a laborious task and we could not define conditions permitting to fix a global threshold to segment un-replicated vs. replicated DNA fragments. The reason for this failure is probably linked to the considerable number of parameters that influence the YOYO-1 signal on combed molecules. We discovered that staining at temperatures higher than the room-temperature speeds up the intercalation process. Indeed, it was reported that reaching binding equilibrium between the YOYO-1 molecules and the target DNA is strongly boosted by the temperature [START_REF] Carlsson | Double bands in DNA gel electrophoresis caused by bisintercalating dyes[END_REF]. Also the YOYO-1 concentration, as well as the buffer composition and the hydrophobic coating influence the YOYO-1 fluorescence. I personally believe that it will be very challenging to ameliorate the YOYO-1-based bubble detection because it results from too many interplaying parameters. However, it should be noticed that we used simple epifluorescence microscopy, which might not be the best choice considering the nature of our substrate (i.e. very few fluorophores emitting very close to the surface). Some other advanced microscopy techniques such as TIRF and confocal microscopy could ameliorate the output, although the major limitation probably consists in the interaction of the fluorophores with the hydrophobic surface. If it is the case there is little hope to improve the technique: the only way to automatically detect replication eyes by step-changes in YOYO-1 detection will be to develop robust software capable of adjusting flexible thresholds for each molecule. The situation is different for nanochannel, where the DNA is not attached to the surface, but confined in an imaging buffer, and imaged with a device capable of high sensibility (see section III.3). For these reasons, and because of the quality of the images, the automated YOYO-1based bubble detection on the nanochannel device seems an achievable task in a near future.

IV.1.4 Challenges in combining fluorescent bubble detection with fluorescent barcoding

Another delicate part of my thesis was to combine the two tools we developed for direct bubble detection and for fluorescent DNA barcoding (see paragraph III.2.4). We expected the two techniques to be compatible, but we did not expect that their combination would take one year of trials. Indeed, after systematic testing of all the factors involved, we realised that an insufficient DNA purity was preventing molecular combing. This result should be taken in account for any kind of DNA combing experiment. The factors involved in determining the DNA combing quality are usually identified as pH choice and salt concentration (both affecting the charge of the DNA, and therefore its ability to bind to the hydrophobic surface). Strikingly, here we show that DNA-interacting proteins massively change the DNA combing behaviour and its stretching. It is sufficient to add very few units of DNA polymerase or restriction enzymes to the big volume (1.5-2.5 mL) of the combing reservoir to completely abolish the possibility to comb. The same is true for very small quantity of classic reaction buffers. This was a surprise and has to be kept in mind for future experiments: in many cases we attribute the inability to properly comb to a poor quality of the hydrophobic coverslips, although it might not be the main reason but DNA purity might be in question.

IV.2 Challenges encountered in adapting the tools for nanofluidic analysis

Once the tools to detect and to map the DNA replication bubbles by DNA combing were developed, we rapidly succeeded in adapting them to a nanochannel device. The challenge was to create agarose plugs ~5 fold more concentrated (thus demanding ~5 fold more Xenopus egg extracts for the replication reaction) in order to feed the 13,000 nanochannels present in each flowcell composing the chip 43 . The major steps were the same as in DNA combing: after incubating DNA in egg extracts, replication intermediates were isolated from pelleted nuclei prior to fluorescent DNA barcoding and YOYO-1 staining. The nick-labelling procedure, however, had to be adjusted to the nanochannel technology. Contrary to DNA combing, it had to be performed outside the agarose plugs. Indeed, little undigested agarose pieces represent a serious threat for the chip since they can "cap" the entrance of the 45 nm-wide nanochannels.

This actual protocol has a limitation: the N50 value 44 of the lengths for the nick-labelled samples (~150 kb) is only ~1/3 of the one measured before the nick-labelling procedure (~450 kb). Thus, the fluorescent barcoding systematically breaks the molecules at about ~1/3 of their length in the nanochannel protocol. We are trying to improve the average molecule length by taking the utmost care in minimising DNA shearing during sample handling. This would result in better alignment scores, although molecules >100 kb are long enough to be unequivocally aligned on the reference genome. In terms of throughput, the results (see section III.3) were already astonishing (~15 Gbp per run), but we think we can still increase of a factor two or three. Based on published data, ~50 Gbp per run per flowcell should be reachable. Unfortunately, we are also facing unexpected problems. Optical aberrations in the nanochannel device imaging result in a misalignment of the replicated tracts (excited in the far-red wavelength, red laser, 637 nm) compared to the DNA backbone (blue laser, 473 nm) and the fluorescent barcoding (green laser, 532 nm). The misalignment is important at the border of a typical field-of-view (512x512 px)

and prevents the replication signal to be automatically attributed to its corresponding DNA molecule. We are currently trying to "re-centre" the different colours using affine matrix transformations. Moreover, the detection of the replication signal requires a careful evaluation of the fluorescent gap distribution as done for DNA combing, in order to discern between real 43 On the Irys setup (BioNano Genomics) each chip is composed of two independent flowcells where two different samples can be run. Each flowcell is composed of 13,000 nanochannels. 44 The N50 of the lengths corresponds to the length value splitting in half the total mass of the sample after ordering molecules on the base of their size: molecules shorter than the "N50 length" represent the 50% of the total mass, the remaining 50% of the mass is composed by molecules that are longer than N50.

gaps and false-gaps. Here it was done by manual analysis, but it should be automated and extended in order to reach higher statistical significance.

IV.3 Antibody-free DNA combing vs. nanofluidics: current potential and limitation

The SM, antibody-free fluorescent visualisation of replication tracts that we developed with the DNA combing technique is a considerable advance in the SM analysis of DNA replication. For many years DNA replication studies using the Xenopus egg extracts system were performed under a "mean-field" hypothesis, meaning that data from all the molecules are averaged whatever their genomic origin. This was justified inasmuch as early studies revealed a lack of sequence specificity for replication initiation in this system, and single-locus analysis Concerning the nanochannel technology, the problem of the misalignment of the different channels is slowing down the automated analysis. However, the same experiment of yeast synchronisation described above for DNA combing is also feasible with the nanochannel technology as readout. This would permit us to reach an acceptable coverage even by manual identification of the strong YOYO-1 tracts. The reasons of this choice are the following:

a) The throughput. The actual throughput (~15 Gbp) correspond to an unprecedented SM coverage of the yeast genome (estimated ~100x).

b) The existing software. Although the automated segmentation of the replicated tracts is currently under development, the existing genomic mapping pipeline will allow to automatically map DNA molecules. If the misalignment between the YOYO-1 vs. barcodes channels will be so important to impair the automated mapping45 , then we would still being able to map molecules by sorting molecules in groups based on their lengths (using the existing software) and selecting discrete peaks corresponding to the lengths of entire yeast chromosomes. Once the entire chromosomes are identified, orientation with respect to their restriction map should be immediate.

c) The quality of the images. By nanochannel, images show higher signal/noise ratio than DNA combing. Molecules are perfectly straightened, which facilitates their analysis.

d) The accuracy of the stretching. With the nanofluidic device, molecules are stretched under a fixed electrophoretic force, thus resulting in a very reproducible stretching factor (~1%) compared to DNA combing (~5-10%).

To conclude, at the time of writing there are two major limitations shared by the DNA combing and the nanochannel technology that currently prevent us to scale-up the DNA replication analysis of higher eukaryotes. The first one is the lack of a software able to automatically identify the strong YOYO-1 tracts and to map them on the reference genome. In the case of DNA combing, this is very challenging because of the characteristics of the images obtained. At present, we only succeeded in reliably detecting the DNA backbone based on the YOYO-1 signal, but we did not succeed in automatically segment the replication bubbles based on the doubling of the YOYO-1 signal. Concerning the nanochannel, we already have scripts able to automatically identify DNA molecules and their fluorescent labels (barcode) and to align molecules on the genome of interest. These scripts are working and usually deployed to detect deletions, insertions, inversions and copy number variations (CNV) in genomes as complicated as the human genome. However, detecting step changes in the YOYO-1 profile along the length of the molecules is currently under development in our laboratory and requires step-by-step validation. The second big limitation at present is the lack of a pulse applicable to other systems than the Xenopus egg extracts one. Developing labelling schemes compatible with cells in culture would significantly increase the overall impact of these game-changing SM tools for various reasons: (a) it would be applicable to cells of various sources, such as yeasts and mammalian cells; (b) it would let us monitor fork progression and measure fork speed, quantify termination events, asymmetric fork progression and fork stalling events, for the first time, genome-wide; (c) it would help in identifying the DNA bubbles through a binary signal (1 or 0: signal is present or not present) rather than through a signal doubling as with YOYO-1 (with more "shades of grey": no signal = background / 1x signal = unreplicated DNA / 2x signal = replicated region).

IV.4 The bacteriophage lambda story: DNA combing vs nanochannel

The tools that we developed were used to construct the first fluorescent replication map of λ-DNA by molecular combing [START_REF] Carli | Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules[END_REF]. The local coverage was slightly higher near Nt.BspQI nicking endonuclease (NE) sites. The normalised replication coverage was homogeneous over the entire λ phage sequence. We mapped the initiation events by considering the midpoint of the segments <5 kb (so as to avoid segments derived from the fusion of multiple replication bubbles). The distribution of initiation events was also homogeneous over λ-DNA.

This result was consistent with the literature describing randomly located initiations in Xenopus egg extracts [START_REF] Hyrien | Plasmid replication in Xenopus eggs and egg extracts: a 2D gel electrophoretic analysis[END_REF][START_REF] Hyrien | Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos[END_REF][START_REF] Mechali | Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast[END_REF]. λ-DNA replicated in egg extracts was also analysed using nanochannel arrays (see paragraph III.3.4). A single run produced an astonishing total 15 Gbp of DNA molecules (i.e.

corresponding potentially to a ~300,000x coverage of the λ genome!). As automated analysis is not working yet, a tiny sample was manually analysed as done for the DNA combing article.

Very surprisingly, although the fibre coverage much resembled the one obtained by DNA When incubated in the egg extracts, λ-DNA is subjected to efficient non-homologous end-joining (NHEJ). Thus, multiple λ-DNA molecules are rapidly joined to form long concatamers. The U-shaped profile suggests that replication starts preferentially at the λ ends.

This might be determined by sequence-specific signals, but this hypothesis is in contrast with the literature [START_REF] Hyrien | Plasmid replication in Xenopus eggs and egg extracts: a 2D gel electrophoretic analysis[END_REF][START_REF] Hyrien | Chromosomal replication initiates and terminates at random sequences but at regular intervals in the ribosomal DNA of Xenopus early embryos[END_REF][START_REF] Mechali | Lack of specific sequence requirement for DNA replication in Xenopus eggs compared with high sequence specificity in yeast[END_REF], although one half of the λ-DNA sequence is GC-rich and the other is AT-rich, and a standard DNA combing study suggested that replication preferentially initiates within the AT-rich moiety [START_REF] Stanojcic | In Xenopus egg extracts, DNA replication initiates preferentially at or near asymmetric AT sequences[END_REF]. This possible preference for AT-rich sequence, however, cannot explain a concentration of initiation events close to both ends of the λ-DNA genome. Another possibility is that the skew between the AT-and the CG-rich moieties that is formed after joining two λ-DNA molecules in a head-to-tail concatamer 46 acts as preferential initiation site.

A more appealing possibility is that joined (NHEJ) or repaired (HR) fragments, present some epigenetic signals able to "call" the replication machinery. This could either involve some proteins implicated in the joining/repair process (KU, PK, ligases... / ATM, RPA, RAD51...) or the modification of histones at the site of joining. It is conceivable that the creation of such [START_REF] Carli | Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules[END_REF]. Unfortunately, in the nanochannel experiment we did not perform the TCA radioactive quantification. As the two experiments (DNA combing vs. nanochannel) were performed with different egg extracts, we cannot exclude that the higher fraction of AF647-dUMP labelled in the nanochannel experiment is due to better replication, although this would not explain the ten-fold lower density of gaps.

46 For two λ-DNA molecules, three possible concatamers can be formed: head-to-head, head-to-tail or tail-to-tail. Application to the genome of other organisms, which cannot uptake fluorescent dNTPs, will require the development of alternative replication labelling techniques and/or a stronger validation of replication bubble detection by YOYO-1 doubling. The utility of barcoding will also be limited to small genomes, for which a significant coverage can be attained with the necessarily limited number of molecules on can detect and handle by DNA combing, due to the lack of efficient software for automatic detection and alignment of such molecules.

Concerning the nanochannel technology, it is already more powerful than DNA combing in the present configuration. This is thanks to the possibility to automatically sort the molecules according to their lengths and to automatically align them to the reference genome.

Automatic detection of replication eyes must still be implemented, but even by manual analysis, the throughput is much higher than with DNA combing, the stretching is more precise and the molecules are strictly linear, which facilitates automatic detection and measurements. The quality of the pictures and the automated mapping makes the technology much less timeconsuming than DNA combing. We therefore plan to perform the above-described experiment Yet, it is inconceivable to analyse genomes bigger than yeast without implementing the automated detection of replication bubbles. This is ongoing work in our laboratory.

IV.5.2 Potential of the technologies

We need to develop new antibody-free replication-labelling protocols to track replication fork progression using either DNA combing or nanochannels. Novel analogs detectable via "click"-chemistry offer potential solutions to this problem. Developing such labelling techniques and applying them to yeast should not take more than a few months.

However, the resolution of the imaging problems on the nanochannel device, as well as the development of the automated software to analyse the replication tracts for both nanochannel and DNA combing, will requires much programming effort. We will concentrate future efforts in developing the nanochannel scripts and validate them using yeast, before testing their applicability to human cells. Hopefully, the study of human cells will bring SM evidence in support to the origin firing cascade model [START_REF] Guilbaud | Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome[END_REF][START_REF] Hyrien | From simple bacterial and archaeal replicons to replication N/U-domains[END_REF] and clarify the replication mechanism of regions difficult to analyse with population-based techniques, such as highly-repetitive regions, centromeres and telomeres.

IV.6 Future of the single-molecule techniques.

IV.6.1 Is the DNA combing era at the end?

DNA combing and related DNA fibre techniques are widely used around the world and, I believe, will continue to be useful for a while. The first reason is the cost. The nanochannel apparatus is much more expensive than a standard DNA combing equipment. Therefore, DNA combing is available at the place of work whereas the cost of a complex nanofluidic device is prohibitive for a single team (although it is not for an institute or a platform). The second reason is that if one is interested in specifically analysing only a peculiar region of the genome, DNA combing coupled to FISH is still a convenient approach. Third, even in the nanochannel era, DNA combing will still be a useful tool to check DNA quality prior to the nanochannel runs and to quickly test labelling protocols "in-house". Fourth, nanochannel technology is today limited in the size of the imaged molecules because the channels are limited in lengths to ~3

Mb, and require large amounts of material. Recent progress in DNA combing showed that is possible to systematically obtain long molecules up to 10 Mb [START_REF] Kaykov | Molecular Combing of Single DNA Molecules on the 10 Megabase Scale[END_REF], and that it is possible to comb picograms of DNA [START_REF] Deen | Genome-wide mapping of human DNAreplication origins: levels of transcription at ORC1 sites regulate origin selection and replication timing[END_REF]. Finally, one last consideration concerns the data analysis. Managing Gbp of molecules (i.e. Terabytes of data) is not an easy task for untrained researcher used to "wet-lab" and demands competent bioinformaticians as well as powerful calculation clusters.

IV.6.2 Meeting the dynamic studies of DNA replication

The nanochannel experiments shown in this thesis exploit a nanofluidic device to obtain "static" images of a dynamic process that is happening in the cell. However, in the last five years huge progresses have been made in visualising the dynamic process of DNA replication Le but de cette thèse a été de développer des outils nouveaux, rapides et efficaces pour cartographier la réplication de l'ADN au niveau de la molécule unique sur l'ensemble du génome.

Il existe plusieurs techniques, dont le peignage moléculaire est sans doute la plus précise, pour étudier la réplication de l'ADN à l'échelle de la molécule unique. Le peignage moléculaire permet d'étirer de l'ADN purifié de façon parallèle et reproductible sur une lamelle de microscope spécialement traitée [START_REF] Bensimon | Alignment and sensitive detection of DNA by a moving interface[END_REF][START_REF] Michalet | Dynamic molecular combing: stretching the whole human genome for high-resolution studies[END_REF]. L'ADN peut être peigné sur diverses surfaces hydrophobes [START_REF] Allemand | pH-dependent specific binding and combing of DNA[END_REF]. On utilise généralement des lamelles silanisées pour des raisons détaillées plus bas. Quand les cellules en culture sont traitées avec des analogues de la thymidine comme la 5-chloro-

Table of illustrations

IV. 1 . 2

 12 Challenges in developing a fluorescent DNA barcoding ............................... IV.1.3 Challenges in developing a direct fluorescent bubble detection .................... IV.1.4 Challenges in combining fluorescent bubble detection with fluorescent barcoding156 IV.2 Challenges encountered in adapting the tools for nanofluidic analysis ................. IV.3 Antibody-free DNA combing vs. nanofluidics: current potential and limitation .. IV.4 The bacteriophage lambda story: DNA combing vs nanochannel ......................... IV.5 Towards genome-wide, single-molecule analysis of eukaryotic DNA replication IV.5.1 Capacity of the technologies at present .......................................................... IV.5.2 Potential of the technologies .......................................................................... IV.6 Future of the single-molecule techniques. .............................................................. IV.6.1 Is the DNA combing era at the end? .............................................................. IV.6.2 Meeting the dynamic studies of DNA replication .......................................... Résumé détaillé de la thèse .................................................................................................... Bibliography ........................................................................................................................... Table of illustrations ............................................................................................................... Table of tables ........................................................................................................................

Figure I- 1 :

 1 Figure I-1: Basic mechanisms of DNA replication. A| The replicon model as proposed by[START_REF] Jacob | On the regulation of DNA synthesis in bacteria: the hypothesis of the replicon][END_REF]: the replicon is a circular structure harbouring two genetic determinants. A trans-acting element, the initiator protein, coded by a "structural gene" (SG1, blue), recognises a specific cis-acting element, the replicator (orange). This interaction allows the beginning of DNA replication. B| DNA fiber autoradiography of mammalian chromosomes (Chinese hamster cells, CH). CH cells were pulse labelled with 3 H-thymidine for 30 min, and chased by non-radioactive thymidine for another 45 min. Cells were collected and lysed. The DNA trapped on dialysis filters was subjected to autoradiography. Replication occurs at several starting sites (i.e. replication origins, indicated by orange arrows) and proceed in a bidirectional fashion from each of these. Adapted from[START_REF] Huberman | On the mechanism of DNA replication in mammalian chromosomes[END_REF].

Figure I- 2 :

 2 Figure I-2: autonomously replicating sequence (ARS) assay.ARS assay allows to identify sequences that behave as replicators (ORIs). A| The genome is fragmented in random pieces that are cloned in a plasmid vector that contains a specific selectable marker. B| The library is transfected to the host cells, which contain all the replication machinery (initiator and additional markers). C| If the cell is able to grow in a selecting media it means that it incorporated a plasmid with the ability to autonomously replicate within the cell. The colony can be isolated and expanded for further ARS characterisation. Alternatively, the cell can gain the ability to grow by integrating the selectable marker into one of its chromosomes by integrative recombination, but this is a rare event.

Figure I- 3 :

 3 Figure I-3: Analysis of replication intermediates by neutral/neutral 2D gels.After digesting the total DNA with a restriction enzyme to isolate the region of interest, the restriction digest is submitted to a first electrophoretic migration (1D, orange arrows) that separate the fragments based on their mass. The unreplicated restriction fragment of interest has a mass of 1n. If the fragment was completely replicated, just before dividing the 2 daughter molecules would have a double mass of 2n. A second electrophoretic migration (2D, blue arrows) is performed at high voltage and agarose percentage in order to separate fragments of equivalent mass but different shapes. The 2D migration is performed in a direction orthogonal to the 1D. Branched molecules have a retarded migration compared to linear molecules, but are quicker than bubbles. Finally, the gel is blotted and probed with specific probes allowing to resolve fragments containing a simple fork (simple Y-shaped molecules), 2 diverging forks (bubbles, replication ORIs) or 2 converging forks (double-Ys, termination). Adapted from[START_REF] Hyrien | Peaks cloaked in the mist: The landscape of mammalian replication origins[END_REF].

Figure I- 4 :

 4 Figure I-4: Population-based replication mapping techniques. Schematic drawing of A| a growing replication bubble, B| nascent strands (NS), C| Okazaki fragments (OK) and D| leading strands. E-G| Representation of the existing techniques to analyse DNA replication by nascent strands abundance or polarity. E| In order to isolate short nascent strands (SNS) without contaminant short single-strands (SSS) due to sheared and nicked DNA, various methods were used. 1)Cells can be lysed directly inside the agarose well to reduce further manipulation. 2) SNS can be purified by recovering BrdU-positive SSS using anti-BrdU antibodies after size selection excluding Okazaki fragments on alkaline gel, neutral sucrose gradient, or isopycnic centrifugation. 3) Finally, since NS are synthetized elongating an RNA primer, SNS can be enriched thanks by a λ-exonuclease digestion removing all the SSS that are not protected by an RNA. F| Okazaki fragments can also be isolated using different approaches. 1) Cells are radio-labelled and selected on alkaline gel prior to elution of 50-300 nt-long SSS. 2) Alternatively, they were isolated by size selection on ligase-deficient yeast cells. 3) Recently, OK were isolated by size-selection on denatured, EdU-labelled DNA. Biotin was coupled to EdU thanks to "click"-chemistry and EdU-positive SSS were captured on streptavidine beads. G| Leading strand bias can also be used to find replication ORIs by isolating BrdU-NS with an isopycnic separation on cells treated with emetine to block lagging strand synthesis. See text for references. Modified from[START_REF] Hyrien | Peaks cloaked in the mist: The landscape of mammalian replication origins[END_REF].

5 A

 5 explain both the U-shaped timing domains and the N-shaped directionality profiles (FigureI--D)[START_REF] Hyrien | From simple bacterial and archaeal replicons to replication N/U-domains[END_REF]. Additional single-molecule studies are required in order to fully elucidate the mechanism governing the replication of these regions[START_REF] Rhind | DNA replication timing[END_REF].

Figure I- 5 :

 5 Figure I-5: Replication fork directionality and models for origin activation. A| Schematic representation of an N-domain, the constitutive unit of skew profiles. Skew (S) is defined as S = STA+SGC where STA = (T-A)/(T+A) and SGC = (G-C)/(G+C)). B| Schematic representation of a Udomain composing the timing profiles. Constant timing regions (CTRs) are linked by transition timing regions (TTRs). The increasing ORI firing rate in S-phase can be explained with two models: C| First model: replication initiates at early firing ORIs. Origins are specified by an open chromatin structure and are independent each other. D| Second: the cascade (or domino) model. Replication initiates at early and highly efficient ORIs that sequentially activate other ORIs (in a more compact chromatin environment) with a domino effect. Blue arrows represent sequential stimulation of ORIs (in purple), the colour fading from green to red marks opened and closed chromatin, respectively. E| Example of Ndomain and F| U-domain on Chr 10 (human). Different timing profiles of different cell lines (green: BG02 ESC, red: K562, blue: GM06990) are shown. G| Replication fork directionality (RFD) profiles determined by OK-Seq on HeLa (orange) and GM06990 (teal blue) cell lines from Petryk et al (2016). N-shaped RFD profiles perfectly match the U-domains of the timing profiles, as well as the N-domains of the nucleotide compositional skew profile. Positive or negative Okazaki RFD values (calculated on the number of reads mapping on the C=Crick and W=Watson strands over a 1 kb window with the formula: RFD = (C-W)/(C+W)) are shown in red and blue, respectively. C and D are adapted from Guilbaud et al (2011), E and F from Hyrien et al (2013) and G is modified from Petryk et al (2016).

  generally more dispersed, and did not associate with open chromatin marks. Strikingly, the two categories of ORIsearly and latereside in specific and dedicated chromatin environments, showing a high correlation between replication timing and chromatin conformation. However, it is still not clear the cause/effect relation between the two components. Interestingly, N/Udomains allowed to propose a cascade model able to explain these observation if early and efficient ORIs locate in open chromatin (at the border of the domains), while random, rather inefficient and dispersed initiation can happen elsewhere. RFD profiles obtained with the recent OK-seq technique agreed with the cascade model and started to depict a sharper picture of the mammalian DNA replication landscape. Nevertheless, definitive validation requires singlemolecule observations on the entirety of the genome.

  genomic DNA or against cloned DNA molecules from the investigated locus. Major concerns about the SNS purification techniques were raised when comparing the different studies. A very low concordance between experiments obtained by different laboratories was observed. When looking at λ-SNS-chip experiments (Hyrien 2015, Urban et al 2015):

  the possibility to guarantee a reproducible and precise replication program assuring faithful genome duplication (see paragraph I.5.2.3). The spatiotemporal program of DNA replication requires single-molecule (SM) visualisation of (ideally) large regions, in order to validate the mechanisms underlying the ORI flexibility. It should be noted that SM methods visualize the instantaneous replication state of single DNA fragments. It is not the case of ORC-ChIP for example, which is only detecting potential initiation sites within a cell population. Thus, SM methods can tackle the important question of cell-to-cell variability 23 , which appears necessary to understand the regulation of DNA replication. tool for single-molecule analysis of DNA replication The first SM studies of DNA replication were possible thanks to the invention of the fibre autoradiography analysis (paragraph I.2.1). SM techniques were substantially improved when replication intermediates of large plasmids started being examined by EM (paragraph I.2.4): precise measurements of replication bubble sizes and ORIs spacing could be done without requiring several months of radio-exposure. In 1993 Parra and Windle developed a method called "DNA fiber spreading" which consisted in lysing cells on a glass slide by adding detergent on one side, and letting the DNA flow down to the other side (Parra & Windle 1993). Specific target loci could be identified hybridising fluorescent in situ hybridisation (FISH) probes onto the fibres. In 1998, Jackson and Pombo managed to label the growing replication bubbles with the thymidine analog 5-Bromo-2'-deoxyuridine (BrdU) or with two consecutive pulses of BrdU and 5-Iodo-2′-deoxyuridine (IdU) in cells synchronized at the beginning of S-

  was rapidly adapted to mammalian cells by labelling replicated tracts with thymidine analogs, such as 5-Chloro-2'-deoxyuridine (CldU) and 5-Iodo-2′-deoxyuridine (IdU) (Norio & Schildkraut 2001)(Figure I-6 B-C). Conversely to digoxigenin-dUTP and biotin-dUTP, CldU

Figure I- 6 :

 6 Figure I-6: Dynamic DNA combing. A| Schematic representation of the dynamic molecular combing technique. A hydrophobic glass coverslip is incubated in a buffered DNA solution (1). Free DNA ends attach to the surface (2) and the coverslip is lifted at a constant speed[START_REF]2 Test the continuity of the fluorescent replication signal[END_REF]. Thanks to the constant force exerted by the meniscus at the solution/air interface, molecules are uniformly stretched along their length and adsorbed on the coverslip. Exemplary λ-DNA (48.5 kb) combed fibres are shown on the top right corner. B| "Classic" labelling scheme: asynchronous cells are pulse-labelled with IdU (blue) for 20 min and with IdU+CldU (blue+green) for another 20 min, the DNA is extracted, purified and combed. A specific DNA probe for the target locus can be hybridised on combed fibres. C| A replicative DNA fibre from the human IGH locus. DNA fibres obtained as explained in (B) from HeLa cells: the three labels (red, blue, green) and the total DNA (white) were revealed with appropriate antibodies. To facilitate the reading, the 4 channels are depicted on top of one another. Top, map position of the probe. Bottom, interpretation. The detected ORI is shown with the purple triangle. Adapted from[START_REF] Guilbaud | Evidence for sequential and increasing activation of replication origins along replication timing gradients in the human genome[END_REF].

Finally

  , the observation of replication tracts on long (i.e. Mb-scale) replicating molecules by high-throughput, SM techniques may clarify the replication landscape of highlyrepetitive regions. So far, because of the difficulty to unequivocally map short reads obtained by NGS techniques along repetitive sequences, some regions of the DNA are not accessible to sequencing-based replication analysis.

  freely permeate the cell membrane to be incorporated in the growing DNA chain. Moreover, unlike BrdU and the other halogenated analogs, EdU visualisation does not require DNA denaturation and antibody detection as it can be covalently coupled to fluorescent azides by a Cu(I)-catalysed [3+2] cycloaddition reaction (i.e. ''click'' chemistry)[START_REF] Salic | A chemical method for fast and sensitive detection of DNA synthesis in vivo[END_REF]. As a result, EdU labelling of newly-synthetized DNA is also compatible with DNA staining using a fluorescent intercalator. Visualisation of EdU-labelled tracks on combed DNA molecules has been achieved in S. cerevisiae, proving its applicability to the SM study of DNA replication[START_REF] Bianco | Analysis of DNA replication profiles in budding yeast and mammalian cells using DNA combing[END_REF]. More recently, 5-Vinyl-2'-deoxyuridine (VdU) was also reported to enter cells, and can be clicked (inverse electron demand Diels-Alder reaction) with fluorescent tetrazines[START_REF] Rieder | Alkene-tetrazine ligation for imaging cellular DNA[END_REF]. Importantly, the VdU and EdU labelling reactions are orthogonal (meaning they are chemically compatible and do not interfere with each other) thus allowing detection of the two pulses in two different colours on the same DNA sample. When replication bubbles are detected by DNA staining with a fluorescent intercalator, pulse labeling with only one nucleotide analog will specifically label the outer parts of the bubble synthesized during the pulse, allowing to measure the speed of each fork.Identifying the locus-of-origin of the molecule was historically done by FISH probing (see paragraph I.7.2). During the last decade, however, novel mapping techniques potentially able to identify every single molecule in the sample (instead of "FISHing" very few candidates) were developed. It is possible to fluorescently "barcode" genomic DNA molecules by introducing fluorescent dyes at specific sequence motifs. Xiao and colleagues were able to fluorescently barcode λ-DNA at specific nicking endonuclease (Nb.BbvCI) sites by incorporating fluorescent ddNTP terminators (cy3-acyclo-T) at the nicks using a DNA

2 .

 2 By fluorescent "barcoding" each molecule at predictable nicking endonucleases sites, is possible to map every molecule on the reference genome by aligning the molecule on the theoretical restriction map. 3. These two tools are compatible and combine in a technique that allows the simultaneous detection and mapping of DNA replication bubbles without the need for antibody detection. 4. Such technique can be coupled to a nanofluidic device, massively increasing the throughput. During my thesis I have been able to visualise the replication bubbles and the barcode of a very large number of DNA molecules by direct fluorescence alone, without any antibody detection. This work lays the groundwork for genome-wide, single-molecule analysis of eukaryotic DNA replication.

  3): 1) Antibody-based detection of the DNA replication signal. We eliminated the complex steps dictated by the metabolic labelling of replication forks and its subsequent detection with antibodies. To do so, we visualised the replication bubbles by two independent yet compatible fluorescent techniques: the increase of YOYO-1 fluorescence (Figure I-7 A) and the incorporation of AF647-dUTP at replicated tracts. This circumvents the use of antibodies to detect thymidine analogs and simplifies the existing SM techniques. 2) FISH-probing to detect the locus-of-origin of the combed molecules. To bypass this second bottleneck, we used a rapid fluorescent labelling of nicking endonucleases (NEs) sites, allowing each molecule to be mapped on the theoretical restriction map thanks to its fluorescent barcode (Figure I-7 B). All the molecules (instead of ~0.05%, as in FISH-probing) are mappable and therefore informative, and the barcode detection is direct (i.e. no antibodies). These are two significant improvements over FISH detection. The two solutions here-proposed are compatible and applicable on the same molecule, permitting the visualisation of replication bubbles and barcode of every molecule by direct fluorescence alone, without any antibody detection.

Figure I- 7 :

 7 Figure I-7: Novel methods for detecting and mapping DNA ORIs at a single-molecule level. A| Direct visualisation of replication bubbles by the strong DNA fluorescence intensity of replicated tracts: a fluorescent bis-intercalator, YOYO-1-iodide, is used to stain replicating DNA intermediates after DNA extraction, prior to DNA combing and epi-fluorescence microscopy. Replicated and unreplicated DNA segments should appear as strong and weak fluorescent tracts, respectively. B| Rapid DNA mapping using fluorescent labelling of nicking endonucleases (NEs) sites: Specific NE sites are made fluorescent by nick-translation in presence of fluorescently-labelled nucleotide precursors. As a result, NE sites can be visualised on the combed molecules as «dots-on-a-string», allowing their consequent alignment on the in silico restriction map.

II. 4

 4 Construction of a lariat DNA structure II.4.1 Design of the experiment To prove that replication bubbles can be detected based on their expected double YOYO-1 intensity, we constructed a lariat DNA structure from λ-DNA. In order to build the lariat DNA structure, we exploited the possibility to displace predictable oligos when two nicks are introduced on the same strand, in close vicinity (≤25 bp), as reported by H. Kuhn and M.D. Frank-Kamenetskii (Kuhn & Frank-Kamenetskii 2008) (Figure II-1).

Figure II- 1 :

 1 Figure II-1: Creation of a 3'-flap at specific sites. After nicking by specific NEs and strand displacement by moderate heating, an oligonucleotide with an additional 3'-flap was hybridised and ligated in place of the displaced oligonucleotide. Adapted from Kuhn and Frank-Kamenetskii (2008).

  by its flap. Nicking and displacement of target oligonucleotide followed by annealing and ligation of this flap-oligo onto the displaced region should result in a lariat DNA structure (Figure II-2).

Figure II- 2 :

 2 Figure II-2: Construction of a lariat DNA structure. See text for details.

  2.5), forms the most important part of the results and coincides with a technical article reporting useful advances that set the ground for future genome-wide analyses. The first four chapters describe the analysis and elucidation of multiple technical problems that had to be solved before tacking a concrete biological question (i.e. paragraph III.2.5: the first fluorescent replication map of the bacteriophage lambda genome,[START_REF] Carli | Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules[END_REF]).

  a) Density of adsorbed DNA fibres; b) Percentage of exploitable fibres; c) Stretching of fibres (i.e. evaluation of the stretching factor); d) Alignment of fibres; e) Uniformity of fibres' fluorescent signal.

  combing on silane surfaces (if pre-cleaned by plasma exposure), while polystyrene requires more acidic conditions (pH 5.5). To my experience, if the "homemade" coverslips are of acceptable quality, no salt addition to the DNA solution is required. Instead, I prefer to play with the buffer concentration in order to obtain the desired molecule density (FigureIII-1).

Figure III- 1 :

 1 Figure III-1: Variability in DNA combing on different surfaces and conditions.

Figure III- 2 :

 2 Figure III-2: Variation of the density of adsorbed DNA molecules depending on the concentration of MES buffer. A| Silane surfaces. The average densities of molecules observed per field were: 19.34 molecules at 50 mM (N=370), 35.87 molecules at 100 mM (N=686), 0 molecules at 250 mM. In all cases, the same preparation of λ DNA (62.5 pg/µL) at pH 6.0 was used. B| Polystyrene surfaces: 11.48 molecules at 50 mM (N=425), 66.56 molecules at 100 mM (N=634), 93.85 molecules at 250 mM (N=894). In all cases, the same preparation of λ DNA (500 pg/µL) at pH 5.5 was used. Both bar graphs show the relative amounts compared to the 50 mM condition.

Figure III- 3 :

 3 Figure III-3: Density of DNA molecules adjusted per DNA concentration.For silane, the average number of molecules per field per DNA quantity (mol/µm 2 /ng) were 309.57 at 50 mM, 573.95 at 100 mM, 0 at 250 mM. For polystyrene: 22.97 at 50 mM, 133.12 at 100 mM, 187.71 at 250 mM. Bars are normalised to the polystyrene 50 mM MES condition.

U

  -shaped and Y-shaped DNA molecules (Figure III-4). While U-shaped molecules consist in fibres that are stretched by the receding meniscus after binding of both DNA ends to the surface (Figure III-4 A, C), the mechanism generating Y-shaped molecules is not clear. They most probably arise from multiple attachments of the inner part of the molecules (in addition to their ends) before lifting the surface from the solution (Figure III-4 B, D). However, they could also derive from pairing of adjacent DNA molecules.

Figure III- 4 :

 4 Figure III-4: Examples of U-shaped and Y-shaped structures. A| Two U-shaped structures on silane. B| Y-shaped structure on silane. C| Two U-shaped structures on polystyrene. D| Y-shaped structure on polystyrene. U-shaped structure can be explained by a mechanism involving the stretching of molecules that were attached to the coverslip with both DNA ends. Yellow arrows point to the DNA molecule ends. Y-shaped structure may arise with different mechanisms. The most probable consists in an attachment of the inner part of the molecules (blue arrows) in addition to their ends, before lifting the surface from the solution. The direction of combing is from left to right. All the images are acquired at 100x magnification. The fields shown (180 x 120 px) correspond to 28.8 x 19.2 µm.

Figure III- 5 :

 5 Figure III-5: Quantitation of adsorbed U-shaped DNA molecules depending on the concentration of MES buffer. A| Silane surfaces.The average frequencies of Y-shaped molecules observed were: 25.68% molecules at 50 mM (N=370), 21.72% molecules at 100 mM (N=686), 0% molecules at 250 mM. In all cases, the same preparation of λ-DNA (62.5 pg/µL) at pH 6.0 was used. B| Polystyrene surfaces: 1.41% molecules at 50 mM (N=425), 2.84% molecules at 100 mM (N=634) and 3.36% at 250 mM (N=894). In all cases, the same preparation of λ-DNA (500 pg/µL) at pH 5.5 was used.

Figure III- 6 :

 6 Figure III-6: Quantitation of adsorbed Y-shaped DNA molecules depending on the concentration of MES buffer. A| Silane surfaces.The average frequencies of Y-shaped molecules observed were: 4.32% molecules at 50 mM (N=370), 32.94% molecules at 100 mM (N=686), 0% molecules at 250 mM. In all cases, the same preparation of λ-DNA (62.5 pg/µL) at pH 6.0 was used. B| Polystyrene surfaces: 0.71% molecules at 50 mM (N=425), 1.89% molecules at 100 mM (N=634) and 21.14% at 250 mM (N=894). In all cases, the same preparation of λ-DNA (500 pg/µL) at pH 5.5 was used.

  discarding the Y-shaped molecules, I measured all the fibres adsorbed on silane and polystyrene surfaces and computed the correspondent sizes (Figure III-7). In all cases, except at 250 mM MES on polystyrene, data tend to cluster at specific sizes (Figure III-7, red squares). These represent full-length, stretched λ-DNA molecules. The dispersion of the molecule sizes is broader when increasing the buffer concentration.Not surprisingly, an important amount of data distribute at lower lengths than fulllengths molecules, while very few molecules are longer. Shorter measurements might represent either broken or under-stretched DNA molecules, while the longer ones could arise from concatamerisation of multiple fibres as well as from over-stretched molecules. Since the stretching force is almost exclusively determined by the meniscus tension (which is constant with a constant lifting speed), there is little probability that molecules experience such a different stretching force explaining the above-described distributions. In my opinion, shorter molecules entirely derive from DNA breakage happening during or before molecular combing, while longer molecules derive from concatamerisation of multiple fibres.

Figure III- 7 :

 7 Figure III-7: Size distributions of combed λ-DNA fibres depending on the concentration of MES buffer. A| Silane surfaces. The distribution of the molecule lengths show two clusters of data both at 50 mM (N=354) and at 100 mM MES (N=460). Very few measurements distribute at values longer than the full-length λ-DNA molecules indicated by red rectangles.In all cases, the same preparation of λ-DNA (62.5 pg/µL) at pH 6.0 was used. B| Polystyrene surfaces. Unlike silane, molecule sizes tend to cluster in three groups when combing at 50 mM (N=422) and at 100 mM (N=622) MES. The longer-size group, correspond to full-length molecules (red rectangles). At 250 mM (N=705), a very dense distribution is observed, and no evident clustering can be recognised. In all cases, the same preparation of λ-DNA (500 pg/µL) at pH 5.5 was used.

Figure III- 8 :

 8 Figure III-8: Size distributions of combed λ-DNA fibres depending on the concentration of MES buffer.A| Silane surfaces. The distribution of the molecule sizes show two distinct peaks at 50 mM (N=354) and at 100 mM (N=460) MES. More than 35% (50 mM) and more than 20% (100 mM) of molecules are specifically localised in a 1 µm bin corresponding to the full-length λ-DNA molecules. In all cases, the same preparation of λ-DNA (62.5 pg/µL) at pH 6.0 was used. B| Polystyrene surfaces. Unlike silane, a tri-phasic distribution can be recognised at 50 mM (N=422) and at 100 mM MES (N=622). The longersize peak, again, correspond to full-length molecules and contains ~20% (50 mM) or >25% of the molecules in a small 1 µm bin. At 250 mM (N=705), no evident full-length peak can be recognised. In all cases, the same preparation of λ-DNA (500 pg/µL) at pH 5.5 was used.

Figure III- 9 :

 9 Figure III-9: Non-linear fit of λ-DNA molecule lengths. A| Silane surfaces: the 50 mM curve (black, N=354) was fit with a Gaussian, G of mean, m=24.90 and standard deviation, SD=0.52 (red). The 100 mM curve (dark grey, N=460) was fit with G(m=22.25; SD=0.53)(orange). B| Polystyrene surfaces: the 50 mM curve (black, N=422) was fit with G(m=26.95; SD=0.39)(red), the 100 mM curve (dark grey, N=622) was fit with G(m=24.79; SD=0.44)(orange), and the 250 mM curve (light grey, N=705) was fit with G(m=17.82; SD=6.85)(green).

I

  measured the angle of each molecule, expressed as the difference (in degrees of angle) between the line containing the DNA contour (3 pixels width) and the horizontal. This angle is positive when right end of the molecule is above the horizontal and negative when below. When the molecule was not perfectly linear, the angle was approximately calculated using the tangent passing by the molecule ends. At all the MES concentrations, except at 250 mM on silane, it was possible to compute the average molecule angles (Figure III-10).

Figure III- 10 :

 10 Figure III-10: Alignment of λ-DNA molecules under various conditions and surfaces.

Figure III- 11 :

 11 Figure III-11: Distributions of the normalised molecule angles. For each condition, molecules' angles were normalised to the average angle value, in order to compare the dispersion of the data. A| Silane surfaces: at 100 mM MES (N=213), the interquartile range (i.e. the range of scores from lower to upper quartile) is taller than at 50 mM MES (N=212), indicating a broader distribution of angles and thus molecules directions. B| Polystyrene surfaces: the 25 th and the 75 th percentiles have the same values at 50 mM (N=255), 100 mM (N=453) and 250 mM (N=582) MES conditions, showing the same statistical dispersion of the angles. The upper and lower whiskers represent the minimum and the maximum angle values, respectively.

Figure III- 12 :

 12 Figure III-12: Distributions of the normalised molecule angles (directions). A| Silane surfaces: at 50 mM MES (N=212), a normal distribution is observed with equal probabilities of the angles to diverge with positive or negative values compare to the mean angle (0°, due to normalisation). When increasing the MES concentration to 100 mM (N=213), the distribution appears asymmetrical with a long and thin left tail and a short but fat right tail. B| Polystyrene surfaces: at all tested concentrations (50 mM N=255, 100 mM N=453, 250 mM N=582), a normal distribution is observed with equal probabilities to diverge in opposite directions compared to the mean combing direction.

  concentrations (Figure III-13). On silane surfaces, the median of the intensity values was slightly increasing with the buffer concentration (4222 at 50 mM MES and 4718 at 100 mM MES). On polystyrene, when stepping from 50 mM to 100 mM MES the median ranged from 15451 to 24113, but it decreased to 22454 in the case of 250 mM MES. In all cases, the medians of the distributions were significantly different each other (Mann-Whitney rank-sum test, Pvalue < 0.0001).

Figure III- 13 :

 13 Figure III-13: Distributions of the mean molecule intensities. A| Silane surfaces: at both 50 mM (N=354) and 100 mM MES (N=460), a narrow distribution of mean fluorescent intensity of the molecules was observed. The median, the lower and the upper quartiles are also shown in red (for 50 mM MES) and orange (for 100 mM MES) colours. B| Polystyrene surfaces: higher and broader distributions were obtained compared to silane. For 50 mM (N=422), 100 mM (N=622) and 250 mM (N=705), medians and interquartile ranges are shown in red, orange and yellow, respectively. All the distributions had significantly different medians when tested by Mann-Whitney rank-sum tests (P-value < 0.0001).

Figure III- 14 :

 14 Figure III-14: Inter-molecule variability of the mean molecule intensities in different combing conditions. The coefficient of variation of the mean molecule intensities (CV, see text for details about the statistic) indicate more variability in the case of polystyrene coverslips compared to silane. At 50 mM MES the difference is more pronounced that at 100 mM MES. At 250 mM the comparison is not possible having no molecule adsorbed on silane surfaces.

Figure III- 15 :

 15 Figure III-15: Distributions of the mean molecule intensities as function of the molecule sizes in different combing conditions. A-B| Silane surfaces: at both 50 mM (A, N=354) and 100 mM (B, N=460) MES, no evident correlation between the mean fluorescent intensity and the molecule size is observed. For each mean fluorescent intensity value (black circles, Im), the standard deviation of the fluorescent intensity is indicated (grey whiskers, std(im)). The medians of the mean fluorescent intensities are also shown as a red line. C-E| Polystyrene surfaces: higher and broader distribution were obtained at 50 mM (C, N=422), 100 mM (D, N=100) and 250 mM (E, N=705) MES compared to silane. Again, no evident correlation was found between mean molecule intensity and molecule size. See text.

Figure III- 16 :

 16 Figure III-16: Distributions of the intra-molecule intensity variability in different combing conditions.The distribution of the coefficient of variation of the intensity calculated within each molecule (cv, see text for details about the statistic) is statistically higher on polystyrene coverslips compared to silane (Mann-Whitney rank-sum test, P-value < 0.0001). At 50 mM MES, the medians were 0.25 (silane, N=354) vs 0.308 (polystyrene, N=422). At 100 mM MES: 0.25 (silane, N=460) vs 0.304 (polystyrene, N=622). At 250 mM: 0.309 (polystyrene, N=705). Medians showed no statistical difference when comparing different conditions within the same surface.

2 kb)

 2 are located on different strands and spaced by only 300 base-pairs (Figure III-17). Thus, when labeling DNA by nick-translation, a double-strand break should be generated in between the two nicks due to the collision of the converging forks travelling on opposite strands from vicinal nicks. The two generated fragments of ~31.0 kb and ~17.5 kb should be resolvable by agarose gel electrophoresis.

Figure III- 17 :

 17 Figure III-17: Sequence motif distribution of Nb.BbvCI on λ-DNA and predicted nick-translation direction.Top| Sequence motif distribution. Bottom| predicted nick-translation direction. Nb.BbvCI is a nicking endonuclease which recognises the 5'-CCTCAGC-3' sequence on a dsDNA substrate but cleaves only one strand. The introduced nicks (indicated by scissors) are therefore specifically located on the top (in blue) or bottom (in grey) strand, defining the nick-translation direction. Note that nicking sites located at 30.9 kb and 31.2 kb are spaced by only 300 bp and reside on opposite strands in convergent orientation of 3'-ends.

Figure

  Figure III-18: Gel electrophoresis (0.7% agarose) of timed aliquots.

  Surprisingly, molecules labelled with Klenow fragment showed no significant fluorescent signal (not shown). In contrast, molecules nick-translated with DNA polymerase I showed intense signals. Nevertheless, residual unincorporated Cy5-dUTPs tended to stick to the surface both on silane-and polystyrene-coated coverslips. For unclear reasons, less background noise was observed on the red channel (Cy5-dUMP signal detection) when combing on silicone surfaces. Such hydrophobic coverslips, which were retrieved on the market, were discarded from the analysis in this dissertation because resulting in a very high variability of YOYO-1 signal (data not shown), a feature to be avoided while developing our techniques. However, we could use them as first indication that fluorescent barcoding of genomic DNA is compatible with molecular combing and visible by standard epifluorescence microscopy without any need for TIRF or FRET.

Figure III- 19 :

 19 Figure III-19: Combing of lambda-phage DNA fragments fluorescently barcoded at Nb.BbvCI sites.After incubation with Nb.BbvCI, λ-DNA was nick-translated with DNA polymerase I in presence of Cy5-dUTP (red) resulting in complete breakage between vicinal nicks (see explanation in the text). The DNA was purified in G-50 microspin columns, stained with YOYO-1 (1 to 20 bp ratio, green), combed on silicone-coated surfaces and immediately visualized by epifluorescence microscopy without mounting. Small fragments were generated, and not all NE sites were 100% labelled, but it was generally possible to assign the labelled fragments to a particular region of the full-length molecule based on its labelling pattern and length. Scale bar: 5 μm (~10 kb). Similar results were obtained with both silane and polystyrene coverslips. In these cases the YOYO-1 signal was more constant along the length of the molecule but the dotty background presumably due to unincorporated Cy5-dUTP was stronger.

Figure III- 20 :

 20 Figure III-20: Gel electrophoresis (0.7% agarose) of timed aliquots.

  Figure III-21).Again, when labelling at 37°C, the time required for EDTA addition and diffusion, was sufficient to accumulate an important fraction of broken molecules in the 0 min samples. At 5 minutes, the full-length bands were already broken into smaller fragments. Thus, reducing the labelling temperature is clearly not a solution to the fragmentation problem, as the drop in labelling efficiency occurred without a substantial drop in molecule breakage.

Figure III- 21 :

 21 Figure III-21: Gel electrophoresis (0.7% agarose) of timed aliquots.

  time in understanding why the molecules labelled by strand-displacement with Klenow fragment did not show appreciable fluorescent signals by DNA combing. Firstly, I developed an electrophoretic alkaline essay that allowed me to confirm that nicks are exclusively introduced at all the expected sites (Figure III-22 A) as predicted by bioinformatics analysis (Figure III-22 C), and that each of these sites served as starting point for nick-labelling (Figure III-22 B).

Figure III- 22 :

 22 Figure III-22: Monitoring AlexaFluor647-dUMP incorporation at individual Nb.BbvCI nicking sites in λ-DNA molecules by alkaline agarose (0.7%) gel electrophoresis. A| DNA detection (SYBR Gold): bands correspond to single stranded DNA fragments generated by Nb.BbvCI, perfectly reflecting its expected nicking-endonuclease pattern (shown in C|). B| AF647-dUMP detection: the AF647-dUMP signal individual indicates the occurred labelling at each attended site. C| Nb.BbvCI sequence motif distribution and predicted nick-translation direction on λ-DNA: the expected single-stranded fragments and their lengths are indicated both for the top and the bottom strand.

Figure III- 23 :

 23 Figure III-23: Restored combing of λ-DNA molecules barcoded at Nb.BbvCI sites by Klenow polymerase after flaps removal with RecJf. A| DNA combing: molecules showing strong fluorescent labels could comb properly after removal of single-stranded flaps generated during nick-labelling procedure. B| Agarose (0.7%) gel electrophoresis, of precipitated DNA combing sample (ethidium bromide): DNA was purified directly from the combing reservoir by classic phenol-chloroform extraction followed by ethanol precipitation. Three bands, reflecting preferential breakage at the "fragility hotspots" can be identified: 31 kb, 17.5-18 kb, 13 kb (see text). C| AF647-dUMP detection confirmed that the fragments contain fluorescent labels. D| Nb.BbvCI sequence motif distribution and predicted double-strand breaks (DSB) on λ-DNA: regions containing at least 2 nicked sites separated by less than 500 base-pairs behave as "fragility hotspots". The expected generated fragments and their lengths are indicated. E| Individual combed λ-DNA molecules (green YOYO-1 fluorescence) labelled by nick-translation with AF647-dUTP at Nb.BbvCI sites (red), aligned under the map of Nb.BbvCI sites along λ-DNA.

Figure III- 24 :

 24 Figure III-24: Breakage of nicked λ-DNA molecules at the "fragility hotspots" due to heatinactivation of the nicking-enzyme.A| Agarose (0.7%) gel electrophoresis (ethidium bromide): Nb.BbvCI-digested λ-DNA directly loaded after the nicking reaction showed slight fragmentation compared to untreated λ-DNA (48.5 kb). In contrast, heat-inactivated samples showed high DNA breakage (orange arrows: 31 kb, 17.5-18 kb, 13 kb). However, if the sample was immediately incubated at 70°C for 10 minutes just after heatinactivation, a high proportion of full-length material was restored (red arrow). Note than neither freezing (-20°C), nor snap-freezing (-80°C), of nicked DNA increase the proportion of broken molecules compared to control nicked λ-DNA. B| Nb.BbvCI sequence motif distribution and predicted doublestrand breaks (DSB) on λ-DNA: "fragility hotspots" regions are indicated (dashed lines) and explain the observed bands corresponding to broken DNA (see text).

2 .

 2 Efficient nick-labelling was associated with abundant double-strand breaks between vicinal nicks; 3. DNA fragmentation needed to be minimized during purification of barcoded molecules. When electrophoresing the same samples in alkaline conditions, we could confirm that the fragmentation of full-length molecules at the fragility hotspots after G-50 purification was not due to pre-existing nicks, since undigested λ-DNA was not broken by spinning in G-50 columns (Figure III-26 C, left). Furthermore, the incubation at 70°C following the heatinactivation step neither perturbed the migration pattern of the nicked sample, nor introduced nicks on the undigested control, as expected (Figure III-26 C, right).I concluded that a good protocol to maintain full-length λ-DNA molecules consists in cutting the DNA with Nt.BspQI, heat-inactivate the enzyme at 80°C for 20 minutes followed by a 10 minutes incubation at 70°C prior to nick-translation in the presence of fluorescent dUTP. The labelled DNA would then to be ligated at existing nicks to avoid breakage during subsequent purification prior to DNA combing.

Figure III- 26 :

 26 Figure III-26: Monitoring the conservation of full-length λ-DNA molecules after Nt.BspQI nicking endonuclease digestion.

Figure III- 27 :

 27 Figure III-27: Monitoring all the barcoding steps (nicking-labelling-repair) by alkaline agarose (0.7%) gel electrophoresis. A| DNA detection (SYBR Gold): single-strand DNA fragments generated by Nt.BspQI digestion are correctly sealed after the repair reaction, restoring the full-length population of λ-DNA (red arrow). B| AF647-dUMP detection: the AF647-dUMP signal indicates the occurred labelling reaction at each attended site. The full-length molecules also show the expected fluorescent signal.

  Figure III-28 shows a collection of barcoded λ-DNA molecules obtained with this protocol combed on silane surfaces.

Figure III- 28 :

 28 Figure III-28: Nick-labeling of λ-DNA molecules at Nt.BspQI nicking sites. Top| Expected distribution of Nt.BspQI nicking sites (red dots) along the map of the λ genome (green). Bottom| Individual combed λ-DNA molecules (green, YOYO-1 fluorescence) labelled by nicktranslation with AF647-dUTP (red) at Nt.BspQI sites, aligned under the map of Nt.BspQI sites along λ-DNA. Blue lines indicate alignment. Note that the extra label at the left hand is likely due to filling of the 3'-recessed cohesive end, where template sequence limits incorporation to 1 dUTP. This suggests that we are able to detect a single AF647 fluorochrome by standard epifluorescence microscopy.

Figure III- 29 :III

 29 Figure III-29: Nuclear assembly and DNA replication in Xenopus egg extracts.

Figure III- 31 :

 31 Figure III-31: Discontinuity of the direct fluorescent replication signal on combed Xenopus sperm DNA. Sperm nuclei were incubated in Xenopus egg extracts for 120 minutes in presence of AF647-dUTP, nuclei were isolated and the DNA purified. The DNA was stained with YOYO-1 and imaged by epifluorescence microscopy. Top| merged image: YOYO-1, green + AF647-dUMP, red. Middle| YOYO-1 channel (total DNA). Bottom| AF647-dUMP (replicative signal). Yellow arrows indicate gaps in the replication signals, while blue arrows point unreplicated molecules. Sale bar: 20 µm (40 kb).

Figure III- 32 :

 32 Figure III-32: Long Range PCR on λ-DNA.Polymerase Chain Reaction was performed on λ-DNA template hybridising different couples of primers to obtain fragments of various sizes up to 30 kb in length. The 9797 bp-long amplicon, in particular, showed a sharp and defined band as the control PCR band (537 bp).

Figure III- 33 :

 33 Figure III-33: Primer extension using different polymerases and different dNTP cocktails: neutral agarose gel electrophoresis.

Figure III- 34 :

 34 Figure III-34: Primer extension using different polymerases: alkaline agarose gel electrophoresis.PCR fragments of 500 bp or 10 kb were purified and hybridised with a primer. The primer was extended with 3 different polymerases (Taq pol, LongAmp or Klenow exo-) and in 3 different conditions: 1) in the presence of all the canonical nucleoside triphosphate (dNTPs), or 2) a combination of all dNTPs + AF647-dUTP, or 3) a combination of dATP+dCTP+dGTP (dACG) + AF647-dUTP. The gel was stained with SYBR Gold and the two channels were acquired at the fluor-imager. A| SYBR Gold: all the products are of the expected lengths. B| AF647-dUMP: a smear is particularly evident in the 10 kb Klenow reactions confirming uncompleted primer extension. LongAmp Taq pol showed the best DNA labelling at 10 kb length.

Figure III- 35 :

 35 Figure III-35: Preparation of the fluorescent fibres for DNA combing by primer extension.PCR fragments of 10 kb were purified and hybridised with a primer that was extended with LongAmp Taq DNA polymerase in 3 different conditions: 1) in the presence of all the canonical nucleoside triphosphate (dNTPs), or 2) a combination of all dNTPs + AF647-dUTP, or 3) a combination of dATP+dCTP+dGTP (dACG) + AF647-dUTP. The gel was stained with Ethidium bromide or SYBR Gold and the two channels were acquired at the fluor-imager. A-B| Neutral Agarose Gel Electrophoresis. A| Ethidium bromide: all the products are of the expected lengths B| AF647-dUMP: a strong 10 kb fluorescent band is evident when both canonical dTTP and fluorescent dUTP are present, but much less fluorescent product is produced in the absence of dTTP. C-D| Alkaline Agarose Gel Electrophoresis. C| SYBR Gold, D| AF647-dUMP: same considerations as for the neutral gel.

Figure III- 36 :

 36 Figure III-36: DNA combing of hemi-fluorescent DNA constructions. 10 kb amplicons carrying only 1 fluorescent strand were combed on silane surfaces. Molecules shown in A| were obtained incorporating AF647-dUTP in absence of canonical dTTP, while molecules shown in B| were obtained incorporating AF647-dUTP in the presence of canonical dTTP. Scale bar: 10 µm (20 kb).

  duplex segment is joined to a circular moiety. During combing this molecule is expected to attach by its unique double-stranded end, and the circular moiety is expected to be stretched resulting in a pair of duplexes (Figure III-37 A). Indeed, we were able to visualize abrupt changes in fluorescence intensity consistent with a lariat DNA molecule (Figure III-37 B-C). The corresponding intensity profile allowed to discern without ambiguity adjacent segments of precisely single-and double-fluorescence intensity as expected for lariat molecules. This observation established the proof-of-principle that the fluorescent bis-intercalator YOYO-1 allows to detect the boundary between tracts containing one or two double-stranded DNA molecules. It should therefore become feasible to detect replicated bubbles on replication intermediates by YOYO-1 fluorescence without thymidine analog incorporation.

Figure

  Figure III-37: Proof-of-principle of the YOYO-1-based replication bubble detection.A| A lariat-DNA structure, can adhere to the surface only using its free end. The two duplexes forming the lariat are therefore paired together during the combing process under the force of the receding meniscus. B-C| Lariat DNA was stained with YOYO-1, combed on polystyrene-coated surfaces and visualized with standard epifluorescence microscopy. The step difference in YOYO-1 intensity permit the identification of the loop (i.e. region with the two double-stranded arms) revealed by the doubling of fluorescence. This is directly visible on the raw image B|, without requiring any further adjusting step like thresholding or smoothing process C|.

Figure III- 38 :

 38 Figure III-38: Combining direct fluorescent labeling of replication tracks and fluorescent barcoding on λ-DNA or sperm nuclei replicated in Xenopus egg extracts. A| λ-DNA: the image shown represent a ~3 fields-of-view panoramic picture. The DNA backbone is stained with YOYO-1 (blue), late-replicating tracts are labelled in yellow, and nicking-endonuclease sites are detected as red dots. B| Sperm nuclei: a collection of the best 10 selected molecules is shown. Scale bars: 10 µm (20 kb).

Figure III- 39 :

 39 Figure III-39: Buffers addition affect DNA combing quality.A control λ-DNA solution (250 pg/µL) was used to test for the quality of DNA combing on silane surfaces. Different amounts of various 10X buffers were sequentially added to the control reservoir A| to mimic the amounts used in the fluorescent barcoding protocol B-C|. Supplementary buffer was also added to further investigate the effect of exceeding amounts D-E|. All the images correspond to 4 fieldsof-view with a 100X objective and a classic 512x512 captor. Scale bar: 10 µm (20 kb).

Figure III- 40 :

 40 Figure III-40: Enzymes addition affect DNA combing quality.A control λ-DNA solution (250 pg/µL) was used to test for the quality of DNA combing on silane surfaces. Different amounts of various enzymes were sequentially added to the control reservoir A| to mimic the amounts used in the fluorescent barcoding protocol B-C|. Supplementary proteins were also added to further investigate the effect of exceeding amounts E-F|. All the images correspond to 4 fieldsof-view with a 100X objective and a classic 512x512 captor. Scale bar: 10 µm (20 kb).

  reaction and DNA combing to take place with minimal DNA manipulation and restored proper DNA combing and simultaneous visualization of the replication bubbles and the fluorescent barcode (Figure III-41). The complete protocol is described in the article (see paragraph III.2.5, De Carli et al (2016)).

Figure III- 41 :

 41 Figure III-41: Combining direct fluorescent labeling of replication tracks and fluorescent barcoding on DNA from sperm nuclei replicated in Xenopus egg extracts. DNA backbone is stained with YOYO-1 (blue), late-replicating tracts are labelled in yellow, and nicking-endonuclease sites are detected as red dots. Scale bar: 10 µm (20 kb).

Figure III- 42 :

 42 Figure III-42: Irys chip scheme and functioning. A| Schematic view of the Irys chip 39 . The DNA in solution is deposited in the well regions. Applying an electric current the DNA in solution is concentrated at the lip region, and by a jump in the potential, is spiked up into the feeder channels. DNA is then untangled in the pillars (from few, big pillars, to many small ones) arriving in microchannels, and finally in nanochannels. B| Example of DNA (green, YOYO-1 stained) untangling inside the Irys chip 40 . All images are propriety of, and adapted from BioNano Genomics.

  We determined the size distribution of fluorescent gaps measured on random DNA fibres totalling 10.6 Mb of DNA (vs. 9.5 Mb for combing). Surprisingly, along the 10.6 Mb of sperm chromatin analysed, only 163 gaps were found, totalling a length of 2.5 Mb. This value is one order of magnitude lower than the amount of gaps measured by DNA combing (1,130; totalling a length of 3.2 Mb), suggesting a more accurate signal detection by the nanochannel technology (most likely reflecting the better illumination source of the machine 41 ). The lower number of gaps explains the stronger fluctuations of their size distribution (compare Figure III-43 A with Figure III-43 B). Interestingly, the percentage of fluorescently-labelled DNA(AF647-dUMP) measured on nanochannel fibres was 75.9%, very close to the 74% of replication extent estimated by TCA measured by DNA combing. These data suggest that the fluorescent gaps measured on nanochannel fibres, indeed, may correspond to real unreplicated segments. However, the nanochannel experiment was performed with a different Xenopus egg extract than the one used for DNA combing and, unfortunately, for this extract we did not perform the TCA assay. Since different egg extracts replicate with different efficiencies, this hypothesis needs to be formally tested.

Figure III- 43 :

 43 Figure III-43: Comparing the size distribution of the gaps in the replication signal by DNA combing vs. nanochannel technology. A| DNA combing. 1,130 gaps (accounting for 3.3 Mb) were measured on total 9.5 Mb of randomly selected DNA fibres. The percentage of AF647-dUMP labelled DNA was 65.4%. Adapted from De[START_REF] Carli | Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules[END_REF]. B| Nanochannel. Only 163 gaps could be measured on random fibres (accounting for 2.5 Mb on total 10.6 Mb of DNA analysed). In this case, the percentage of fluorescently labelled DNA was 75.9%.

Figure III- 45 :

 45 Figure III-45: Comparison of the replication analysis of λ-DNA using molecular combing vs. nanochannel techniques. A| Map position and DNA fibre coverage of λ-DNA replicating molecules obtained by DNA combing (blue), and B| by nanochannel (orange). Molecules were ordered from left to right. The black line represent the local total coverage, which is higher in regions containing the nicking endonuclease sites (indicated by red dots on the schematized λ-DNA restriction map located above the coverage profiles). C| Comparison of the normalised replication coverage of the λ-DNA genome (48.5 kb) as obtained by DNA combing (blue) vs. nanochannel (orange) techniques. The normalised coverage is almost flat with DNA combing, but reveals higher replication extents at the λ-DNA extremities with nanochannel.

  present in ~50-60 µM concentrations. It should be noticed that this is already a considerable improvement of the combing technique, especially for laboratories that study DNA replication taking advantage of the Xenopus egg extracts system. A typical DNA combing experiment takes at least one day less than the "classic" antibody-based protocols and significantly less handson time, while eliminating the antibody incubations and the washes. The most important advantage, however, is the possibility to reliably detect the DNA molecules thanks to the YOYO-1 staining instead of relying on the anti-ssDNA antibody signal, which is often dotty and difficult to interpret.Nevertheless, the low continuity of the fluorescent replicative signal on combed molecules remains a limitation. AlexaFluor-dUMP signal turned out to be more discontinuous than expected (see paragraphIII.2.3.2). This problem was controlled in the article here described (De Carli et al 2016), concluding that fluorescent gaps that are ≤7 px (~2.24 kb) are non-significant due to technical limitations. We tried to construct by primer extension hemisubstituted (i.e. hemi-fluorescent) dsDNA fragments mimicking the replication product obtained by semi-conservative DNA replication in egg extracts. Neutral and alkaline agarose gel electrophoresis suggested that 10 kb-long hemi-fluorescent amplicons could be obtained, but when imaged by DNA combing these molecules only showed rare and short fluorescent tracts, possibly due to the presence of residual canonical dTTP in the reaction mix. This prevented us to determine whether long, continuous stretches of fluorescent dUTP incorporation show a continuous or discontinuous signal after DNA combing. Therefore, the reason why replicated molecules that are presumably homogeneously substituted with fluorescent dUMP during incubation in egg extracts show discontinuities after DNA combing cannot be ascertained. Preliminary results, however, suggest that labelling patterns are much more continuous when visualized in nanochannels rather than by DNA combing (see paragraphIII.3.2). Comparing the distribution of fluorescence gaps of the same sample of Xenopus sperm chromatin replicative intermediates imaged by DNA combing or in the nanochannel device, will help us discern between the biological and the technical explanations (discussed in details in De[START_REF] Carli | Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules[END_REF], in paragraph III.2.5). At any rate, our statistical analysis show that fluorescent gaps larger than ~2 kb almost surely represent true gaps rather than technical artefacts. This size threshold is already low enough to allow reliable analysis of most replication intermediates.

  required FISH to probe any locus of interest. Comparing replication parameters over many loci simultaneously was therefore impossible. Here, fluorescent DNA barcoding allows us, in principle, to identify the locus of origin of all the DNA molecules combed on a single coverslip and to start to investigate whether replication parameters are indeed homogeneous over the entire genome or show some variation between different loci.At present we only applied the technique to a simple model, the replication of λ-DNA (48.5 kb genome) in egg extracts. Manual alignment of the DNA molecules was challenging because of the formation of concatamers of λ-DNA in the egg extracts. However, I believe that an analysis of the yeast genome (12.1 Mb), especially if collecting longer DNA molecules, would still feasible by manual analysis. Yeast chromosomes (n=16) span from 230 kb to 1.5 Mb, lengths that are in the range of the DNA combing technique.Even though we did not define a "global" threshold value for the YOYO-1 signal, the experiment would still be feasible by synchronising and releasing the yeast cell population into the S-phase: the strong (~double) YOYO-1 tracts should confirm the replication initiation at the known ARSs, providing an internal control. This would be the first single-molecule replication study covering the whole-genome of a eukaryote. It would also allow, for the first time, to rigorously test whether DNA replication initiation can also happen outside the known ARSs (which may have major roles in guaranteeing the genome stability upon replication stress or when a given ARS fails to fire).

  combing (with higher local coverage at the regions containing the NE sites), the normalised replication coverage had a different profile. The replication profile is not flat, but U-shaped, with increased replication at both ends of the λ-DNA sequence. It is not clear why DNA combing and nanochannels gave different results. The difference might reflect fluctuations due to insufficient coverage, but statistical testing argues against this possibility (see paragraphIII.2.5, De Carli et al (2016)).

  epigenetic signals is transitory and variable between experiments, which could explain the apparent discrepancy between the DNA combing and the nanochannel experiments. Analysis of a large number of molecules from repeated experiments is required to elucidate this point.In order to compare the fluorescent replication signals provided by the two different technologies, a limited analysis of the fluorescent replication gaps was also performed on the nanochannel data (see paragraphIII.3.2). When plotting the fluorescent gap distribution measured on sperm chromatin that was replicated in Xenopus egg extracts, the size distribution was similar to the one obtained by DNA combing, but the density was ten-fold lower.Strikingly, although in both cases the same amount of DNA was analysed (~10 Mb), only 163 gaps were counted on the nanochannel-imaged molecules vs. 1,130 on DNA combing-imaged ones. This is suggesting a better detection of the replication signal by the nanochannel technology than by DNA combing. Consistently, the fraction of AF647-labelled DNA was higher in the nanochannel than in the DNA combing sample (75.9% vs. 65.4%). For the DNA combing experiment, the fraction of AF647-labelled (65.4%) was lower than the fraction of replicated DNA measured by a 32 P-dATP incorporation and TCA precipitation (74.2%)(De

  The antibody-free DNA combing technology we presented (De Carli et al 2016) is, in my opinion, especially useful for laboratories handling the Xenopus egg extracts system. These can combine the YOYO-1-based bubble detection and the pulse incorporation of fluorescent nucleotide precursors, in order to reciprocally validate the two signals by overlap analysis.

(

  see paragraph IV.3) on yeast and to combine manual and automated analysis to address the following questions: a) Confirmation of the ARSs of the whole-genome, at SM level; b) Evaluation of potential initiation events outside canonical ARSs; c) Estimation of the stochasticity of ORI activation, ORI-by-ORI, genome-wide; d) Modelisation of the ORI initiation based on SM and genome-wide data.

  at a SM level by tracking specific components of the replication machinery. The major limitation that always impeded this kind of observations was the concentration barrier in SM imaging. Briefly, imaging of single fluorescent proteins is incompatible with the physiological concentrations of the proteins. The fluorescence background is too high and prevents identification of individual molecules. Pioneering works by J.C. Walter and A.M. van Oijen's laboratories showed that it is possible to visualise in real time the replication of λ-DNA that was immobilised on a glass substrate and flown with Xenopus egg extracts supplemented with a fluorescent version of flap endonuclease 1 (Fen1), a protein implicated in the Okazaki fragments processing and in base excision repair (Loveland et al 2012), or with a fluorescent Introduction La réplication de l'ADN est le procédé fondamental qui assure une duplication fidèle du génome au cours du cycle cellulaire. La réplication de l'ADN des cellules eucaryotes démarre en de nombreuses origines de réplication dont la position et le moment d'activation sont précisément régulés. Chez les mammifères, ce programme de réplication est encore mal connu et controversé (Hyrien 2015, Urban et al 2015). Par exemple, certaines données suggèrent que la réplication démarre à des sites choisis aléatoirement au sein de grandes zones (Petryk et al 2016), alors que d'autres données suggèrent l'existence de sites ponctuels efficaces associés à des éléments génétiques précis (Picard et al 2014). De plus, l'analyse en masse d'intermédiaires de réplication purifiés à partir de populations cellulaires ne montre qu'une image moyenne de la réplication dans la population qui masque la variabilité intercellulaire. C'est pourquoi, les évènements d'initiation dispersés sont probablement cachés dans des profils pangénomiques en raison des effets de moyenne (Lebofsky & Bensimon 2003). S'affranchir de ces effets de moyenne nécessite l'examen d'un grand nombre de molécules d'ADN réplicatives individuelles, qui permet d'évaluer la variabilité intercellulaire et de déceler les évènements rares comme les évènements récurrents. Cependant, les techniques en molécule unique actuelles sont lentes et de faible débit (Hyrien 2015, Lebofsky et al 2006). L'analyse d'un locus d'1-2 Mb requiert au minimum 6 mois pour n'obtenir que quelques dizaines à quelques centaines de molécules informatives, ce qui limite la puissance statistique. Il serait souhaitable de pouvoir couvrir la totalité du génome et d'augmenter la couverture de chaque locus.

2 '

 2 -désoxyuridine (CldU) et la 5-iodo-2'-désoxyuridine (IdU), ceux-ci sont incorporés dans l'ADN néosynthétisé qui peut alors être visualisé après détection par des anticorps fluorescents appropriés sur les molécules d'ADN peignées[START_REF] Jackson | Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells[END_REF][START_REF] Norio | Visualization of DNA replication on individual Epstein-Barr virus episomes[END_REF]. De plus, l'hybridation fluorescente in situ (FISH) avec des sondes d'ADN permet de révéler les molécules d'ADN qui portent ces séquences[START_REF] Pasero | Single-molecule analysis reveals clustering and epigenetic regulation of replication origins at the yeast rDNA locus[END_REF]. Malheureusement, cette technique est lente et laborieuse.La détection par anticorps introduit des étapes d'incubation et de lavage complexes, durant lesquelles les molécules ont tendance à se détacher de la lamelle. Ce problème est minimisé, mais non supprimé, par l'utilisation de lamelles silanisées, qui sont plus difficiles à préparer que d'autres types de surfaces.De plus, la détection est souvent imparfaite et génère un marquage pointillé plutôt que continu, ce qui limite la résolution de la technique et risque de faire prendre une région répliquée, mais mal détectée, pour une région non répliquée. L'utilisation de la FISH introduit des étapes d'hybridation et de lavage plus compliquées que celles requises pour la détection des segments néorépliqués, et limite sévèrement la longueur totale des régions génomiques qui peuvent être étudiées dans une seule expérience. Au moins deux sondes de couleurs différentes ou de tailles inégales sont nécessaires pour identifier et orienter les fibres qui contiennent le locus recherché. En raison de la taille habituelle des fibres (0.1 -1.0 Mb), il faut préparer environ 25 sondes pour étudier une région de 1 Mb, (0,03% du génome humain), ce qui prend un temps important. Enfin, un examen méticuleux des lamelles est nécessaire pour trouver les rares fibres qui contiennent le locus recherché.Des méthodes simples pour visualiser les bulles de réplicationNous avons éliminé les étapes complexes imposées par la détection par anticorps du marquage métabolique des fourches de réplication[START_REF] Carli | Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules[END_REF]. Nous avons montré qu'on peut visualiser les bulles de réplication par l'incorporation directe de dUTPs fluorescents au sein des régions répliquées.L'élimination des étapes intermédiaires de détection par des anticorps permet en principe de ne pas se restreindre à l'utilisation de lamelles silanisées. Nous avons donc exploré les propriétés de peignage de plusieurs types de surface, moins chères et plus accessibles, comme le polystyrène, la silicone et le téflon et effectué une comparaison approfondie des paramètres suivants pour le silane et le polystyrène : a) la densité des fibres d'ADN adsorbées. Le silane s'est révélé plus efficace que le polystyrène, spécialement à des concentrations faible du tampon MES (tampon standard de peignage). En utilisant les mêmes concentrations d'ADN, les surfaces de silane ont pu adsorber jusqu'à 13,5 fois plus de molécules d'ADN que les surfaces de polystyrène. Néanmoins, il restait possible d'obtenir une densité de fibres d'ADN acceptable avec du polystyrène. b) le pourcentage des fibres exploitables. En plus des molécules linéaires, on observe en routine des molécules en forme de U ou de Y lors d'une expérience de peignage. Les U proviennent de l'accrochage des deux extrémités de la molécule à la surface, et restent analysables. L'origine des Y est obscure et ces molécules sont écartées de l'analyse. Sur du silane, on observe < 5% de Y à 50 mM MES mais > 30% à 100 mM. Sur du polystyrène, on observe < 2% de Y à des concentrations de MES ≤ 100 mM, mais > 20% à 250 mM. A toutes les concentrations de tampon, on observe > 20% de U sur le silane, mais seulement < 5 % dans le cas du polystyrène.En conclusion, les conditions de peignage acceptables pour nos études sont MES ≤ 50 mM sur du silane ou ≤ 100 mM sur du polystyrène. c) le degré d'étirement des molécules. Sur le silane comme le polystyrène, le degré d'étirement diminue quand la concentration de MES augmente, et n'est plus mesurable au dessus de 100 mM en raison de l'absence de molécules (silane) ou de leur cassure trop fréquente (polystyrène).Nous trouvons 1 μm = 1,95 kb à 50 mM de MES pour le silane et 1 μm = 1,80 kb pour le polystyrène. Ces valeurs sont très proches des valeurs rapportées dans la littérature[START_REF] Allemand | pH-dependent specific binding and combing of DNA[END_REF][START_REF] Michalet | Dynamic molecular combing: stretching the whole human genome for high-resolution studies[END_REF]. La résolution spatiale est donc légèrement plus élevée avec le polystyrène que le silane. d) l'alignement des fibres. Un alignement très reproductible des molécules étirées (déviation standard <1 degré d'angle) a été observé avec les deux surfaces, ce qui facilite l'identification de molécules uniques. e) l'uniformité du signal fluorescent sur les fibres. L'intensité moyenne du signal fluorescent en YOYO-1 de chaque molécule est moins variable (entre molécules) avec le silane qu'avec le polystyrène. De même, la variabilité du signal le long de chaque molécule (variabilité intramoléculaire) est moindre avec le silane qu'avec le polystyrène. Ce point est important pour la discrimination des segments répliqués et non répliqués basée sur leur différence d'intensité. J'ai conclu de l'ensemble de ces tests que le peignage sur du silane à 50 mM de MES était le meilleur choix pour nos expériences. Une fois ces conditions définies, nous avons répliqué de la chromatine de noyaux de spermatozoïdes de xénopes (Xenopus laevis) dans des extraits d'oeufs de xénope en présence de dUTP fluorescent (AF647-dUTP), et extrait et peigné l'ADN ainsi marqué. Ce système in vitro permet une réplication très efficace de ce substrat. Nous avons constaté qu'après une incubation longue, ou la réplication atteignait 74% du maximum (mesure de l'incorporation de dNTP radioactif par précipitation au TCA) les molécules d'ADN montraient un signal très net d'AF647-dUMP le long de la majeure partie des molécules. Néanmoins, le signal n'était pas totalement continu mais interrompu par des segments non marqués de taille hétérogène. La fraction totale d'ADN marqué s'élevait à 65.4%, valeur inférieure à celle mesurée en TCA. Pour vérifier si des dUTPs fluorescents incorporés de manière homogène le long d'une molécule d'ADN donnent naissance à un signal fluorescent homogène après peignage, nous avons tenté de construire in vitro des molécules d'ADN entièrement substituées sur un des deux brins par du AF647-dUTP, mais ces expériences ont donné des résultats non concluants. Nous avons fait l'hypothèse que la différence de degré de réplication mesurée en TCA et en fluorescence était attribuable à l'imperfection du marquage fluorescent. Nous avons mesuré précisément la distribution de taille des segments non marqués et supposé que les trous les plus petits étaient dus à un marquage imparfait, alors que les plus grands reflétaient de véritables segments non répliqués. La différence 74% -65.4% = ~9% peut être entièrement comblée en supposant que tous les trous < 2,24 kb sont dus à un marquage imparfait et ne doivent donc pas être pris en considération, alors que les trous plus longs que ce seuil correspondent à des vrais segments non répliqués. Ce seuil détermine la résolution actuelle de la technique et est en accord avec la résolution du peignage d'ADN conventionnel (Michalet et al 1997). Pour prouver que les bulles de réplication peuvent aussi être détectées en se basant sur leur double intensité en YOYO-1, nous avons construit une structure d'ADN en forme de lasso à partir de l'ADN de lambda. Dans cette structure, un segment de double brin d'ADN est attaché à une portion circulaire. Durant le peignage, cette molécule devrait s'attacher par son unique terminaison double brin, et la portion circulaire devrait s'étirer en donnant une paire de doubles brins. En effet, nous avons été capables de visualiser des changements abrupts dans l'intensité de fluorescence en accord avec le lasso d'ADN. Le profil d'intensité correspondant permet de discerner sans ambiguïté des segments adjacents d'intensité de fluorescence unique et double comme attendu pour les molécules lassos. Cette observation a établi la preuve de concept que le bis-intercalant fluorescent YOYO-1 permet de détecter la limite entre les portions contenant un ou deux doubles brins d'ADN.Puis, nous avons prouvé qu'on peut détecter les bulles de réplication grâce à la seule fluorescence du YOYO-1 sur de véritables intermédiaires de réplication préparés dans les extraits d'oeufs de xénope en présence d'AF647-dUTP. Nous avons constaté une remarquable coïncidence des régions avec une forte intensité de fluorescence YOYO-1 avec les régions ayant incorporé le AF647-dUMP pendant la réplication. Les bulles de réplication peuvent donc être détectées sur de l'ADN peigné par deux techniques fluorescentes indépendantes mais compatibles : l'augmentation de fluorescence de YOYO-1 et l'incorporation de AF647-dUTP. Vu que la méthode YOYO-1 ne requiert pas de marquage métabolique, elle devrait être applicable pour détecter les bulles de réplication dans l'ADN de n'importe quel organisme.Une approche rapide pour cartographier les intermédiaires de réplicationLa disponibilité d'endonucléases qui coupent un seul brin de leur séquence cible (Nicking Endonucleases, NEs) et de dNTPs fluorescents permet de marquer chaque molécule d'ADN par un codebarre fluorescent spécifique, en utilisant une ADN polymérase pour incorporer sur une courte distance des dNTPs fluorescents au niveau de chaque site de coupure. Ce code-barre permet en principe d'identifier le locus dont provient chaque molécule. Nous avons développé des techniques électrophorétiques permettant de contrôler la coupure complète et le marquage de l'ADN par fluorescence directe. En introduisant une étape de réparation de la coupure après le marquage, destinée à minimiser la cassure accidentelle à ces sites, nous avons réussi à définir un protocole qui permet de coder de manière fluorescente les molécules d'ADN à des motifs de séquence précis tout en minimisant leur cassure. L'application de cette techniques à des intermédiaires de réplication a nécessité la mise au point d'un protocole de purification précis pour éviter la perturbation du peignage par les impuretés introduites au moment de la réalisation du code-barre. Comme première application de cette technique, nous avons construit la première carte de réplication fluorescente de l'ADN lambda répliqué dans les extraits d'oeufs de xénope. Les segments correspondant à des portions entières ou partielles du génome du bactériophage lambda ont pu être facilement reconnus à partir de leur « barcode » dans les molécules peignées et les signaux de réplication localisés le long de la séquence d'ADN de lambda. Nous avons confirmé l'absence de préférence de séquence pour l'initiation de la réplication dans ce système expérimental. Augmenter le débit : quand la biologie moléculaire rencontre la nanofluidique La mise en oeuvre de la technique de peignage simplifiée qui a été proposée dans cette thèse permet de visualiser le profil de réplication et la carte de restriction d'intermédiaires de réplication en fluorescence directe, sans utilisation d'anticorps. Nous avons couplé ces outils à un dispositif nanofluidique (Irys Instrument, BioNano Genomics Inc.), qui permet d'augmenter massivement le débit et la précision de l'étirement de l'ADN. Ce dispositif a été créé pour générer automatiquement des cartes physiques du génome complet à partir de molécules d'ADN individuelles barcodées comme précédemment. L'ADN est conduit électrophorétiquement à travers des milliers de nanocanaux parallèles et imagé automatiquement. Nous montrons ici que des intermédiaires de réplication obtenus comme précédemment dans des extraits d'oeufs de xénope peuvent facilement entrer dans les nanocanaux et être visualisés dans trois couleurs différentes (ADN, code-barre et signal réplicatif) avec un débit extraordinaire. Nous pouvons collecter en une seule expérience de quelques heures plus de 15000 Mb d'ADN. Ceci correspond, par exemple, à 300000 fois la couverture du génome lambda de 48,5 kb ! De plus, ce qui est important, nous avons observé avec cette technique dix fois moins de petits trous dans le signal de réplication fluorescent (AF647-dUMP) que par peignage, ce qui laisse espérer une amélioration de la résolution. La coïncidence entre le marquage réplicatif AF647-dUTP et les segments d'intensité accrue du signal YOYO-1 est remarquable. La détection de bulles de réplication basée seulement sur l'intensité du signal YOYO-1 est donc compatible avec la technologie des nanocanaux. Enfin, nous avons manuellement analysé des images de l'ADN du bactériophage lambda répliqué dans les extraits d'oeufs de Xénope en présence d'AF647-dUTP, comme fait par peignage. Contrairement à l'expérience de peignage, les données suggèrent une initiation de la réplication plus facile aux jonctions formées entre molécules d'ADN lors de l'incubation dans les extraits l'ADN. La raison de cette différence n'est pas encore comprise et nous devrons vérifier si cette observation est reproductible d'un extrait à l'autre.

Figure I- 1 :

 1 Figure I-1: Basic mechanisms of DNA replication. ............................................................. Figure I-2: autonomously replicating sequence (ARS) assay. ............................................ Figure I-3: Analysis of replication intermediates by neutral/neutral 2D gels. ................. Figure I-4: Population-based replication mapping techniques. ......................................... Figure I-5: Replication fork directionality and models for origin activation. ................. Figure I-6: Dynamic DNA combing. ..................................................................................... Figure I-7: Novel methods for detecting and mapping DNA ORIs at a single-molecule level. ......................................................................................................................................... Figure II-1: Creation of a 3'-flap at specific sites. ............................................................... Figure II-2: Construction of a lariat DNA structure. ......................................................... Figure III-1: Variability in DNA combing on different surfaces and conditions. ............ Figure III-2: Variation of the density of adsorbed DNA molecules depending on the concentration of MES buffer. ................................................................................................ Figure III-3: Density of DNA molecules adjusted per DNA concentration. ..................... Figure III-4: Examples of U-shaped and Y-shaped structures. ......................................... Figure III-5: Quantitation of adsorbed U-shaped DNA molecules depending on the concentration of MES buffer. ................................................................................................ Figure III-6: Quantitation of adsorbed Y-shaped DNA molecules depending on the concentration of MES buffer. ................................................................................................ Figure III-7: Size distributions of combed λ-DNA fibres depending on the concentration of MES buffer. ........................................................................................................................ Figure III-8: Size distributions of combed λ-DNA fibres depending on the concentration of MES buffer. ........................................................................................................................ Figure III-9: Non-linear fit of λ-DNA molecule lengths. .................................................... Figure III-10: Alignment of λ-DNA molecules under various conditions and surfaces. .Figure III-11: Distributions of the normalised molecule angles. ....................................... Figure III-12: Distributions of the normalised molecule angles (directions). ................... Figure III-13: Distributions of the mean molecule intensities. .......................................... Figure III-14: Inter-molecule variability of the mean molecule intensities in different combing conditions. ................................................................................................................ Figure III-15: Distributions of the mean molecule intensities as function of the molecule sizes in different combing conditions. ................................................................................... Figure III-16: Distributions of the intra-molecule intensity variability in different combing conditions. ................................................................................................................ Figure III-17: Sequence motif distribution of Nb.BbvCI on λ-DNA and predicted nicktranslation direction. .............................................................................................................. Figure III-18: Gel electrophoresis (0.7% agarose) of timed aliquots. ...............................Figure III-19: Combing of lambda-phage DNA fragments fluorescently barcoded at Nb.BbvCI sites. .......................................................................................................................Figure III-20: Gel electrophoresis (0.7% agarose) of timed aliquots. ...............................Figure III-21: Gel electrophoresis (0.7% agarose) of timed aliquots. ...............................Figure III-22: Monitoring AlexaFluor647-dUMP incorporation at individual Nb.BbvCI nicking sites in λ-DNA molecules by alkaline agarose (0.7%) gel electrophoresis. ........ Figure III-23: Restored combing of λ-DNA molecules barcoded at Nb.BbvCI sites by Klenow polymerase after flaps removal with RecJf. .........................................................Figure III-24: Breakage of nicked λ-DNA molecules at the "fragility hotspots" due to heat-inactivation of the nicking-enzyme. ...........................................................................

Figure III- 11 :

 11 Figure I-1: Basic mechanisms of DNA replication. ............................................................. Figure I-2: autonomously replicating sequence (ARS) assay. ............................................ Figure I-3: Analysis of replication intermediates by neutral/neutral 2D gels. ................. Figure I-4: Population-based replication mapping techniques. ......................................... Figure I-5: Replication fork directionality and models for origin activation. ................. Figure I-6: Dynamic DNA combing. ..................................................................................... Figure I-7: Novel methods for detecting and mapping DNA ORIs at a single-molecule level. ......................................................................................................................................... Figure II-1: Creation of a 3'-flap at specific sites. ............................................................... Figure II-2: Construction of a lariat DNA structure. ......................................................... Figure III-1: Variability in DNA combing on different surfaces and conditions. ............ Figure III-2: Variation of the density of adsorbed DNA molecules depending on the concentration of MES buffer. ................................................................................................ Figure III-3: Density of DNA molecules adjusted per DNA concentration. ..................... Figure III-4: Examples of U-shaped and Y-shaped structures. ......................................... Figure III-5: Quantitation of adsorbed U-shaped DNA molecules depending on the concentration of MES buffer. ................................................................................................ Figure III-6: Quantitation of adsorbed Y-shaped DNA molecules depending on the concentration of MES buffer. ................................................................................................ Figure III-7: Size distributions of combed λ-DNA fibres depending on the concentration of MES buffer. ........................................................................................................................ Figure III-8: Size distributions of combed λ-DNA fibres depending on the concentration of MES buffer. ........................................................................................................................ Figure III-9: Non-linear fit of λ-DNA molecule lengths. .................................................... Figure III-10: Alignment of λ-DNA molecules under various conditions and surfaces. .Figure III-11: Distributions of the normalised molecule angles. ....................................... Figure III-12: Distributions of the normalised molecule angles (directions). ................... Figure III-13: Distributions of the mean molecule intensities. .......................................... Figure III-14: Inter-molecule variability of the mean molecule intensities in different combing conditions. ................................................................................................................ Figure III-15: Distributions of the mean molecule intensities as function of the molecule sizes in different combing conditions. ................................................................................... Figure III-16: Distributions of the intra-molecule intensity variability in different combing conditions. ................................................................................................................ Figure III-17: Sequence motif distribution of Nb.BbvCI on λ-DNA and predicted nicktranslation direction. .............................................................................................................. Figure III-18: Gel electrophoresis (0.7% agarose) of timed aliquots. ...............................Figure III-19: Combing of lambda-phage DNA fragments fluorescently barcoded at Nb.BbvCI sites. .......................................................................................................................Figure III-20: Gel electrophoresis (0.7% agarose) of timed aliquots. ...............................Figure III-21: Gel electrophoresis (0.7% agarose) of timed aliquots. ...............................Figure III-22: Monitoring AlexaFluor647-dUMP incorporation at individual Nb.BbvCI nicking sites in λ-DNA molecules by alkaline agarose (0.7%) gel electrophoresis. ........ Figure III-23: Restored combing of λ-DNA molecules barcoded at Nb.BbvCI sites by Klenow polymerase after flaps removal with RecJf. .........................................................Figure III-24: Breakage of nicked λ-DNA molecules at the "fragility hotspots" due to heat-inactivation of the nicking-enzyme. ...........................................................................

Figure III- 25 :

 25 Figure III-25: Sequence motif distribution of Nt.BspQI on λ-DNA and predicted nicktranslation direction. ............................................................................................................ Figure III-26: Monitoring the conservation of full-length λ-DNA molecules after Nt.BspQI nicking endonuclease digestion. ......................................................................... Figure III-27: Monitoring all the barcoding steps (nicking-labelling-repair) by alkaline agarose (0.7%) gel electrophoresis. ....................................................................................Figure III-28: Nick-labeling of λ-DNA molecules at Nt.BspQI nicking sites. .................Figure III-29: Nuclear assembly and DNA replication in Xenopus egg extracts. ..........Figure III-30: Time-course of the percentage of nuclei having started DNA replication. ................................................................................................................................................ Figure III-31: Discontinuity of the direct fluorescent replication signal on combed Xenopus sperm DNA. ........................................................................................................... Figure III-32: Long Range PCR on λ-DNA. ...................................................................... Figure III-33: Primer extension using different polymerases and different dNTP cocktails: neutral agarose gel electrophoresis. .................................................................. Figure III-34: Primer extension using different polymerases: alkaline agarose gel electrophoresis. ..................................................................................................................... Figure III-35: Preparation of the fluorescent fibres for DNA combing by primer extension. ............................................................................................................................... Figure III-36: DNA combing of hemi-fluorescent DNA constructions. .......................... Figure III-37: Proof-of-principle of the YOYO-1-based replication bubble detection. Figure III-38: Combining direct fluorescent labeling of replication tracks and fluorescent barcoding on λ-DNA or sperm nuclei replicated in Xenopus egg extracts. .Figure III-39: Buffers addition affect DNA combing quality. ......................................... Figure III-40: Enzymes addition affect DNA combing quality. ....................................... Figure III-41: Combining direct fluorescent labeling of replication tracks and fluorescent barcoding on DNA from sperm nuclei replicated in Xenopus egg extracts. Figure III-42: Irys chip scheme and functioning. .............................................................. Figure III-43: Comparing the size distribution of the gaps in the replication signal by DNA combing vs. nanochannel technology. ...................................................................... Figure III-44: Doubling of YOYO-1 fluorescence intensity at the replicated tracts in nanochannel. ......................................................................................................................... Figure III-45: Comparison of the replication analysis of λ-DNA using molecular combing vs. nanochannel techniques. .................................................................................

Figure III- 39 :

 39 Figure III-25: Sequence motif distribution of Nt.BspQI on λ-DNA and predicted nicktranslation direction. ............................................................................................................ Figure III-26: Monitoring the conservation of full-length λ-DNA molecules after Nt.BspQI nicking endonuclease digestion. ......................................................................... Figure III-27: Monitoring all the barcoding steps (nicking-labelling-repair) by alkaline agarose (0.7%) gel electrophoresis. ....................................................................................Figure III-28: Nick-labeling of λ-DNA molecules at Nt.BspQI nicking sites. .................Figure III-29: Nuclear assembly and DNA replication in Xenopus egg extracts. ..........Figure III-30: Time-course of the percentage of nuclei having started DNA replication. ................................................................................................................................................ Figure III-31: Discontinuity of the direct fluorescent replication signal on combed Xenopus sperm DNA. ........................................................................................................... Figure III-32: Long Range PCR on λ-DNA. ...................................................................... Figure III-33: Primer extension using different polymerases and different dNTP cocktails: neutral agarose gel electrophoresis. .................................................................. Figure III-34: Primer extension using different polymerases: alkaline agarose gel electrophoresis. ..................................................................................................................... Figure III-35: Preparation of the fluorescent fibres for DNA combing by primer extension. ............................................................................................................................... Figure III-36: DNA combing of hemi-fluorescent DNA constructions. .......................... Figure III-37: Proof-of-principle of the YOYO-1-based replication bubble detection. Figure III-38: Combining direct fluorescent labeling of replication tracks and fluorescent barcoding on λ-DNA or sperm nuclei replicated in Xenopus egg extracts. .Figure III-39: Buffers addition affect DNA combing quality. ......................................... Figure III-40: Enzymes addition affect DNA combing quality. ....................................... Figure III-41: Combining direct fluorescent labeling of replication tracks and fluorescent barcoding on DNA from sperm nuclei replicated in Xenopus egg extracts. Figure III-42: Irys chip scheme and functioning. .............................................................. Figure III-43: Comparing the size distribution of the gaps in the replication signal by DNA combing vs. nanochannel technology. ...................................................................... Figure III-44: Doubling of YOYO-1 fluorescence intensity at the replicated tracts in nanochannel. ......................................................................................................................... Figure III-45: Comparison of the replication analysis of λ-DNA using molecular combing vs. nanochannel techniques. .................................................................................

I.5.2 Genome-wide analysis of DNA replication: what did we learn? I.5.2.1 Replication timing: contributions from genome-scale methods (CTRs, TTRs)

	The advent of genome-scale studies strongly boosted our knowledge of the replication
	timing. The Repli-chip and Repli-seq techniques consist in sorting asynchronous, BrdU-pulse-
	labelled cells according to their total DNA content, into 2-6 successive stages of the S-phase
	and then hybridising the immunoprecipitated Br-DNA to microarray (Repli-chip) or submit it
	to deep-sequencing (Repli-seq) to draw a comprehensive picture of the DNA replication
	temporal organisation (see Rhind and Gilbert (2013) for a review). Repli-chip profiles obtained
	after sorting cells in two (early and late) compartments of S-phase showed megabase-sized,
	early or late constant timing regions (CTRs) separated by 100-600 kb timing transition regions
	(TTRs)(Figure I-5 B). Because of their large size, CTRs were presumed to contain multiple
	synchronous replicons, whereas TTRs were presumed to replicate by unidirectional progression
	of a single fork travelling from an earlier-to a later-replicating domain (Farkash-Amar et al
	2008, Norio et al 2005, Ryba et al 2010). Repli-seq profiles obtained from 4-6 compartments
	of S-phase questioned the CTR/TTR dichotomy because a broad distribution of replication
	timing gradients was observed genome-wide, suggesting that TTRs can also replicate by
	sequential firing of multiple ORIs, known as the cascade model (Frum et al 2009, Guilbaud et
	al 2011, Hyrien et al 2013)(see paragraph I.5.2.5 for further details).
	The size of the replication timing domains is variable among eukaryotes depending on
	genome complexity. The typical scale ranges from [75-250 kb] in Drosophila to [hundreds of
	kb-several Mb] in mammals. Despite these different sizes, replication profiles from different
	organisms share the same features and are qualitatively similar

.2.3 Spatiotemporal program of DNA replication: missing elements?

	correlate with early-and late-replicating domains (Lieberman-Aiden et al 2009, Ryba et al
	2010). Later Hi-C studies with an increased resolution revealed that chromatin is organised in
	self-interacting domains called topologically associating domains 15 (TADs). Very interestingly,
	TADs often coincides with the replication timing domains (Pope et al 2014).
	I.5DNA replication timing is a 2-step mechanism: the location and the firing time of ORIs
	is specified in G1-phase, but this program is only executed later in S-phase. One interesting
	discovery is that some DNA replication factors are limiting for ORI firing. In yeasts, initiation
	factors such as Cdc45, Sld2/3 and DDK were shown to be less abundant than the number of
	available ORIs and therefore to limit the rate of ORI firing (Mantiero et al 2011, Patel et al
	2008, Wu & Nurse 2009). It is proposed that limiting factors are first captured by the most avid
	ORIs then can be recycled to lower efficiency ORIs only after firing of the most avid ones.

  competitor DNA or limiting ORC concentrations. Thus, it is possible that replication may, at some low frequency, initiate elsewhere than at bona fide ARSs in the yeast genome. These rare and possibly dispersed initiation events may have escaped detection in population studies but may become visible in future high-throughput, SM studies.

	The possibility to validate the cascade model for N/U-domain replication is very
	appealing. SM data can precisely quantify the number of initiation within and between the
	predominant initiation zones identified by OK-seq, and discriminate whether descending RFD
	segments replicate passively, by merging of forks emanating from adjacent ascending RFD
	segments, or actively, by internal initiations following a temporal cascade. Zones of constant
	and high │RFD│ values replicate with forks predominantly moving in the same direction. SM

et al

(2014)

showed that yeast replicators are not essential for plasmid origin function in vitro, while

[START_REF] On | Prereplicative complexes assembled in vitro support origin-dependent and independent DNA replication[END_REF]

demonstrated that replication becomes origin-dependent only in the presence of analysis can discriminate between (1) passive replication, (2) active replication by unidirectional forks that fired within the zone, and (3) active replication by asymmetrical forks (i.e. forks that fired as sequential but bidirectional ORIs within the zone). Finally, SM data can also quantify initiation and termination events within zones of null RFD (that replicate by random initiation and termination).

OK-seq provided a precise, high-resolution replication map of the genome allowing to quantitate replication initiation and termination preponderance genome-wide

[START_REF] Mcguffee | Quantitative, genome-wide analysis of eukaryotic replication initiation and termination[END_REF][START_REF] Petryk | Replication landscape of the human genome[END_REF]

. However, the number of detected initiation zones in OK-seq profiles is clearly lower than the total number of initiation events in a single S-phase estimated by SM techniques. Most likely, a large proportion of initiation events occur in a too dispersive manner to create detectable changes in the RFD profile. SM analysis may provide direct evidence for such events, and permit their precise quantification.

3 Fluorescent imaging of replicating nuclei in Xenopus egg extracts

		Demembranated Xenopus sperms (sperm chromatin) incubated in egg extracts (t=0 min)
	II.2.1 were collected at different time-points. Nuclei were sedimented on 12 mm, round coverslips Neutral agarose gel
	(Marienfeld GmbH & Co.KG) coated with polylysine (SIGMA-Aldrich) through a sucrose Neutral agarose gels (0.5% -1%, depending on the fragments to be separated) were cushion (0.7 M Sucrose in PBS) to remove impurities. 24 samples could be purified at once by prepared by boiling ultrapure agarose-LE (low electroendosmosis, Ambion) in Tris-Borate-spinning in a 24-wells plate (at the bottom of each well, there was a polylysine coverslip EDTA (TBE), then supplemented with 0.2 µg/mL ethidium bromide and solidified at room covered by 1 mL of sucrose cushion, at the top of which was carefully deposed the sample). temperature. Five volumes of sample were mixed to one volume of 6x neutral loading buffer Sedimented nuclei were washed twice in PBS, fixed in 4% formaldehyde in PBS, stained in 2 [10 mM Tris-HCl pH 7.6, 0.03% bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol 60 µg/mL Hoechst 33342 in PBS (30 min, room temperature, dark) and washed again. The mM EDTA] and loaded onto the gel. Electrophoreses were performed at 5-10 V/cm in TBE buffer containing 0.2 µg/mL ethidium bromide. coverslips were mounted in Vectashield anti-bleaching media (Vector Laboratories) and images
	II.2.3	Signal acquisition
		Ethidium bromide or SYBR Gold and, eventually, AlexaFluor647-aha-dUMP signals
	(if present), were detected using a Typhoon FLA 9500 laser scanner (GE Healthcare) equipped
	with appropriate bandpass filters.

II.2.2 Alkaline agarose gel

For alkaline gel electrophoresis, agarose-LE (0.5% -1%, depending on the fragments to be separated) was boiled in water and, once the solution cooled to ~55°C, supplemented with final 1x alkaline electrophoresis buffer [50 mM NaOH, 1 mM EDTA pH 8.0]. The gel was solidified at room temperature. 5 volumes of samples were mixed to one volume of 6x alkaline loading buffer [300 mM NaOH, 0.15% bromocresol green, 0.25% xylene cyanol FF, 18% Ficoll 400, 6 mM EDTA] prior to sample loading. Alkaline gels were run at 4°C and low voltage (<3.5

V/cm) to avoid overheating of the gel. The gel was neutralised by soaking 15 min in TBE, followed by 10 min staining (in agitation) in 0.5x SYBR Gold (ThermoFisher Scientific) in TBE.

II.

were acquired with an inverted Nikon Eclipse Ti microscope equipped with a 60X PlanApo oil objective (N.A. 1.4; W.D. 0.13 mm, Nikon), a mercury vapor short-arc lamp (Excelitas Technologies), appropriate filter sets (Semrock) and a CoolSNAP HQ 2 camera (Photometrics).

The automated counting of replicating nuclei (positive for AlexaFluor555-aha-dUMP or AlexaFluor647-aha-dUMP) was done with a custom written pipeline implemented in CellProfiler (Broad Institute).

Table II -1: Representative targets that can be displaced within λ-phage genome.

 II

In eukaryotes, the word "replicon" refers to the DNA replicated from a single origin.

kb = kilobase-pairs, 10 3 base-pairs (bp).

Mb = Megabase-pairs, 10 6 base-pairs (bp).

rRNA genes in eukaryotes are organised in ~300-400 tandem repeats to form clusters (on different chromosomes). These clusters are commonly called "ribosomal DNA" (rDNA), while coding for rRNAs.

The mid-blastula transition (MBT) is a specific moment during the blastula stage of embryonic development in which the zygotic gene transcription is activated.

« Omics » refers to the fields in biology research ending with the "-omic" suffix. Omics experiments are all characterised by studying pools of biological molecules, ideally the totality of these molecules, thanks to their high-throughputs.

« Next-generation sequencing » refers to non-Sanger-based high-throughput DNA sequencing technologies. In a typical experiment, millions to billions of molecules can be sequenced in parallel.

Replication "bubbles" or "eyes" refer to the open, unwound region where DNA replication occurs. Replication bubbles are isolated in agarose: after cell lysis and proteins digestion they can be trapped in the agarose matrix thanks to their shape.

Short nascent strands (SNS) purification consists in isolating the small fragments of single-stranded DNA (ssDNA) of 1-1.5 kb that are synthetized at the replication forks immediately after the ORI firing. They are usually purified on a sucrose gradient.

Okazaki fragments are short, ssDNA fragments that are synthetized at the lagging strand during DNA replication. They are ~1000-2000 nucleotides (nt) long in prokaryotes but only ~100-200 nt in eukaryotes. Okazaki fragments are also purified on sucrose gradient.

DNA (or molecular) combing is a technique to produce an array of stretched DNA that allows the investigation of DNA replication on single DNA molecules. Further described in paragraph I.7.2.

Hi-C is an extension the chromosome conformation capture (3C) technique coupled to high-throughput sequencing. Hi-C is capable of identifying long-range interactions, genome-wide.

Topologically associating domains (TADs) can be thought as "chromosome neighborhoods". They can range from thousands to millions of DNA bases.

G-quadruplexes are nucleic acids structures formed at sequences that are rich in guanine. Four guanine bases associate to form a planar and squared structure (guanine tetrad). ≥2 stacked tetrads form a G-quadruplex.

CpG islands are regions with rich in "CpG" sites usually longer than 200 bp. CpG sites are regions where a cytosine nucleotide is followed by a guanine nucleotide (5'-C-phosphate-G-3').

In a quantification of λ-exo efficiency,[START_REF] Cadoret | Genome-wide studies highlight indirect links between human replication origins and gene regulation[END_REF] showed that almost all (~99%) of SSS DNA is eliminated from DNA isolated from HeLa cells.

The ENCODE (Encyclopaedia of DNA Elements) Consortium is an international collaboration of research groups with the goal to build a comprehensive list of the functional elements of the human genome.

When observing DNA extracted from a population of cells for SM analysis, the probability that two given molecule come from the same cell is very low. This probability depends mainly on the amount of starting materials and on the throughput of the technology, but in vast majority of cases the equation: "molecule-by-molecule" = "cell-by-cell" is true.

The original DNA (molecular) combing is a biophysics technique invented in 1994 at the Ecole Normale Supérieure (ENS), in Paris, which was afterwards developed by the A. Bensimon group at the Pasteur Institute.

A silanated coverslip consists in a glass coverslips covered with a monolayer of silane molecules exposing a vinyl end group (-CH = CH2).

Below the pKa of the ionizable groups, ensuring that they are charged enough to interact with the ends of DNA.

The resolution of DNA combing, a priori, is only determined by the diffraction limit of the microscope in use. It is usually estimated around 1-2 µm, although this is much probably an overestimation of its potential. See the Results section (III).

To study a region of ~1 Mb, ~25-30 PCR probes (~5 kb each) or at least 3 BAC probes (~100 kb) are required.

1) "Computer aided scoring and analysis" (CASA) software, developed in 2011 for fibre spreading analysis by Wang and Chastain (http://dnafiberanalysis.com); 2) "IDeFIx" software, developed at the Montpellier DNA Combing Facility by Gostan (http://www.igmm.cnrs.fr/spip.php?rubrique87); 3) "FiberStudio®", propriety of the French company Genomic Vision, holding the licence to commercialise DNA combing for biomedical applications (http://www.genomicvision.com/fr/produits/plateforme-de-peignage-moleculaire/logiciel/#top).

SYTOX Orange (Molecular Probes) is a nucleic acid stain capable of increasing the fluorescence intensity >500-fold upon nucleic acid binding.

The average intensity within the molecule was calculated as the sum of the grey values of all the pixels of the molecule divided by the number of pixels. If the molecule was not perfectly linear, a spline was adopted to fit the DNA molecule.

The nanochannel device is powered by strong dedicated lasers, while on the DNA combing technique we simply used epi-fluorescence microscopy connected to a standard mercury arc lamp.

Which should not be the case, considering that in our experiments the misalignment is particularly evident only in the red channel (replication), while between the blue (YOYO-1, DNA) and the green channels (barcodes) it is not.

Acknowledgements

II. Methods

Interphase egg extracts and demembranated sperm nuclei (6.6x10 4 nuclei/mL) were prepared and frozen as described in [START_REF] Marheineke | Use of DNA combing to study DNA replication in Xenopus and human cell-free systems[END_REF].

All the protocols regarding the replication of sperm nuclei or λ-DNA in Xenopus egg extracts, the quantitation of the DNA synthesis (trichloroacetic acid (TCA) precipitation assay), the preparation of silanated hydrophobic coverslips, the DNA purification, the fluorescent barcoding (of both unreplicated and replicated DNA) and the DNA combing are detailed in the paper [START_REF] Carli | Single-molecule, antibody-free fluorescent visualisation of replication tracts along barcoded DNA molecules[END_REF] in paragraph III.2.5.

Other methods not described in the article are described in this chapter.

III.2.2.2

The problem of the adjacent nicks (i.e. "fragility hotspots")

Although useful to demonstrate the efficient nick-translation reaction, the breakage of full-length molecules at vicinal nicks is a serious problem for a functional mapping technique.

I restricted the labelling time to less than 5 minutes (note that the gel shown in 36 These bands also results from the crossing of vicinal nicks (~400 bp apart) with the progress of the labelling reaction, reflecting the different nicking endonuclease pattern. 37 AlexaFluor647 (AF647) has spectral characteristics similar to Cyanine5 (Cy5), and was adopted because resulting in a lower background noise on DNA combing experiments.

III.2.2.3 Definition of a working protocol (Nt.BspQI + Taq pol + PreCR repair)

Inspired by some novel papers, I altered the barcoding protocol keeping in mind the above-described goals. Firstly, I replaced the Nb.BbvCI nicking endonuclease with Nt.BspQI.

When cutting with Nt.BspQI ( 5 (cutting on average every 9 kb), allowed this technique to collect and align to the human genome ~900,000 molecules >150 kb in length, resulting in a ~70 fold coverage of 93% of the genome [START_REF] Cao | Rapid detection of structural variation in a human genome using nanochannel-based genome mapping technology[END_REF].

Here below I present the results that we obtain in collaboration with Valérie Barbe and Wahiba Berrabah (Genoscope, Evry, France), showing that this device, indeed, is compatible with our tools to detect and map replication bubbles.

III.3.3 Evaluating the YOYO-1-based bubble detection

In order to check whether is possible to reliably detect the replication bubbles based on the strong YOYO-1 tracts also in the nanochannel device, we imaged replication intermediates from Xenopus egg extracts. AF647-dUTP was added to the extract before adding sperm chromatin. The reaction was stopped at 35 minutes and the replicating molecules were extracted as for DNA combing, and prepared as described (see paragraph II.

Table of tables