I dedicate my work and this thesis to two people whom I would never be able to thank in person, as they have already left this world. One of them is Professor Andrey Perevozchikov, my first ever group leader. He accepted me to his team for an internship when I was a high school student, naïve and ignorant. He opened a wide world of biological research before me, like a wonderland. I will never forget his kindness, his sense of humour, his introductions into molecular biology, and first scientific papers that I read upon his recommendation.

Another is my granny. I always knew I had her love and her support, whatever happens. She always believed in me. I know she would have been happy and proud for me if she had known. She will not see it, she will not read it, but I want to leave it here for her, to let her know and thank her. Granny, your trust in me helps me even after you are gone.

I would like to thank all people who helped during my time here. I would have never managed without them.

First of all, I would like to express my sincere gratitude to my supervisor, Dr. Evi Soutoglou for everything that happened in this four years. Thank you for accepting me to your lab and letting me work on my project.

Thank you for putting your trust in me. Thank you for being so kind and gentle all this years. Thank you for being always ready to help with solving problems with my project or give more ideas for it. Thank you for being willing to listen to my ideas and letting me try them, and to explain yours when I could not understand them. Thank you for making me believe I can think, and for teaching to do it. Thank you for being so positive and motivating. Thank you for your support and your patience. Thank you for being a model for me, sometimes not only in science but also in life. I would like to thank Dr. Massimo Lopes, Dr. Petra Hajkova, Dr. Valerie Schreiber and Dr. Anna Poetsch for their kind agreement to become jury members for my defense. I am also very grateful to Dr. Anna Poetsch for her interest to my project, for taking time for discussion and the feedback I have got from her.

I would like to express my deep gratitude to my midthesis committee members: Dr. Thomas Sexton, Dr. Massimo Lopes and Dr. Maria-Elena Torres Padilla for their help and feedback for my project. I thank Dr.

Maria-Elena Torres-Padilla for her kindness and support, for believing in me and for encouraging me. I do not know if I would be where I am without it.

I want to thanks all lab members, past and present, for this great four years, for support and help in science and in life. Audrey, Duygu, Indrajeet, Sylvain, Ophelie, Ioanna, Michalis, Alex, Ujjwal, Alkmini, Katerina, Lucile, Celine, Kostas, thank you! Thank you, Audrey, for the time we were working togetherit was a wonderful time. Thank you for always, from the very first day, taking care of me, thank you for helping me to solve my household issues and problems with documents all the times. I would have never managed it all on my own. Thank you for being so uncommonly nice. Thank you, Duygu, for the joy of working together with you. Thank you for being kind and patient. And thank you for helping me with translating all forms and documents from and for university for me. I do not know what I would do without you. Thank you, Indrajeet, for the fun time. Thank you for being there for me, for your support and for your advice. Thank you for being almost like a brother. Thank you, Sylvain, for being so positive, thank you for the fun. Thank you for your advice and for being ready to help me with troubleshooting and with translations to French. Thank you, Ophelie, for being such a great neighbour in the lab. Thank you for your help with my project. Thank you for taking over it: I am happy I leave it in such reliable hands. Thank you, Ioanna, for being patient with my attempts to read in Greek.

Thank you, Alex and Michalis, for the time you were working with me as trainees. It was a great experience.

Thank you, Ujjwal, for being such a miscellaneous interlocutor and for your patience while teaching me how to do ChIP. Thank you, Alkmini, for being sincere, and fun as well, and for teaching me Greek tong twisters.

Thank you for teaching me at the time when I just came to the lab and had to learn everything. Thank you, Lucile, for the pleasure of working with you and getting to know your absolutely amazing personality. Thank you, Celine, for your honesty and liveliness. Thank you, Katerina, for teaching me so many protocols at the beginning. Thank you, Kostas, for being a nice colleague, even for a short period of time.

I would like to thank Le Programme Investissements d'Avenir and Association pour la Recherche sur le

Cancer for funding my work.

I want to thank my friends here for making my life here bright and colourful, and for so many wonderful moments. Mumin, thank you for always being there for me, for being the friend to whom I can come with any trouble and with any reason for happiness however stupid they are. Thank you, Olga, Rocio and Sushil, for your support, for being there to listen to me, to cheer me up or to have fun. Monica, thank you for always reminding me of bright sides of life, for being a local little sun that brings light and warmth into lives of everybody around, and mine as well. Thank you, Salvatore, for fun we had and for support you gave me, and for keeping my wheels rolling even when I could not do so on my own. Iskander, thank you for teaching me how to play football and for your help with troubleshooting my experiments, thank you for the fun times and for supporting me when it was not so much fun. Alexey and Sergey, thank you for the possibility to discuss everything from art to literature and from music to philosophy. Annabela, Giovanni, Margarita, Nemanja, Marta, Xieyang, Dimitra, Paul, thank you for the happiness of being friends with you.

I would like to thank people without who my work would much more difficult, if not impossible. I thank Betty, Amelie, Marion, Patricia and the whole cell culture facility. I thank Claudine and Murielle for their help with flow cytometry. I want to thank Armelle, France, Francine and Annick for their help with all kind of documents and administrative issues. I would like to express my gratitude to all IGBMC personnel, whose work is often invisible to us but so important for us.

I want to thank Ines and Cedric for always keeping my spirits up. I thank my friends who are far from me but whose support helped me to get through all troubles that appear on the way: Michael, Peter, Denis, Michael, Ekaterina, Michael, Gustavo, Monika and Yaarub. I thank Andrew, who was always there, the closest one, even being so far physically.

And of course, thank you, mom! None of it would have been possible without you always being on my side, being ready to listen, and comfort, and support, and encourage me. I am what I am, and I am where I am thanks to you.

List of abbreviations

Thesis summary

Thousands of acts of DNA damage happen in multicellular organisms every day. This makes the process of DNA repair, particularly of double-strand breaks, extremely important to study. Evidence grows to support the hypothesis that chromatin organization plays a notable role in repair pathway choice. It has been shown by multiple groups that the local chromatin organization around the site of a double-strand break (DSB) can influence the repair outcome. For example, transcriptionally active chromatin marks such as trimethylation of the lysine 36 of histone H3 (H3K36me3) and acetylation of a lysine 16 of the histone H4 (H4K16ac) has been observed to promote homologous recombination (HR). Moreover, it has recently been demonstrated that in case of DSBs induced in transcrtiptionally active regions in G1, where HR is not possible due to the absence of a sister chromatid that should serve as a template, repair can even be postponed till G2 phase where HR is enabled. On the other hand, it has been shown that the presence of repressive chromatin marks, such as mono-and dimethylation of the lysine 20 of histone H4 (H4K20me1 and H4K20me2) favours the choice of non-homologous end joining (NHEJ) as the main repair pathway.

Results obtained in our laboratory a few years ago showed that in lamina-associated domains (LADs) that represent highly repressed chromatin type HR is absolutely prohibited and cells even use potentially deleterious microhomology-mediated end-joining (MMEJ) instead. It has also been observed in the lab that in pericentric heterochromatin, another case when HR can potentially be deleterious, there are certain precautions cells take to prevent undesirable consequences of its use by translocating breaks to the periphery of a chromocenter for repair. DSB repair in heterochromatin, especially constitutive, is much better studied than in euchromatin or facultative heterochromatin. It has been shown that euchromatin is more susceptible to DNA damage and generally promotes HR but overall it requires more detailed studies.

One of the chromatin types, relatively abundant in stem cells, that has yet never been studied in the field of DNA repair is so-called bivalent chromatin. Bivalent domains is a name for chromatin stretches marked with both permissive and repressive histone modifications. At first, the combination of H3K4me3 and H3K27me3 was observed, which is now also named 'classic bivalent domains'; later on H3K4me3 and H3K9me3 or H3K36me2/3 and H3K9me3 containing bivalent domains were found in human mesenchymal stem cells and preadipocytes and HEK293 cells respectively. For some years after their discovery, they were considered a cell culture artifact by many scientists. However, within several last years, they were observed in vivo by several groups, both during normal development and in cancer. All in all, we consider it an interesting phenomenon, worth thorough investigation.

In our laboratory we are working on various aspects of DSB repair regulation by the chromatin context, using various chromatin types, experimental approaches, and model organisms. In my project, I used mouse ESCs as a model to study stem cell-specific features of chromatin influence over the DNA repair process and study the specific features of DNA repair in bivalent chromatin.

The main questions I wanted to answer are the following: Do bivalently marked regions represent a distinct chromatin type in terms of DNA repair? Does chromatin structure affect repair of the same loci in the same cell line during differentiation?

Are there ES-specific features of DNA repair in the chromatin context?

The goal of my project was to study the kinetics and mechanisms of DSB repair and its relation to the chromatin context that the breaks are induced. To this end, I was using CRISPR-Cas9 to induce doublestrand breaks (DSBs) in various chromatin contexts in ESCs or differentiated cells. I have generated stable mES cell lines expressing wtCas9 fused to GFP and to a destabilisation domain (DD) that leads to constant degradation of the wtCas9. In the presence of the chemical molecule called Shield1, Cas9 is stabilized. I have observed that 8-10h after shield addition, Cas9GFP reaches its max levels and 10h after shield withdrawal it drops almost to the levels observed in the absence of Shield1.

To induce DSBs at the different chromatin contexts, I use plasmids or in vitro transcribed guide RNAs that target genomic locations decorated by different chromatin modifications. As one of the chromatin types of our interest is bivalent domains that have not been studied before in terms of DNA repair and are often observed at developmental regulators and thought to enable fast differentiation, we chose to induce breaks at several bivalently marked genes. These genes (Pax6, Zic1, and Ngn2) are reported to be expressed at very low levels in ESCs and in high levels in some differentiated cell types and regulate neuronal development. These loci were compared with genes that are considered to be markers of pluripotency (Nanog, Pou5f1, Tfcp2l1, Zfp42) that are expressed highly in mESCs and are shut down during differentiation and with housekeeping genes (Actb, Gapdh) that are expressed all the time, and genes that are reported to be totally repressed by H3K27me3 in ES cells (Hoxb1,Tdrd1,Mc4r). We also sought to include some genes that belong to LADs in ES cells (Sox6, Ptn, Nrp1) into the comparison.

To this end, we designed two guide RNAs per gene to introduce DSBs into their promoters. We used the Cas9-expressing NIH 3T3 cell line previously established in the lab to get a comparison with a differentiated cell type.

To measure NHEJ efficiently as well as repair fidelity of the breaks induced in the above-described chromatin contexts in mESCs and NIH3T3 cells we have employed a method named TIDE (Tracking of Indels by Decomposition). TIDE is a sequencing-based method allowing us to quantify a percentage of incorrectly repaired sequences in the population, as well as a percentage of particular insertions or deletions. Our results from TIDE showed that ESCs are using non-homologous end joining (NHEJ) or other error-prone pathways more than expected from the literature. Interestingly, we also observed that the relative frequency of erroneous repair varies depending on a type of gene where the break was introduced. ESCs are using error-prone repair less than 3T3 cells for housekeeping genes and developmental regulators but, surprisingly, not for pluripotency markers, suggesting that both the chromatin structure as well as the levels of transcription influence TIDE efficiency. The possibility that disrupting a promoter of a pluripotency regulator we interfere with the cell cycle and increase the proportion of cell in G1 leading to increase of NHEJ use was discharged as we could show that there are no changes at the ESCs cell cycle profiles in all conditions.

We also noticed although the overall TIDE efficiency depends on the cell type, the pattern of insertions and deletions (indels) is quite similar between ESCs and 3T3 cells and depends on guide RNAs per se. As deletions less than 10nt are considered products of NHEJ and more than 10nt alternative end-joining (AltEJ), our results suggest that the NHEJ/AltEJ balance does not largely depend on chromatin structure or cell type.

Unfortunately, the TIDE method did not allow us to distinguish between the DNA that was not cut or repaired using HR. To measure HR efficiency, we needed to modify the method, taking advantage of the TIDE sensitivity of measuring small insertion efficiency after a break induction. To this end, for each guide RNA, we designed a specific HR template that consists of 1000bp homology arms (500bp at each side of the break) and a 9 bp unique DNA that is inserted by HR at the break site. ESCs and 3T3 cells that stably express Cas9 were co-transfected with a guide RNA together with a template, and the locus was amplified by PCR and subjected to TIDE. The frequency of 9 bp insertion at each genomic location indicated the HR frequency. Using this modified TIDE method (which we called HR-TIDE) we were able to get a comparison of HR efficiency between different chromatin types, as well as between 3T3 and ES cells. We could see that HR frequency in 3T3 cells was generally low, and never exceeded 20%. ES cells demonstrated greater variability, from as low as 3% in some inactive up to 40% in active genes. In general, using HR-TIDE we could not confirm previous reports that 80% of DSBs are repaired using HR, although it was giving a greater contribution than in differentiated cells, which goes in line with current ideas in the field.

Comparing HR efficiency between different chromatin contexts we could confirm the observation that transcriptionally active chromatin is rather promotive for it, whereas facultative heterochromatin represents a repressive environment. We also observed that bivalent domains show intermediate levels of HR, supporting the idea that they represent an intermediate state between active and repressed chromatin. As for genes located in LADs, we saw some variability, with some genes totally repressed and some demonstrating higher HR proficiency, which might be explained by their location inside or at a border of domains.

Seeing differences in HR frequency we bared in mind that the cell cycle differs between ES and 3T3 cells, the former having a much shorter G1 and thus a larger percentage of cells capable of this way of repair.

To address this question, we first decided to block 3T3 cells in the G2 phase of the cell cycle using RO inhibitor. In accordance with previous reports, we could see a considerable increase in the percentage of HR. However, we required complementary proof that extending the G1 phase of ES cells would lead to an HR efficiency drop. In order to model such a situation without driving cells into a commitment for differentiation, we decided to use 2i medium. 2i medium for culturing ES cells is serum-free and includes a cocktail of two inhibitors (MEK inhibitor and GSK-3 inhibitor, that block MEK/ERK and Wnt/b-Catenin signalling pathways respectively). However, we could not observe a significant drop in HR efficiency in active genes, which indicates that cell cycle differences between cell types cannot be on their own accountable for differences in a repair pathway choice. At the same time, we have noticed a decrease in HR usage in inactive genes, which goes in line with the fact that bivalency is lost in 2i conditions.

All in all, we have established an inducible and degradable system to assess DNA double-strand break repair and we have put forwards several assays to study DNA repair pathway choice. Our data shed light on the role of bivalent chromatin and facultative heterochromatin in the process of DNA repair pathway choice. At the same time, we have proposed and optimized an easy and quick method of accessing HR proficiency of a particular locus in a cellular context, which can be of practical use in designing knock-ins using the CRISPR-Cas9 system.

As future perspectives, several experiments can be planned. Taking advantage of the shield inducible and degradable Cas9 system I described at the beginning it is possible to perform ChIP experiments monitoring the kinetics of appearance and disappearance of several DDR factors (gH2AX and 53BP1) at different times after shield addition and withdrawal. As above, these experiments should be performed in ES cells and 3T3 cells. To correlate actual break induction and repair with DDR mounting and switch off at these breaks, LM-PCR should be performed at the same time points after shield addition and withdrawal in all breaks and cell types.

However, to get a broader picture of the chromatin influence over DNA repair pathway choice, it would be advantageous to perform a larger-scale experiment. Having optimized template design and cloning process on the one hand, and with the availability of data on genome-editing efficiencies of different guides genome-wide on the other hand, it can be possible to combine these two approaches. For this, a library containing guides and corresponding templates could be cloned into non-integrating viral vectors and used for infection of ES or differentiated cells and subsequent analysis of a repair profile using NGS.

Thesis summary in French

Des milliers de dommages à l'ADN se produisent chaque jour au sein des organismes multicellulaires. Dans notre laboratoire, nous étudions comment le contexte chromatinien affecte la régulation des mécanismes de réparation des DSB. Dans mon projet, j'ai étudiée l'influence des caractéristiques spécifiques de la chromatine bivalente au sein de cellules souches de souris (Embryonic Stem Cell soit ESC en anglais) sur le processus de réparation de l'ADN.

Au cours de ma thèse, j'ai essayé de répondre aux questions telles que : les régions bivalentes représentent elles un type de chromatine distinct en termes de réparation de l'ADN ? Le rôle de la structure de la chromatine lors de la réparation des mêmes loci est-il important au cours de la différenciation cellulaire ? Il y a-t-il des caractéristiques spécifiques aux ESC lors de la réparation de l'ADN dans le contexte de la chromatine ? A big variety of DNA damaging factors leads to different kinds of damage.

DNA damage

As I previously mentioned, DNA damage can be classified by its origin as endogenous and exogenous.

Endogenous DNA damage is the one that our organism faces the most frequently, and it cannot be avoided.

One of the major sources of endogenous damage is reactive oxygen species (ROS). They form as by-products of normal cellular processes such as cellular respiration and at low levels are involved in cellular homeostasis as messengers in redox signalling reactions [START_REF] Friedberg | DNA repair and mutagenesis[END_REF]. However, at higher concentrations, they can react with DNA bases causing damage by reacting with double bonds, methyl groups, or sugar residues [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Winterbourn | Reconciling the chemistry and biology of reactive oxygen species[END_REF]. Influence of ROS species, such as electrophilic -OH radicals, lead to residues chemical modification, such as thymine glycol residue generation of formamidopyrimidine formation [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Friedberg | DNA repair and mutagenesis[END_REF] or 8-oxoguanine formation [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Kasai | Hydroxylation of deoxyguanosine at the C-8 position by ascorbic acid and other reducing agents[END_REF]. ROS can also break the DNA backbone and induce single-strand break (SSB) formation [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Henner | gamma Ray induced deoxyribonucleic acid strand breaks. 3' Glycolate termini[END_REF].

Another common endogenous cause of DNA damage is DNA replication. It can lead to base mismatches due to replicative polymerase errors (which happens at rates between 10 -6 and 10 -8 per cell per generation) [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Kunkel | Evolving Views of DNA Replication (In) Fidelity[END_REF][START_REF] Kunkel | DNA Replication Fidelity[END_REF], or replication fork stalling or collapse [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Viguera | Replication slippage involves DNA polymerase pausing and dissociation[END_REF], which can lead to double-strand break (DSB)

formation. DNA can also be mutilated by various topoisomerase enzymes that act to remove superhelical tension or other inappropriate DNA structures by introducing nicks or DSBs [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Pommier | Repair of Topoisomerase I-Mediated DNA Damage[END_REF][START_REF] Wang | Cellular roles of DNA topoisomerases: A molecular perspective[END_REF].

Finally, spontaneous base deamination and DNA methylation (or rather, removal of methylated DNA bases) can threaten genome integrity and need mechanisms for correction [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Lindahl | Repair of endogenous DNA damage[END_REF][START_REF] Lindahl | Instability and decay of the primary structure of DNA[END_REF][START_REF] Yonekura | Generation, Biological Consequences and Repair Mechanisms of Cytosine Deamination in DNA[END_REF].

Exogenous DNA damaging agents can be more or less commonly encountered, and it is not possible to completely avoid their influence during the lifespan. Perhaps the most abundant exogenous cause of DNA damage is ultraviolet (UV) radiation. UV radiation is capable of affecting biological molecules in two ways:

by direct absorption and by energy transfer. In the case of absorption, energy received by a molecule can cause photochemical alterations. Otherwise, UV energy is absorbed by molecules called photosensitizers and then transferred to nearby molecules. Both ways could lead to DNA damage [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF]. One of the main outcomes is a covalent link formation between two adjacent pyrimidines (so-called bulky dimers), primarily cyclobutane pyrimidine dimers and pyrimidine-pyrimidone (6-4) photoproducts [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Davies | Ultraviolet radiation damage in DNA[END_REF]. Another possible outcome of UV exposure is DNA-protein crosslinks and SSBs [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Friedberg | DNA repair and mutagenesis[END_REF].

Ionizing radiation of various kinds, alpha, beta, gamma, neutrons or X-rays, is also abundant in the environment, and can both direct (SSB occurrence) and indirect (by ROS production such as water radiolysis)

DNA damage [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Desouky | Targeted and non-targeted effects of ionizing radiation[END_REF][START_REF] Friedberg | DNA repair and mutagenesis[END_REF]. SSBs caused by ionizing radiation (IR) have are unique as they tend to have 3' phosphate or 3'phosphoglycolate and not 3'-OH ends. Also, fragmented sugar derivatives can accumulate around break sites, additionally complicating the repair process. Such modified ends mutt be processed by endonucleases such as Apurinic-apirimidinic (AP) endonucleases, Polynucleotide kinase 3'-phospate (PNKP) or Tyrosyl-DNA phosphodiestherase 1 (TDP1) prior to repair [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] El-Khamisy | TDP1 facilitates repair of ionizing radiation-induced DNA single-strand breaks[END_REF][START_REF] Friedberg | DNA repair and mutagenesis[END_REF][START_REF] Jilani | Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3'-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage[END_REF][START_REF] Zhou | Deficiency in 3ʹ-phosphoglycolate processing in human cells with a hereditary mutation in tyrosyl-DNA phosphodiesterase (TDP1)[END_REF]. IR can also cause DSBs by inducing multiple damage events close to each other at a short interval [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Hutchinson | Chemical Changes Induced in DNA by Ionizing Radiation[END_REF].

Food, tobacco smoke, industrial pollution, and byproducts from burning fuel can contain exogenous agents that are harmful to DNA while microorganisms and fungi can produce natural toxins. Together, these factors can lead to various DNA mutilations [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF]. Exemplarily, alkylating agents present as by-products of tobacco smoke or organic material burning, as well as in food or medication, could result in adducted DNA base formation [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Friedberg | DNA repair and mutagenesis[END_REF][START_REF] Singer | Chemical mutagenesis[END_REF] and aromatic amines present in tobacco, colourants, fuels, etc. lead to base substitution and frameshift mutations [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Skipper | Monocyclic aromatic amines as potential human carcinogens: Old is new again[END_REF]. Additionally, environmental stress factors such as oxidative stress, hypoxia, heat or cold could result in DNA damage [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Gafter-Gvili | Oxidative Stress-Induced DNA Damage and Repair in Human Peripheral Blood Mononuclear Cells: Protective Role of Hemoglobin[END_REF][START_REF] Kantidze | Heat Stress-Induced DNA Damage[END_REF][START_REF] Luoto | Tumor hypoxia as a driving force in genetic instability[END_REF][START_REF] Neutelings | Effects of Mild Cold Shock (25°C) Followed by Warming Up at 37°C on the Cellular Stress Response[END_REF]. Reversal of DNA damage in an error-free way is possible with UV photolesions by non-enzymatic lightinduced photoreversal and with alkylated bases by specialized enzymes. These enzymes belong to two families: O 6 -alkylguanine-DNA alkyltransferase that repairs O-alkylated and AlkB-related a-ketoglutaratedependent dioxygenases that repair N-alkylated DNA lesions [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Friedberg | DNA repair and mutagenesis[END_REF].

Base excision repair (BER) pathway is used to correct lesions that do not cause large distortion of DNA helix such as abasic sites and some base modifications. It begins with removing a modified base by DNA glycosylases to create an abasic site by cleaving N-glycosylic bond linking the base to a sugar phosphate backbone and proceeds with the cleavage of this apurinic-apyrimidinic (AP) site by an AP nuclease that generates an SSB by cleaving the phosphodiester bond 5' to it. The created SSB is then repaired with either the short-patch or long-patch repair pathway. An abasic site gets removed and the gap is filled by DNA polymerase beta (Polb) and is followed by DNA ligase 1 (LIG1) -mediated ligation. BER is predominantly used in the G1 phase of the cell cycle [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Dianov | Mammalian base excision repair: The forgotten archangel[END_REF].

Nucleotide excision repair (NER) acts to repair bulky lesions and is divided into two mechanisms: global genome NER (GG-NER) and transcription-coupled NER (TC-NER). They differ in a recognition step: while in GG-NER a complex of XPC (Xeroderma Pigmentosum, complementation group C), RAD23b and CETN2 (Centrin2) scans genome for the presence of transient ssDNA caused by DNA helix unwinding at the place of damage [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Nishi | Centrin 2 Stimulates Nucleotide Excision Repair by Interacting with Xeroderma Pigmentosum Group C Protein[END_REF], TC-NER is initiated by PolII and involves CSA and CSB proteins (Cockayne Syndrome proteins A and B) [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Friedberg | DNA repair and mutagenesis[END_REF][START_REF] Marteijn | Understanding nucleotide excision repair and its roles in cancer and ageing[END_REF]. Following repair process is the same for both pathways and involves pre-incision complex (consisting of TFIIH ( [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Friedberg | DNA repair and mutagenesis[END_REF].

Mismatch repair (MMR) is a post-replicative repair mechanism active in S and G2 phases of the cell cycle. It is used to correct replication errors and thus plays a role in replication fidelity and genome maintenance through generations [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Kunkel | Evolving Views of DNA Replication (In) Fidelity[END_REF]. It acts to correct mismatches that occur due to replication errors as well as insertion-deletion loops at repetitive regions [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Friedberg | DNA repair and mutagenesis[END_REF]. Various MSH (MutS homolog) proteins act to recognise lesions and initiate

Exonuclease 1 (EXO1)-mediated excision. Resulting gaps are processed by Pold, RFC (Replication factor C), HMGB1 (high mobility group box 1 protein), and Lig1 [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF].

Interstrand crosslink (ICL) repair is required when a covalent bond is formed between bases of two complementary strands. It is mediated by FA (Fanconi anemia) proteins. FA family contains 21 functional complementation groups, which are involved in ICL resistance [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Clauson | Advances in understanding the complex mechanisms of DNA inter strand cross-link repair[END_REF]. Damage sites are recognised by FANCM protein together with FAAP24 (FA associated protein of 24 kDA) and Forkhead box protein C2 (MFH) [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Ciccia | Identification of FAAP24, a Fanconi Anemia Core Complex Protein that Interacts with FANCM[END_REF][START_REF] Clauson | Advances in understanding the complex mechanisms of DNA inter strand cross-link repair[END_REF]. MFH stimulates fork remodelling and FANCM is responsible for Holliday junction migration and creation of ssDNA gaps [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Clauson | Advances in understanding the complex mechanisms of DNA inter strand cross-link repair[END_REF][START_REF] Gari | The Fanconi Anemia Protein FANCM Can Promote Branch Migration of Holliday Junctions and Replication Forks[END_REF][START_REF] Huang | The FANCM/FAAP24 complex is required for the DNA interstrand crosslink-induced checkpoint response[END_REF]. Presence of ssDNA results in RPA recruitment and ATR (Ataxia telangiectasia and Rad3-related protein) signalling activation. In the context of the FA pathway, it leads to FANCE, FANCD2, and FANCI activation by ATR target checkpoint kinase 1 (CHK1) as well as Mre11-Rad50-Nbs1 (MRN) complex assembly (discussed in details later) [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Clauson | Advances in understanding the complex mechanisms of DNA inter strand cross-link repair[END_REF][START_REF] Duquette | CtIP Is Required to Initiate Replication-Dependent Interstrand Crosslink Repair[END_REF][START_REF] Smogorzewska | Identification of the FANCI Protein, a Monoubiquitinated FANCD2 Paralog Required for DNA Repair[END_REF]X. Wang et al., 2007). Other FA pathway core components get recruited to the lesion and stimulate the excision of the DNA strand of the lesion by structure-specific endonucleases [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Clauson | Advances in understanding the complex mechanisms of DNA inter strand cross-link repair[END_REF]. In replicating cells following repair is carried out by translesion synthesis polymerases Pol i,k,n, and Rev1-like terminal deoxycytidyl transferase (REV1). These polymerases are capable of carrying synthesis through aberrant DNA fragments, although with lower fidelity [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Clauson | Advances in understanding the complex mechanisms of DNA inter strand cross-link repair[END_REF][START_REF] Minko | Role for DNA polymerase κ in the processing of N2-N 2-guanine interstrand crosslinks[END_REF][START_REF] Räschle | Mechanism of Replication-Coupled DNA Interstrand Crosslink Repair[END_REF][START_REF] Yamanaka | Novel enzymatic function of DNA Polymerase v in translesion DNA synthesis past major groove DNApeptide and DNA-DNA cross-links[END_REF]. In non-replicating cells, it depends on both GG-NER and TC-NER pathways and TLS polymerases [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Clauson | Advances in understanding the complex mechanisms of DNA inter strand cross-link repair[END_REF].

Translesion synthesis (TLS) is performed by a highly-conserved Y-family of DNA polymerases (consisting of Pol i,k,n, and REV1) or some polymerases belonging to other families (B, X or A), such as Pol q,m,l, or z.

These polymerases are capable of carrying replication through DNA lesions but have considerably lower fidelity as damaged bases often provide a misleading template [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Sale | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF]. It has been shown that despite in some cases it is possible for a single polymerase to bypass a lesion [START_REF] Johnson | Efficient bypass of a thymine-thymine dimer by yeast DNA polymerase[END_REF][START_REF] Sale | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF] the bypass might also involve cooperation of different polymerases [START_REF] Sale | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF][START_REF] Shachar | Two-polymerase mechanisms dictate error-free and error-prone translesion DNA synthesis in mammals[END_REF]. Two models have been proposed for this phenomenon. The first one, the polymerase switch model, suggests that TLS polymerases come sequentially in a two-step process, where an inserter enzyme (usually Pol h, i, or k) incorporates a nucleotide at the place of the DNA lesion and then is replaced by an extender enzyme (Pol z) [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Korzhnev | Targeting the Translesion Synthesis Pathway for the Development of Anti-Cancer Chemotherapeutics[END_REF][START_REF] Washington | Human DINB1-encoded DNA polymerase κ is a promiscuous extender of mispaired primer termini[END_REF]. The second one, the gap-filling model, implies that ssDNA stretches are left by replicative polymerases and are subsequently filled by TLS polymerases [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Quinet | Translesion synthesis mechanisms depend on the nature of DNA damage in UV-irradiated human cells[END_REF][START_REF] Sale | Timing matters: Error-prone gap filling and translesion synthesis in immunoglobulin gene hypermutation[END_REF]. As translesion synthesis is a highly mutagenic process it must be tightly regulated.

In mammalian cells it is achieved by concentrating them in replication factories [START_REF] Sabbioneda | Effect of Proliferating Cell Nuclear Antigen Ubiquitination and Chromatin Structure on the Dynamic Properties of the Y-family DNA Polymerases[END_REF][START_REF] Sale | Translesion DNA synthesis and mutagenesis in prokaryotes[END_REF]. As previously mentioned, TLS polymerases are also known to play a role in other repair pathways including NER, BER, and FA pathway, which further emphasises the importance of this phenomenon [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF].

SSBs are repaired via three different pathways. First is the long patch single strand break repair (SSBR) pathway where SSBs are detected by poly (ADP-ribose) polymerase 1 (PARP1) which is poly(ADP)ribosylated and quickly dissociates [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] D'amours | Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions[END_REF], and the ends are further processed by APE1 (apurinic-apyrimidinic endonuclease 1), PNKP (polynucleotide kinase 3'phosphate) and APTX (aprataxin). Subsequently, Flap endonuclease 1 (FEN1) removes the mutilated 5' end and the resulting ssDNA gap is filled by Polb and Pold/e and ligated by Lig1. In the short patch SSBR pathways, breaks are recognised by APE1, and following steps converge with the long patch SSB pathway.

Another particularity of this pathway is the fact that gap-filling is performed exclusively by Polb and ligation by Lig3 [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Mckinnon | DNA strand break repair and human genetic disease[END_REF]. And the third one, the DNA topoisomerase 1 (TOP1)-SSB pathway is a modification of the long-patch SSB repair where end processing is performed by the TDP1, which acts to remove TOP1 (Keith W. [START_REF] Caldecott | Single-strand break repair and genetic disease[END_REF][START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF].

Double-strand breaks are considered to be among the most deleterious and toxic kinds of DNA lesions as in that case the second strand is not available as a repair template. Failure to repair them might lead to severe consequences such as cancer or ageing, and it is therefore absolutely essential for cells to mend them efficiently [START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF][START_REF] Mladenov | DNA double-strand-break repair in higher eukaryotes and its role in genomic instability and cancer: Cell cycle and proliferation-dependent regulation[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. Despite they pose a serious threat to a cell in particular and to an organism in general, DSBs are quite abundant. They could result from exposure to IR, UV, or small molecules (for example chemotherapy drugs), from environmental stresses such as hyperosmotic stress, hypoxia or heat shock, and from replication stress, or be created on purpose during lymphocyte maturation (V(D)J recombination of class-switch recombination) [START_REF] Fillingham | γH2AX and its role in DNA double-strand break repair[END_REF][START_REF] Lamarche | The MRN complex in double-strand break repair and telomere maintenance[END_REF][START_REF] Ohnishi | DNA double-strand breaks: Their production, recognition, and repair in eukaryotes[END_REF]. As my work was focusing on DSB repair, I will discuss mechanisms of their recognition and repair in more details in the next section.

DSB recognition and DNA damage response

The process of DSB repair can be conceptually divided into three sequential steps: DSB recognition and DNA damage response (DDR) signalling activation, repair pathway choice, and repair itself. We shall consider all of them one by one.

DSB sensing and DDR activation

Unlike other kinds of DNA lesions, DSB recognition is thought to be based on the altered chromatin structure rather than on the recognition of mutilated DNA by sensor proteins. Chromatin relaxation, an essential step for DSB repair, is promoted by certain covalent histone modifications, as well as by ATPdependent chromatin remodelling allowing repair factors to assess the damage site [START_REF] Murr | Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of DNA double-strand breaks[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF][START_REF] Van Attikum | Crosstalk between histone modifications during the DNA damage response[END_REF]. ATM (Ataxia telangiectasia mutated) activation seems to be uniformly seen as the initial step of DDR. However, the exact mechanism of this event remains controversial [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. It has been proposed that it may result from changes in chromatin structure due to relieving topological constraints caused by supercoiling [START_REF] Bakkenist | DNA damage activates ATM through intermolecular autophosphorylation and dimmer association[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF].

It has been observed that in eukaryotes DDR proteins often accumulate in conglomerates called ionizing radiation-induced foci (IRIF) as they were first observed in cells treated with IR. IRIFs are considered to be an indication of ongoing repair of one or more DSB [START_REF] Bekker-Jensen | Spatial organization of the mammalian genome surveillance machinery in response to DNA strand breaks[END_REF][START_REF] Carney | The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: Linkage of double-strand break repair to the cellular DNA damage response[END_REF][START_REF] Fernandez-Capetillo | Focusing on foci[END_REF][START_REF] Van Attikum | Crosstalk between histone modifications during the DNA damage response[END_REF][START_REF] Vignard | Ionizing-radiation induced DNA double-strand breaks: A direct and indirect lighting up[END_REF]. Among others, components of MRN (MRE11-RAD50-NBS1) complex appear to accumulate at IRIFs (Fernandez-Capetillo et al., 2003;[START_REF] Van Attikum | Crosstalk between histone modifications during the DNA damage response[END_REF].

It is widely accepted that in higher eukaryotic cells DSBs are sensed by MRN complex (Ji Hoon [START_REF] Lee | ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex[END_REF][START_REF] Ohnishi | DNA double-strand breaks: Their production, recognition, and repair in eukaryotes[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF][START_REF] Van Attikum | Crosstalk between histone modifications during the DNA damage response[END_REF]. Despite influencing the repair pathway choice at later stages by favouring one of the pathways, homologous recombination (HR), it has been shown that MRN complex is involved at the earliest steps of DSB recognition and is essential for DDR activation in all repair pathways. It binds to free DNA ends at the break site and promotes ATM activation [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Carney | The hMre11/hRad50 protein complex and Nijmegen breakage syndrome: Linkage of double-strand break repair to the cellular DNA damage response[END_REF][START_REF] Dupré | Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex[END_REF][START_REF] Lee | Direct Activation of the ATM Protein Kinase by the Mre11/Rad50/Nbs1 Complex[END_REF][START_REF] Lou | MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals[END_REF][START_REF] Stucki | MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks[END_REF][START_REF] Van Attikum | Crosstalk between histone modifications during the DNA damage response[END_REF]. Along with two other phosphoinositide 3-kinase (PI3K)-related kinases (PIKKs), ATR (ATM and Rad3-related kinase), and DNA-PKcs (DNA-dependent protein kinase catalytic subunit), ATM plays a major role in DSB repair [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF].

In an inactive form, ATM exists as a catalytically inactive homodimer [START_REF] Bakkenist | DNA damage activates ATM through intermolecular autophosphorylation and dimmer association[END_REF]. Its activity is modulated by post-translational modifications. Autophosphorylation at S1981 and acetylation at K3016 acetylation by Tip60 (60 kDa Tat-interactive protein) have been shown to lead to its dissociation into active monomers [START_REF] Bakkenist | DNA damage activates ATM through intermolecular autophosphorylation and dimmer association[END_REF][START_REF] Dupré | Two-step activation of ATM by DNA and the Mre11-Rad50-Nbs1 complex[END_REF][START_REF] Sun | A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM[END_REF][START_REF] Sun | DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF].

However, the role of ATM autophosphorylation is controversial, which will be discussed later. Ribosylation of ATM is required for its further activation and is performed by PARP1 in response to DNA damage.

Impairing this process may lead to delays in phosphorylation of ATM targets and abolished ATM foci formation [START_REF] Aguilar-Quesada | Interaction between ATM and PARP-1 in response to DNA damage and sensitization of ATM deficient cells through PARP inhibition[END_REF][START_REF] Haince | Ataxia telangiectasia mutated (ATM) signaling network is modulated by a novel poly(ADP-ribose)-dependent pathway in the early response to DNA-damaging agents[END_REF][START_REF] Núñez | DNA damage and prediction of radiation response in lymphocytes and epidermal skin human cells[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. Thus, the whole range of reactions from DDR signal spreading and DSB repair to an induction of a cell cycle block is triggered by a pioneering sensory complex binding and ATM activation (fig. 2). gH2AX and MDC1 foci formation ATM, ATR, and DNA-PKcs phosphorylate and thus activate a number of repair and checkpoint proteins but one of its most important targets is the serine residue S139 of histone variant H2AX [START_REF] Burma | ATM Phosphorylates Histone H2AX in Response to DNA Double-strand Breaks[END_REF][START_REF] Fillingham | γH2AX and its role in DNA double-strand break repair[END_REF][START_REF] Hanasoge | H2AX phosphorylation after UV irradiation is triggered by DNA repair intermediates and is mediated by the ATR kinase[END_REF][START_REF] Wang | Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors[END_REF][START_REF] Ward | Histone H2AX Is Phosphorylated in an ATR-dependent Manner in Response to Replicational Stress[END_REF]). The roles of ATM and DNA-PKcs in phosphorylating H2AX are largely overlapping. However, there some aspects specific for each kinase (to be discussed later) [START_REF] Stiff | ATM and DNA-PK Function Redundantly to Phosphorylate H2AX after Exposure to Ionizing Radiation[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. This phosphorylated form is called gH2AX and it is one of the main signalling hallmarks of a DSB, independently of the way of its induction and on whether it was caused by a hostile environment or induced in a controlled manner as a part of cell homeostasis [START_REF] Fillingham | γH2AX and its role in DNA double-strand break repair[END_REF][START_REF] Tang Chen | Response to RAG-mediated V(D)J cleavage by NBS1 and γ-H2AX[END_REF][START_REF] Nazarov | Dephosphorylation of histone γ-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A[END_REF][START_REF] Petersen | AID is required to initiate Nbs1/γ-H2AX focus formation and mutations at sites of class switching[END_REF][START_REF] Rogakou | DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139[END_REF][START_REF] Rogakou | Megabase chromatin domains involved in DNA double-strand breaks in vivo[END_REF][START_REF] Tomilin | Visualization of focal nuclear sites of DNA repair synthesis induced by bleomycin in human cells[END_REF]. In mammalian cells, it forms large domains, up to megabases size [START_REF] Rogakou | DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139[END_REF][START_REF] Van Attikum | Crosstalk between histone modifications during the DNA damage response[END_REF]. gH2AX foci arise quickly, within minutes after DNA damaging event has taken place, but continue to expand further for one to several hours according to different studies (Y. [START_REF] Lee | Development of a high-throughput γ-H2AX assay based on imaging flow cytometry[END_REF][START_REF] Löbrich | γH2AX foci analysis for monitoring DNA double-strand break repair: Strengths, limitations and optimization[END_REF][START_REF] Sharma | Chapter 40 Histone H2AX Phosphorylation: A Marker for DNA Damage[END_REF][START_REF] Staszewski | Kinetics of c-H2AX Focus Formation Upon Treatment of Cells With UV Light and Alkylating Agents[END_REF]. Although H2AX-deficient mice are viable, they show increased genome instability and phenotypical abnormalities, underlining its importance for DNA repair [START_REF] Celeste | Genomic instability in mice lacking histone H2AX[END_REF][START_REF] Celeste | Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF][START_REF] Weyemi | Histone H2AX deficiency causes neurobehavioral deficits and impaired redox homeostasis[END_REF]. Another histone that is phosphorylated in the course of DDR signalling activation is H2B. H2B phosphorylation on S14 is induced by IR and progresses to form foci. They colocalise with those of gH2AX, although accumulating slower [START_REF] Fernandez-Capetillo | Phosphorylation of histone H2B at DNA doublestrand breaks[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. It has recently been shown that kinases responsible for this process are Mst1 and 2 [START_REF] Bitra | Rassf proteins as modulators of Mst1 kinase activity[END_REF][START_REF] Pefani | MST2 kinase suppresses rDNA transcription in response to DNA damage by phosphorylating nucleolar histone H2B[END_REF].

As DDR progresses, gH2AX is bound by the Mediator of DNA damage checkpoint protein 1 (MDC1), which serves as a platform for a repair machinery assembly [START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. It directly interacts with multiple proteins involved in DSB repair, such as ATM, MRN complex, 53BP1, and Structural maintenance of chromosomes protein 1 (Smc1) [START_REF] Stewart | MDC1 is a mediator of the mammalian DNA damage checkpoint[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. H2AX phosphorylation, as well as MDC1 recruitment, also acts as a signal for activation of intra-S and G2-M checkpoints and a cell cycle arrest, notably for phosphorylation of checkpoint kinases Chk1 and Chk2, KRAB-associated protein 1 (Kap1) and Smc1 [START_REF] Celeste | Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks[END_REF][START_REF] Fernandez-Capetillo | DNA damage-induced G2-M checkpoint activation by histone H2AX and 53BP1[END_REF][START_REF] Stewart | MDC1 is a mediator of the mammalian DNA damage checkpoint[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. It has been shown that phosphorylation and foci formation of H2AX and MDC1 is interdependent [START_REF] Stewart | MDC1 is a mediator of the mammalian DNA damage checkpoint[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. Direct interaction of MDC1 and gH2AX has been proven by co-crystallization, and the relevance of this interaction has been demonstrated by analysing MDC1 mutant or null cells, which have impaired accumulation of other DDR factors including 53BP1, NBS1 (Nijmegen breakage syndrome protein 1), and phosphorylated ATM [START_REF] Lou | MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals[END_REF][START_REF] Stucki | MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks[END_REF][START_REF] Van Attikum | Crosstalk between histone modifications during the DNA damage response[END_REF].

Therefore, MDC1 can be considered an early DDR factor, important for both recruitment and retention of other players [START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF][START_REF] Van Attikum | Crosstalk between histone modifications during the DNA damage response[END_REF].

Role of parylation in DDR

Another important process involved in DDR is poly-ADP-ribosylation, or parylation, a post-translational modification crucial for DSB recognition. This modification is catalysed by PARP (Poly-ADP-Ribosyl

Polymerase) enzymes that covalently link poly-ADP-ribosyl chains to protein substrates. PARP1 and PARP2

are involved in both SSB and DSB recognition, whereas PARP3 specifically responds to DSBs [START_REF] Beck | Poly(ADP-ribose) polymerases in double-strand break repair: Focus on PARP1, PARP2 and PARP3[END_REF][START_REF] Boehler | Poly(ADP-ribose) polymerase 3 (PARP3), a newcomer in cellular response to DNA damage and mitotic progression[END_REF][START_REF] Boulton | Interactive effects of inhibitors of poly(ADP-ribose) polymerase and DNA-dependent protein kinase on cellular responses to DNA damage[END_REF][START_REF] Caldecott | Protein ADP-ribosylation and the cellular response to DNA strand breaks[END_REF][START_REF] Rulten | PARP-3 and APLF function together to accelerate nonhomologous end-joining[END_REF]. PARP enzymes mediate DDR activation in several ways. First, some DDR factors have a PAR motif and are therefore recruited to damage sites in a PAR-dependent manner. Second, parylation of DDR and downstream repair factors might influence their catalytic activity or capability to bind to DNA or their interaction factors. In particular, parylation facilitates the recruitment of MRN complex and ATM kinase to a break site [START_REF] Beck | Poly(ADP-ribose) polymerases in double-strand break repair: Focus on PARP1, PARP2 and PARP3[END_REF][START_REF] Bryant | PARP is activated at stalled forks to mediate Mre11-dependent replication restart and recombination[END_REF]. Further on repair process, parylation plays a role in a pathway choice by promoting end resection, and therefore favouring homologous recombination and microhomology-mediated end joining [START_REF] Beck | PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways[END_REF][START_REF] Langelier | PARP-2 and PARP-3 are selectively activated by 5' phosphorylated DNA breaks through an allosteric regulatory mechanism shared with PARP-1[END_REF][START_REF] Luijsterburg | PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining[END_REF]. However, PARP3 is involved in non-homologous end-joining by limiting end-resection as well as by promoting X-ray repair cross-complementing protein 1 (XRCC4)-Lig4 mediated ligation [START_REF] Beck | PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways[END_REF][START_REF] Caldecott | Protein ADP-ribosylation and the cellular response to DNA strand breaks[END_REF][START_REF] Fenton | The PARP3-and ATM-dependent phosphorylation of APLF facilitates DNA double-strand break repair[END_REF][START_REF] Rulten | PARP-3 and APLF function together to accelerate nonhomologous end-joining[END_REF]. Therefore, PARP enzymes play an important and complex role in DSB repair from the first to the last steps of this process.

Signal transduction and amplification. DDR kinases

As I mentioned before, break recognition leads to an activation of ATM, ATR, and DNA-PKcs kinases that all belong to the phosphatidylinositol-3-kinase family. These kinases mediate signal transduction and amplification via gH2AX foci formation and play a role in downstream DNA repair proteins activation.

ATM is the central kinase involved in DDR. It was identified in 1995 a single mutated gene responsible for a rare genetic disorder, ataxia telangiectasia occurrence [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Savitsky | A single ataxia telangiectasia gene with a product similar to PI-3 kinase[END_REF].

Ataxia telangiectasia is characterised by dilated blood vessels, progressive neurodegeneration, and immunodeficiency [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF]. The activation of ATM proceeds as dissociation of catalytically inactive homodimers into active monomers [START_REF] Bakkenist | DNA damage activates ATM through intermolecular autophosphorylation and dimmer association[END_REF][START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. Among post-translational modifications that lead to its activation autophosphorylation at S1981 was the first to be discovered [START_REF] Bakkenist | DNA damage activates ATM through intermolecular autophosphorylation and dimmer association[END_REF]. However, its role remains controversial as on the one hand it has been reported to be the process initiated as the very first step of DDR stimulated by chromatin changes upon the DSB occurrence, and on the other hand, it has been shown dispensable for ATM functioning [START_REF] Bakkenist | DNA damage activates ATM through intermolecular autophosphorylation and dimmer association[END_REF][START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Kozlov | Involvement of novel autophosphorylation sites in ATM activation[END_REF][START_REF] Pellegrini | Autophosphorylation at serine 1987 is dispensable for murine Atm activation in vivo[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. Another possible mechanism for early ATM activation is acetylation by Tip60, which is attracted to the break site by exposed H3K9me3 nucleosomes, on K3016 [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Sun | A role for the Tip60 histone acetyltransferase in the acetylation and activation of ATM[END_REF][START_REF] Sun | DNA Damage-Induced Acetylation of Lysine 3016 of ATM Activates ATM Kinase Activity[END_REF]. It is certain, however, that whatever causes its initial activation, it is later enhanced by ATM direct interaction with the C-terminal domain of Nbs1, a component of MRN complex. This interaction is stabilised in the presence of free DNA ends [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Falck | Conserved modes of recruitment of ATM, ATR and DNA-PKcs to sites of DNA damage[END_REF]. Nevertheless, MRN-independent mechanisms of ATM activation have also been shown

in multiple studies [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Guo | ATM activation by oxidative stress[END_REF][START_REF] Olcina | Replication stress and chromatin context link ATM activation to a role in DNA replication[END_REF]. Activated ATM phosphorylates a vast variety of targets, influencing the process of DSB repair at all levels: break recognition and DDR activation, DDR enhancement, repair pathway choice and stimulation, cell cycle checkpoint activation, and apoptosis. Its role in break recognition and DDR activation has already been discussed.

Further on ATM promotes DDR by phosphorylating H2AX into gH2AX and MDC1, the major gH2AX reader, promoting its dimerization and stabilisation [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Burma | ATM Phosphorylates Histone H2AX in Response to DNA Double-strand Breaks[END_REF][START_REF] Jungmichel | The molecular basis of ATM-dependent dimerization of the Mdc1 DNA damage checkpoint mediator[END_REF][START_REF] Liu | Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. MDC1 further recruits the MRN complex, thus promoting additional ATM accumulation and H2AX phosphorylation, thus providing a positive feedback loop for repair foci formation [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF]. Moreover, ATM has also been shown to be involved in chromatin relaxation and remodelling around the break [START_REF] Goodarzi | ATM Signaling Facilitates Repair of DNA Double-Strand Breaks Associated with Heterochromatin[END_REF][START_REF] Moyal | Requirement of ATM-Dependent Monoubiquitylation of Histone H2B for Timely Repair of DNA Double-Strand Breaks[END_REF][START_REF] Ziv | Chromatin relaxation in response to DNA double-strand breaks is modulated by a novel ATM-and KAP-1 dependent pathway[END_REF]. ATM also promotes recruitment downstream repair proteins to the break site. It was shown to predominantly stimulate HR by phosphorylating CtBP-interacting protein (CtIP), a major MRN interactor that promotes end-resection [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF][START_REF] Shibata | Factors determining DNA double-strand break repair pathway choice in G2 phase[END_REF].

However, ATM is also able to stimulate DSB repair in G1 in a way that requires ATM, DNA-PK, MRN complex and Artemis activity and by promoting NHEJ due to its redundant functions with XRCC4-like factor (XLF) [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Riballo | A pathway of double-strand break rejoining dependent upon ATM, Artemis, and proteins locating to γ-H2AX foci[END_REF][START_REF] Zha | ATM damage response and XLF repair factor are functionally redundant in joining DNA breaks[END_REF]. Additionally, it phosphorylates Chk2 and p53 causing a cell cycle block at G2/M transition [START_REF] Banin | Enhanced phosphorylation of p53 by ATM in response to DNA damage[END_REF][START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Matsumura | H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation[END_REF][START_REF] Siliciano | DNA damage induces phosphorylation of the amino terminus of p53[END_REF].

The existence of DNA-PK was presupposed in 1985 when it was observed that the addition of doublestranded DNA (dsDNA) into Xenopus and Arbacia egg extracts promoted phosphorylation of several proteins and named DNA-dependent protein kinase [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Walker | Double-stranded DNA induces the phosphorylation of several proteins including the 90 000 mol. wt. heat-shock protein in animal cell extracts[END_REF]. This kinase was purified five years later [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Carter | A DNA-activated protein kinase from HeLa cell nuclei[END_REF][START_REF] Jackson | GC box binding induces phosphorylation of Sp1 by a DNA-dependent protein kinase[END_REF][START_REF] Lees-Miller | Human cells contain a DNA-activated protein kinase that phosphorylates simian virus 40 T antigen, mouse p53, and the human Ku autoantigen[END_REF]. DNA-PKcs is recruited to DNA damage sites by Ku70/Ku80 (Ku autoantigen protein p70/80 homolog) heterodimers and form DNA-PK complex that enhances the catalytic activity of the kinase [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Gell | Mapping of protein-protein interactions within the DNA-dependent protein kinase complex[END_REF][START_REF] Singleton | The C Terminus of Ku80 Activates the DNA-Dependent Protein Kinase Catalytic Subunit[END_REF]. DNA-PKcs acts cooperatively with ATM to promote gH2AX signal spreading and DDR activation [START_REF] Firsanov | H2AX phosphorylation at the sites of DNA double-strand breaks in cultivated mammalian cells and tissues[END_REF][START_REF] Stiff | ATM and DNA-PK Function Redundantly to Phosphorylate H2AX after Exposure to Ionizing Radiation[END_REF][START_REF] Wang | Complex H2AX phosphorylation patterns by multiple kinases including ATM and DNA-PK in human cells exposed to ionizing radiation and treated with kinase inhibitors[END_REF]. It also plays a crucial role in NHEJ, and multiple target proteins have been suggested [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Enriquez-Rios | DNA-PKcs, ATM, and ATR interplay maintains genome integrity during neurogenesis[END_REF]. Some prominent NHEJ players as Artemis, are among the confirmed targets [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Jiang | Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and endligation during nonhomologous end-joining[END_REF][START_REF] Malu | Artemis C-terminal region facilitates V(D)J recombination through its interactions with DNA Ligase IV and DNApkcs[END_REF]. While DNA-PKcs phosphorylation by ATM leads to end-processing by promoting Artemis recruitment, additional autophosphorylation is necessary for it to act on other targets. The exact target proteins are unknown but the importance of DNA-PKcs is affirmed by the fact that its knock-out is lethal in mice during embryonic development by E16.5 and on the cellular level leads to an inability to multiple NHEJ defects similar to XRCC4 and XLF deficient cells [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Jiang | Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and endligation during nonhomologous end-joining[END_REF]. Particularly, it was shown to play an important role in regulating end-ligation by promoting the transition between two synaptic complexes [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Graham | Two-Stage Synapsis of DNA Ends during Non-homologous End Joining[END_REF][START_REF] Jiang | Differential phosphorylation of DNA-PKcs regulates the interplay between end-processing and endligation during nonhomologous end-joining[END_REF]. At the same time, DNA-PKcs phosphorylates RPA and activates the S-phase cell cycle checkpoint [START_REF] Ashley | DNA-PK phosphorylation of RPA32 Ser4/Ser8 regulates replication stress checkpoint activation, fork restart, homologous recombination and mitotic catastrophe[END_REF]. Activated RPA mediates the recruitment of PALB2 (Partner and localizer of BRCA2), thus promoting DSB repair progression [START_REF] Murphy | Phosphorylated RPA recruits PALB2 to stalled DNA replication forks to facilitate fork recovery[END_REF]. On the other hand, it has also been shown that Chk1 promotes DNA-PK activity in order to stimulate fast repair by NHEJ and prevent cell cycle block [START_REF] Goudelock | Regulatory interactions between the checkpoint kinase Chk1 and the proteins of the DNA-dependent protein kinase complex[END_REF][START_REF] Shimura | DNA-PK Is Involved in Repairing a Transient Surge of DNA Breaks Induced by Deceleration of DNA Replication[END_REF].

ATR is an analogue of the yeast protein Mec1 (Mitosis entry checkpoint 1) that was first discovered as Rad3 in a radiosensitivity screen and later as being essential for cell cycle checkpoints [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Nasim | Genetic control of radiation sensitivity in diploid yeast[END_REF][START_REF] Weinert | Mitotic checkpoint genes in budding yeast and the dependence of mitosis on DNA replication and repair[END_REF]. A human ATR protein was cloned independently by two different groups [START_REF] Bentley | The schizosaccharomyces pombe rad3 checkpoint gene[END_REF][START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Cimprich | cDNA cloning and gene mapping of a candidate human cell cycle checkpoint protein[END_REF]. ATR kinase plays a crucial role in replication stress response. It gets recruited ssDNA in response to various kinds of genotoxic stress. Therefore, it accumulates at DSB sites after resection but also at stalled replication forks. Interaction of ATR with RPA allows an ATRIP (ATR-interacting protein) cofactor recruitment and binding to ATR [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Zou | Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes[END_REF]. ATR-ATRIP heterodimer formation is essential for ATR activation. ATR recruitment alone is not sufficient for the optimal response and requires cofactors such as TopBP1 (DNA topoisomerase 2-binding protein 1) or ETAA1 (Ewing's tumor-associated antigen 1) [START_REF] Bass | ETAA1 acts at stalled replication forks to maintain genome integrity[END_REF][START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Feng | Ewing tumor-associated antigen 1 interacts with replication protein A to promote restart of stalled replication forks[END_REF][START_REF] Haahr | Activation of the ATR kinase by the RPA-binding protein ETAA1[END_REF][START_REF] Kumagai | TopBP1 activates the ATR-ATRIP complex[END_REF][START_REF] Mordes | TopBP1 activates ATR through ATRIP and a PIKK regulatory domain[END_REF]. Activated ATR promotes gH2AX signal spreading and leads to Chk1 phosphorylation and activation, which also causes cell cycle block in the G2/M phase [START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Hui | ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1[END_REF][START_REF] Liu | Chk1 is an essential kinase that is regulated by Atr and required for the G2/M DNA damage checkpoint[END_REF]. ATR has also some unique targets related to replication stress management, including FANCI protein, which promotes dormant origin firing and FANCD2 mono-ubiquitylation and therefore FA pathway progression [START_REF] Andreassen | ATR couples FANCD2 monoubiquitination to the DNAdamage responsey[END_REF][START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF]Y. H. Chen et al., 2015;[START_REF] Ishiai | FANCI phosphorylation functions as a molecular switch to turn on the Fanconi anemia pathway[END_REF].

In G1/S checkpoint is thought to be mainly regulated by ATM, whereas intra-S and G2/M are controlled by both ATM and ATR cooperatively [START_REF] Adams | Recruitment of ATR to sites of ionising radiationinduced DNA damage requires ATM and components of the MRN protein complex[END_REF][START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF][START_REF] Cuadrado | ATM regulates ATR chromatin loading in response to DNA double-strand breaks[END_REF][START_REF] Jazayeri | ATM-and cell cycledependent regulation of ATR in response to DNA double-strand breaks[END_REF][START_REF] Myers | Rapid activation of ATR by ionizing radiation requires ATM and Mre11[END_REF]. All PIKKs are strictly regulated by cofactors required for DNA binding: Nbs1, ATRIP, and Ku80 for ATM, ATR, and DNA-PKcs respectively. Therefore their appropriate involvement is achieved throughout the cell cycle and three DNA repair PIKKs are acting cooperatively in the process of DSB repair (fig. 3).

Role of ubiquitylation and SUMOylation in DSB repair

Histone ubiquitylation and SUMOylation (Small ubiquitin-like modifier) play a prominent role in DRR and repair progression. As it was mentioned before, gH2AX recruits MDC1, which plays a role in both signal spreading by creating a positive feedback loop with ATM and DNA repair progression by serving as a platform for downstream repair complexes assembly. Multiple ubiquitylation steps are performed to ensure this process. As a first step, H2AX acetylation enabled by Tip60 facilitated H2AX monoubiquitylation at K118/119. Not all players involved in this process are known to date but Ubc13 (ubiquitin-conjugating enzyme 13) E2 ligase have been shown to play a role in it [START_REF] Ikura | DNA Damage-Dependent Acetylation and Ubiquitination of H2AX Enhances Chromatin Dynamics[END_REF][START_REF] Jiang | Acetylation of H2AX on lysine 36 plays a key role in the DNA double-strand break repair pathway[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF][START_REF] Zhao | A Critical Role for the Ubiquitin-Conjugating Enzyme Ubc13 in Initiating Homologous Recombination[END_REF].

Also, PRC1 (Polycomp repressive complex 1) gets recruited to DSBs and its subunits, Rnf2 (RING finger protein 2) and Bmi1 (B cell-specific Moloney murine leukemia virus integration site 1), have been shown to mediate the monoubiquitylation of H2A and H2AX on K119 [START_REF] Ismail | BMI1-mediated histone ubiquitylation promotes DNA double-strand break repair[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. This modification plays a role in the recruitment of DDR proteins and IRIFs formation. RNF2/BMI1 complex controls DDR in an ATM-dependent manner and their knockdown leads to increased sensitivity to IR and compromised DSB repair [START_REF] Facchino | BMI1 confers radioresistance to normal and cancerous neural stem cells through recruitment of the DNA damage response machinery[END_REF][START_REF] Pan | Monoubiquitination of H2AX protein regulates DNA damage response signaling[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF].

The best-established E3 ligases involved in DSB repair are RNF8 (RING finger protein 8), CHFR (checkpoint protein with FHA and RING domain), and RNF168 (RING finger protein 168). All three of them use Ubc13 an E2 ligase (Panier & Durocher, 2009;[START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. RNF8 recruitment is facilitated by MDC1 [START_REF] Huen | RNF8 Transduces the DNA-Damage Signal via Histone Ubiquitylation and Checkpoint Protein Assembly[END_REF][START_REF] Mailand | RNF8 Ubiquitylates Histones at DNA Double-Strand Breaks and Promotes Assembly of Repair Proteins[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. RNF8 ubiquitylates histones H1 and H2A, H2AX, and H2B

to promote binding of repair factors such as 53BP1 (TP53-binding protein1), RAP80 (Receptor-associated protein 80) and BRCA1 (Breast cancer type 1 susceptibility protein) [START_REF] Huen | RNF8 Transduces the DNA-Damage Signal via Histone Ubiquitylation and Checkpoint Protein Assembly[END_REF][START_REF] Mandemaker | DNA damage-induced histone H1 ubiquitylation is mediated by HUWE1 and stimulates the RNF8-RNF168 pathway[END_REF][START_REF] Sobhian | RAP80 targets BRCA1 to specific ubiquitin structures at DNA damage sites[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF].

Another E3-ligase, RNF168, is recruited in an RNF8-dependent manner to enhance the signal by ubiquitylation of H1 and H2A histones [START_REF] Mandemaker | DNA damage-induced histone H1 ubiquitylation is mediated by HUWE1 and stimulates the RNF8-RNF168 pathway[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. Upon irradiation, RNF168 gets stabilized by HERC2 (HECT domain and RCC1-like domain-containing protein 2) (Bekker-Jensen [START_REF] Bekker-Jensen | Assembly and function of DNA double-strand break repair foci in mammalian cells[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]. It colocalizes with gH2AX at damage sites and promotes amplification of the ubiquitylation established by RNF8 and PRC1 complex [START_REF] Doil | RNF168 Binds and Amplifies Ubiquitin Conjugates on Damaged Chromosomes to Allow Accumulation of Repair Proteins[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF].

Finally, CHFR (checkpoint protein with FHA and RING domain) E3 ligase, which has considerable structural similarity to RNF8, has been shown to act synergistically with it in promoting ATM activation and cell cycle checkpoints activation [START_REF] Bothos | The Chfr mitotic checkpoint protein functions with Ubc13-Mms2 to form Lys63-linked polyubiquitin chains[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF]J. Wu et al., 2011). Ubiquitination-dependent DDR promotion is limited by deubiquitylation enzymes. One of the enzymes involved in this process is BRCC36 (BRCA-containing complex 36), a member of the RAP80-ABRA1-BRCA1-BARD1-BRCC36 complex [START_REF] Ng | The Lys63-deubiquitylating enzyme BRCC36 limits DNA break processing and repair[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF][START_REF] Wang | Ubc13/Rnf8 ubiquitin ligases control foci formation of the Rap80/Abraxas/Brca1/Brcc36 complex in response to DNA damage[END_REF], Other prominent players such as USP3, USP11 and USP16 (ubiquitin-specific proteases 3, 11 and 16) help to regulate ubiquitylation-mediated signalling and fine-tune the extent of DDR activation [START_REF] Nicassio | Human USP3 Is a Chromatin Modifier Required for S Phase Progression and Genome Stability[END_REF][START_REF] Thompson | Recognition, signaling, and repair of DNA double-strand breaks produced by ionizing radiation in mammalian cells: The molecular choreography[END_REF][START_REF] Wiltshire | Sensitivity to poly(ADP-ribose) polymerase (PARP) inhibition identifies ubiquitin-specific peptidase 11 (USP11) as a regulator of DNA doublestrand break repair[END_REF][START_REF] Zhang | The histone H2A deubiquitinase USP16 interacts with HERC2 and fine-tunes cellular response to DNA damage[END_REF].

DDR activation consequences

The presence of mutilated DNA regions is unfavourable in the course of a cell cycle progression. Therefore, cell cycle arrest mechanisms, known as cell cycle checkpoints have developed in cells. Such precautions ensure that existing DNA damage is repaired before the transition to the next stage. However, if DNA damage is too profound or impossible to fix, cells undergo senescence or apoptosis.

Cell cycle arrest is one of the main and immediate DDR activation consequences. In the course of a cell cycle that constitutes from post-mitotic gap (G1) DNA replication (S), post-growth (G2), and mitotic (M) phases, all transitions are tightly orchestrated by cyclins and cyclin-dependent kinases (Cdks) [START_REF] Schäfer | The Cell Cycle: A Review[END_REF]. Cell cycle checkpoints are signaling pathways that are intended to sense DNA damage or other mistakes such as erroneous chromosome segregation and delay cell cycle progression to facilitate the repair or eliminate cells that are beyond it (Bartek & Lukas, 2003;[START_REF] Zhou | The DNA damage response: putting checkpoints in perspective[END_REF]. There are three checkpoints. G1/S checkpoint is activated after ATM-dependent Chk2 phosphorylation (Bartek & Lukas, 2003;[START_REF] Falck | The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis[END_REF][START_REF] Matsuoka | Linkage of ATM to cell cycle regulation by the Chk2 protein kinase[END_REF]. ATM and Chk2 in turn mediate Cdc25A (M-phase inducer phosphatase 1) phosphorylation and sequential degradation which promotes p53 stabilisation followed by p21 expression and leads to Cdk inhibition and cell cycle progression abrogation (Bartek & Lukas, 2003;[START_REF] Di Fagagna | Living on a break: Cellular senescence as a DNA-damage response[END_REF][START_REF] Falck | The ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA synthesis[END_REF]. Intra-S checkpoint is activated by ATR-dependent Chk1 phosphorylation (Bartek & Lukas, 2003;[START_REF] Hui | ATR-Mediated Checkpoint Pathways Regulate Phosphorylation and Activation of Human Chk1[END_REF]. It results in inhibition of origin firing and therefore slowing down the replication process in unperturbed conditions to avoid replication stress [START_REF] Ge | Chk1 inhibits replication factory activation but allows dormant origin firing in existing factories[END_REF][START_REF] Moiseeva | An ATR and CHK1 kinase signaling mechanism that limits origin firing during unperturbed DNA replication[END_REF][START_REF] Sørensen | Chk1 regulates the S phase checkpoint by coupling the physiological turnover and ionizing radiationinduced accelerated proteolysis of Cdc25A[END_REF]. Activation of G2/M checkpoint relies on ATMand ATR-dependent phosphorylation for Chk2 and Chk1 [START_REF] Ahn | Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for efficient activation of Chk2 in response to ionizing radiation[END_REF][START_REF] Bahassi | Priming phosphorylation of Chk2 by polo-like kinase 3 (Plk3) mediates its full activation by ATM and a downstream checkpoint in response to DNA damage[END_REF]Bartek & Lukas, 2003;[START_REF] Gatei | Ataxia-telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on Ser-317 in response to ionizing radiation[END_REF][START_REF] Xiao | Chk1 mediates S and G2 arrests through Cdc25A degradation in response to DNA-damaging agents[END_REF]. Activated Chk1 blocks Cdc2/CyclinB activation through targeting Cdc25C to prevent mitosis [START_REF] Stanford | Changes in Regulatory Phosphorylation of Cdc25C Ser287 and Wee1 Ser549 during Normal Cell Cycle Progression and Checkpoint Arrests[END_REF].

It has been shown that too prolonged checkpoint activation is perceived as unrepairable damage by a cell.

One of the proteins activated by ATM is p53, which has a dual role in balancing between repair and apoptosis depending on the degree and continuance of activation (D'Adda Di Fagagna, 2008).

Double-strand break repair pathways

As discussed above, DNA damage and particularly DSBs can represent a considerable danger for a cell, and therefore repair should be guaranteed, so several pathways have been developed in mammalian cells to ensure it. After a break recognition and DDR activation one of either two major pathways, non-homologous end joining (NHEJ), homologous recombination (HR), or two minor pathways, microhomology-mediated end joining (MMEJ), also sometimes referred to as alternative end joining (Alt-EJ), or single-strand annealing (SSA) can be employed [START_REF] Sallmyr | Repair of DNA double-strand breaks by mammalian alternative end-joining pathways[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF].

Non-homologous end joining

Non-homologous end joining, which is also called classical non-homologous end-joining (cNHEJ) in contrast to alternative end-joining (Alt-EJ), is considered to be a predominantly used pathway in higher eukaryotes [START_REF] Ferguson | The nonhomologous end-joining pathway of DNA repair is required for genomic stability and the suppression of translocations[END_REF][START_REF] Karanjawala | The nonhomologous DNA end joining pathway is important for chromosome stability in primary fibroblasts[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF]. It has been reported that approximately 80% of DSBs are repaired using this mechanism [START_REF] Tichy | Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks[END_REF]. NHEJ is initiated by Ku70/Ku80 heterodimer binding to free DNA ends [START_REF] Britton | A new method for high-resolution imaging of Ku foci to decipher mechanisms of DNA double-strand break repair[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF]. Ku proteins are abundant in the nucleus and have a high affinity to free DNA ends which allows them to bind to a break site within 5 seconds after its occurrence [START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF][START_REF] Mari | Dynamic assembly of end-joining complexes requires interaction between Ku70/80 and XRCC4[END_REF]. It serves a dual role of, on the one hand, protecting DNA ends from degradation and keeping them together for further repair, and on the other hand, preventing resection [START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF][START_REF] Mimitou | S1-seq Assay for Mapping Processed DNA Ends[END_REF]. It also serves as a platform for DNA repair machinery assembly [START_REF] Costantini | Interaction of the Ku heterodimer with the DNA ligase IV/Xrcc4 complex and its regulation by DNA-PK[END_REF][START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF][START_REF] Hsu | Defining interactions between DNA-PK and ligase IV/XRCC4[END_REF][START_REF] Rivera-Calzada | Structural model of full-length human Ku70-Ku80 heterodimer and its recognition of DNA and DNA-PKcs[END_REF][START_REF] Yano | Ku recruits XLF to DNA double-strand breaks[END_REF]. Another important function of Ku70/80 heterodimer is in DDR regulation by modulating ATM activity and in preventing apoptosis by binding and inhibiting a proapoptotic factor Bax [START_REF] Amsel | Regulation of the proapoptotic factor Bax by Ku70dependent deubiquitylation[END_REF][START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF][START_REF] Tomimatsu | Ku70/80 modulates ATM and ATR signaling pathways in response to DNA double strand breaks[END_REF]; X. Y. [START_REF] Zhou | Ku affects the ATM-dependent S phase checkpoint following ionizing radiation[END_REF]. DNA-PKcs binds to Ku70/80 heterodimer to form an active DNA-PK complex. DNA-PKcs mediates tethering of broken ends [START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF][START_REF] Hammel | Ku and DNA-dependent protein kinase dynamic conformations and assembly regulate DNA binding and the initial non-homologous end joining complex[END_REF]. DNA-PK autophosphorylates its own subunits and phosphorylates several other target molecules including Artemis, PNKP, XRCC4, XLF, and DNA ligase IV [START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF][START_REF] Goodarzi | DNA-PK autophosphorylation facilitates Artemis endonuclease activity[END_REF][START_REF] Hammel | Ku and DNA-dependent protein kinase dynamic conformations and assembly regulate DNA binding and the initial non-homologous end joining complex[END_REF][START_REF] Wang | Phosphorylation and regulation of DNA ligase IV stability by DNA-dependent protein kinase[END_REF][START_REF] Yu | DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination[END_REF][START_REF] Yu | DNA-PK and ATM phosphorylation sites in XLF/Cernunnos are not required for repair of DNA double strand breaks[END_REF]. In case end-processing is required before ligation it can be performed by various enzymes including PNKP (polynucleotide kinase/phosphatase), Artemis, Exo1, Tdp1, and others in order to remove 3' phosphate ends, 3ʹ-phosphoglycolates, or 5ʹ-hydroxyl groups and generate 5' phosphate ends required for further ligation [START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF][START_REF] Mahaney | Repair of ionizing radiation-induced DNA double-strand breaks by non-homologous end-joining[END_REF]. The next step is ligation, which is performed by a ligase complex of DNA Ligase IV with XRCC4 and XLF, co-factors required for its stabilisation and stimulation [START_REF] Ahnesorg | XLF interacts with the XRCC4-DNA Ligase IV complex to promote DNA nonhomologous end-joining[END_REF][START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF][START_REF] Grawunder | Activity of DNA ligase IV stimulated by complex formation with XRCC4 protein in mammalian cells[END_REF][START_REF] Mcelhinny | Ku Recruits the XRCC4-Ligase IV Complex to DNA Ends[END_REF]. However, it has recently been shown that a small subset of breaks undergoes resection-dependent NHEJ repair enabled by Artemis and CtIP cooperation and resection performed by MRE11 (Meiotic recombination 11 homolog 1) and EXO1 [START_REF] Biehs | DNA Double-Strand Break Resection Occurs during Non-homologous End Joining in G1 but Is Distinct from Resection during Homologous Recombination[END_REF][START_REF] Shibata | The pendulum of the Ku-Ku clock[END_REF]. Finally, Ku dimers have to be removed from repaired DNA. The exact mechanism of how that happens remains unclear. However, two mechanisms have been proposed: ubiquitylation-driven degradation or DNA nicking that would allow Ku to escape [START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF][START_REF] Langerak | Release of Ku and MRN from DNA ends by Mre11 nuclease activity and Ctp1 is required for homologous recombination repair of double-strand breaks[END_REF][START_REF] Postow | Ku80 removal from DNA through double strand break-induced ubiquitylation[END_REF]. [START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF].

Homologous recombination

The homologous recombination repair pathway uses a sister chromatid as a template for restoring mutilated DNA. Therefore, this pathway is error-free but restricted to S and G2 phases of the cell cycle [START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Takata | Homologous recombination and non-homologous end-joining pathways of DNA doublestrand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells[END_REF]. HR is promoted by MRN complex and resection is initiated by its subunit MRE11(T. [START_REF] Liu | DNA End Resection: Facts and Mechanisms[END_REF][START_REF] Shibata | DNA Double-Strand Break Repair Pathway Choice Is Directed by Distinct MRE11 Nuclease Activities[END_REF][START_REF] Stracker | The MRE11 complex: Starting from the ends[END_REF]. MRN complex also recruits CtIP (CTBP interacting protein) that further promotes end-resection [START_REF] Escribano-Díaz | A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice[END_REF][START_REF] Sartori | Human CtIP promotes DNA end resection[END_REF][START_REF] Stracker | The MRE11 complex: Starting from the ends[END_REF]. CtIP recruits BRCA1 to prevent binding of RIF1 (Rap1interacting factor 1 homolog), a protein involved in NHEJ initiation [START_REF] Escribano-Díaz | A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice[END_REF][START_REF] Liu | DNA End Resection: Facts and Mechanisms[END_REF]. End-resection is then performed by EXO1 (Exonuclease1) by 5' to 3' digestion of DNA ends, which leads to the formation of single-strand DNA (ssDNA) overhangs [START_REF] Cannavo | Relationship of DNA degradation by Saccharomyces cerevisiae Exonuclease 1 and its stimulation by RPA and Mre11-Rad50-Xrs2 to DNA end resection[END_REF][START_REF] Eid | DNA end resection by CtIP and exonuclease 1 prevents genomic instability[END_REF] T. [START_REF] Liu | DNA End Resection: Facts and Mechanisms[END_REF]. ssDNA is then bound by RPA (replication protein A) heterotrimer that protects it from degradation [START_REF] San Filippo | Mechanism of eukaryotic homologous recombination[END_REF][START_REF] Van Komen | Functional cross-talk among Rad51, Rad54, and replication protein A in heteroduplex DNA joint formation[END_REF]. MRN complex promotes Bloom syndrome helicase (BLM) recruitment, which together with DNA2 (DNA replication ATP-dependent helicase/nuclease DNA2) promotes extended resection [START_REF] Daley | Enhancement of BLM-DNA2-Mediated Long-Range DNA End Resection by CtIP[END_REF][START_REF] Nimonkar | BLM-DNA2-RPA-MRN and EXO1-BLM-RPA-MRN constitute two DNA end resection machineries for human DNA break repair[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF]. Rad51

recombinase must be recruited to a break site by interacting with RPA, and later replace it as to HR could proceed [START_REF] San Filippo | Mechanism of eukaryotic homologous recombination[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF]. To that end, BRCA2 competes with RPA for binding ssDNA and interacts with RAD51 monomers and BRCA1 via its partner PALB2 to promote RAD51 filament formation [START_REF] Jensen | Purified human BRCA2 stimulates RAD51-mediated recombination[END_REF][START_REF] Jensen | BRCA2 is epistatic to the RAD51 paralogs in response to DNA damage[END_REF][START_REF] Prakash | Homologous Recombination and Human Health[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Stark | Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF][START_REF] Xia | Control of BRCA2 Cellular and Clinical Functions by a Nuclear Partner, PALB2[END_REF][START_REF] Yang | The BRCA2 homologue Brh2 nucleates RAD51 filament formation at a dsDNA-ssDNA junction[END_REF][START_REF] Zhang | PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2[END_REF]. RAD51 nucleoprotein filaments stabilized by RAD54 are involved in the homology search process that is a key step of HR facilitated by BRCA1-BARD1 (BRCA1-associated RING domain protein 1) [START_REF] Sanchez | Combined optical and topographic imaging reveals different arrangements of human RAD54 with presynaptic and postsynaptic RAD51-DNA filaments[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF][START_REF] Zhao | BRCA1-BARD1 promotes RAD51-mediated homologous DNA pairing[END_REF]. RAD51 nucleofilaments invade a sister chromatid in a RAD54-dependent manner and form a three-strand helix intermediate which is further resolved into a heteroduplex formed by an invading strand and a complementary strand (Z. Chen et al., 2008;[START_REF] Mazina | Human Rad54 protein stimulates DNA strand exchange activity of hRad51 protein in the presence of Ca2+[END_REF][START_REF] Van Der Heijden | Homologous Recombination in Real Time: DNA Strand Exchange by RecA[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]. The dissociated DNA strand is displaced into a displacement-loop (D-loop) and protected by RPA [START_REF] Daley | Regulation of DNA Pairing in Homologous Recombination[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]. At this step RAD51 filament is disassembled in an ATP-dependent manner [START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Van Der Heijden | Homologous Recombination in Real Time: DNA Strand Exchange by RecA[END_REF]. Invading strand extension is performed by DNA polymerase d, although translesion synthesis DNA polymerases could also be involved in this process [START_REF] Mcvey | Eukaryotic DNA Polymerases in Homologous Recombination[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF]. PCNA (Proliferating cell nuclear antigen) and its loader RFC1-5 has been shown to be required for the initiation of DNA synthesis by Pold during HR (X. [START_REF] Li | PCNA Is Required for Initiation of Recombination-Associated DNA Synthesis by DNA Polymerase δ[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]. The final steps of HR can be conducted in three different ways. Synthesisdependent strand annealing pathway (SDSA), where only one strand undergoes invasion. This results in the annealing of the non-invading strand with the displaced strand once D-loop is unwound and promotes HR termination. This is a non-crossover and therefore preferable pathway as it does not involve a Holliday junction formation [START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Westmoreland | Coincident Resection at Both Ends of Random, γ-Induced Double-Strand Breaks Requires MRX (MRN), Sae2 (Ctp1), and Mre11-Nuclease[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]. Another pathway is double Holliday junction formation which happens due to the second strand invasion and can be resolved with or without crossover [START_REF] Osman | Generating crossovers by resolution of nicked Holliday junctions: A role for Mus81-Eme1 in meiosis[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF][START_REF] Wyatt | The SMX DNA Repair Trinuclease[END_REF]. Resected DNA is protected by RPA, which is further displaced by Rad51, resulting in RAD51 nucleofilament formation.

After RAD51-mediated strand invasion, facilitated by BRCA1 and BARD1, nascent strand is synthesised by either SDSA, Holliday junction formation, or LTGC. Adapted from [START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF].

Finally, during replication long-tract gene conversion (LTGC) and break-induced replication (BIR) might be deployed, in which DNA synthesis induced by HR proceeds to a large distance from the break sites. Those pathways are highly mutagenic and are therefore suppressed in mammals and could mostly be observed if one of the key HR players is dysfunctional [START_REF] Chandramouly | BRCA1 and CtIP suppress long-tract gene conversion between sister chromatids[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Willis | Mechanism of tandem duplication formation in BRCA1-mutant cells[END_REF].

Microhomology-mediated end joining MMEJ (or Alt-EJ) pathway was for a long time considered as a backup for the major ones. It is initiated by end resection and requires short (2-20 bp) 3' ssDNA overhangs [START_REF] Seol | Microhomology-mediated end joining: Good, bad and ugly[END_REF][START_REF] Welcker | Involvement of very short DNA tandem repeats and the influence of the RAD52 gene on the occurrence of deletions in Saccharomyces cerevisiae[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]. It is Ku-independent and require proteins known to be a part of HR end resection machinery and is more prominent if NHEJ or HR is impaired (e.g. in KU or BRCA1-deficient cells) and utilizes microhomologies in close proximity to a break site [START_REF] Boboila | Alternative end-joining catalyzes class switch recombination in the absence of both Ku70 and DNA ligase[END_REF][START_REF] Ceccaldi | Homologousrecombination-deficient tumours are dependent on Polθ -mediated repair[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]. This pathway leads to small deletions so it was also assumed that other mechanisms, which are error-free or cause minimal error would be preferable. Break recognition is thought to be mediated by PARP1 that competes with Ku dimer [START_REF] Badie | BRCA 1 and Ct IP promote alternative non-homologous end-joining at uncapped telomeres[END_REF][START_REF] Mansour | The alternative end-joining pathway for repair of DNA doublestrand breaks requires PARP1 but is not dependent upon microhomologies[END_REF][START_REF] Mansour | The absence of Ku but not defects in classical non-homologous end-joining is required to trigger PARP1-dependent end-joining[END_REF].

After break recognition, the initial steps of MMEJ are common with HR. It is also dependent on Mre11 and CtIP resection activity [START_REF] Badie | BRCA 1 and Ct IP promote alternative non-homologous end-joining at uncapped telomeres[END_REF][START_REF] Truong | Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF][START_REF] Xie | Role of mammalian Mre11 in classical and alternative nonhomologous end joining[END_REF]. However, BLM/Exo1 complex that promotes long-range resection is inhibiting MMEJ [START_REF] Daley | Biochemical mechanism of DSB end resection and its regulation[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]Y. Wu et al., 2015). Mechanisms of microhomology search are not yet clear, but once it is found ssDNA gaps are filled by Polq [START_REF] Kent | Mechanism of microhomology-mediated end-joining promoted by human DNA polymerase θ[END_REF][START_REF] Mateos-Gomez | The helicase domain of Polθ counteracts RPA to promote alt-NHEJ[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF].

Polq contains a Rad51 domain that allows it to inhibit Rad51-mediated recombination as well as misplaces RPA that is known to promote HR and negatively regulate MMEJ [START_REF] Ahrabi | A role for human homologous recombination factors in suppressing microhomology-mediated end joining[END_REF][START_REF] Ceccaldi | Homologousrecombination-deficient tumours are dependent on Polθ -mediated repair[END_REF] S. K. [START_REF] Deng | RPA antagonizes microhomologymediated repair of DNA double-strand breaks[END_REF][START_REF] Mateos-Gomez | Mammalian polymerase θ promotes alternative NHEJ and suppresses recombination[END_REF][START_REF] Carroll | Genome editing: Past, present, and future[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]. The final ligation step is performed by the XRCC1-Lig3 complex [START_REF] Audebert | Involvement of poly(ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double-strand breaks rejoining[END_REF][START_REF] Okano | Spatial and Temporal Cellular Responses to Single-Strand Breaks in Human Cells[END_REF][START_REF] Okano | Translocation of XRCC1 and DNA ligase IIIα from centrosomes to chromosomes in response to DNA damage in mitotic human cells[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]. Single-strand annealing Finally, another pathway that could perform a repair of resected ends is single-strand annealing (SSA). This pathway is carried out through annealing of homologous regions of 3' ssDNA on the same chromosome (i.g. within repetitive regions). Therefore SSA is considered to be a highly deleterious pathway [START_REF] Iliakis | Necessities in the processing of DNA double strand breaks and their effects on genomic instability and cancer[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF]. In the course of SSA resection could be carried to a considerable distance, sometimes longer than during HR [START_REF] Iliakis | Necessities in the processing of DNA double strand breaks and their effects on genomic instability and cancer[END_REF][START_REF] Ochs | 53BP1 fosters fidelity of homologydirected DNA repair[END_REF]. Although the mechanisms are poorly understood in mammals, it has been shown that SSA initiation requires PARP1 and resection that precedes it relies on CtIP and RPA [START_REF] Grimme | Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52-ssDNA complexes[END_REF][START_REF] Iliakis | Necessities in the processing of DNA double strand breaks and their effects on genomic instability and cancer[END_REF][START_REF] Sullivan-Reed | Simultaneous Targeting of PARP1 and RAD52 Triggers Dual Synthetic Lethality in BRCA-Deficient Tumor Cells[END_REF][START_REF] Truong | Microhomology-mediated End Joining and Homologous Recombination share the initial end resection step to repair DNA double-strand breaks in mammalian cells[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF][START_REF] Xie | Role of mammalian Mre11 in classical and alternative nonhomologous end joining[END_REF]. Annealing of ssDNA after resection is mediated by Rad52 [START_REF] Grimme | Human Rad52 binds and wraps single-stranded DNA and mediates annealing via two hRad52-ssDNA complexes[END_REF][START_REF] Iliakis | Necessities in the processing of DNA double strand breaks and their effects on genomic instability and cancer[END_REF][START_REF] Iyama | DNA repair mechanisms in dividing and non-dividing cells[END_REF][START_REF] Rothenberg | Human Rad52-mediated homology search and annealing occurs by continuous interactions between overlapping nucleoprotein complexes[END_REF][START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF].

It has been shown that non-homologous 3' end is removed by a NER-associated nuclease complex ERCC1/XPF [START_REF] Ahmad | ERCC1-XPF Endonuclease Facilitates DNA Double-Strand Break Repair[END_REF][START_REF] Al-Minawi | The ERCC1/XPF endonuclease is required for efficient single-strand annealing and gene conversion in mammalian cells[END_REF][START_REF] Iyama | DNA repair mechanisms in dividing and non-dividing cells[END_REF]. However, downstream polymerases and ligases remain unknown [START_REF] Iyama | DNA repair mechanisms in dividing and non-dividing cells[END_REF].

Double-strand break repair pathway choice

The availability of multiple pathways for DSB repair leads to the necessity to choose between them, preferably selecting the most appropriate one. This choice is very complex and it is regulated at multiple levels. In most of the cases, NHEJ and HR are considered to be predominant pathways (exceptions to be discussed later), and the backup pathways step in occasionally when two preferential pathways failed to complete repair (Jachimowicz, Goergens, et al., 2019;[START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF].

The crucial decision point is the decision for resection as Ku70/80 binding to ssDNA is weak, therefore predisposing such breaks to be repaired by HR or backup pathways, and its binding to dsDNA is strong, so they would be likely repaired by NHEJ [START_REF] Mimori | Mechanism of interaction between Ku protein and DNA[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF]. As previously mentioned, one of the key factors in the repair pathway choice is the cell cycle state. This arises from HR requiring a sister chromatid and therefore is limited to S and G2 phases of the cell cycle. Therefore many of the resection-initiating factors that are essential for commitment to HR are regulated by CDKs [START_REF] Aylon | The CDK regulates repair of double-strand breaks by homologous recombination during the cell cycle[END_REF][START_REF] Ira | DNA end resection, homologous recombination and DNA damage checkpoint activation require CDK1[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Tomimatsu | Phosphorylation of EXO1 by CDKs 1 and 2 regulates DNA end resection and repair pathway choice[END_REF]. The competition between Ku70/80 binding and repair machinery is ensured by multiple proteins, the 53BP1 and CtIP being the major players. CtIP phosphorylation is essential for MRE11 endonuclease activity initiation and BRCA1 binding, whereas 53BP1

suppresses resection and promotes NHEJ [START_REF] Anand | Phosphorylated CtIP Functions as a Co-factor of the MRE11-RAD50-NBS1 Endonuclease in DNA End Resection[END_REF][START_REF] Bunting | 53BP1 inhibits homologous recombination in brca1-deficient cells by blocking resection of DNA breaks[END_REF]Jachimowicz, Goergens, et al., 2019;[START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF][START_REF] Xie | Distinct Roles of Chromatin-Associated Proteins MDC1 and 53BP1 in Mammalian Double-Strand Break Repair[END_REF]; X. [START_REF] Yu | DNA Damage-Induced Cell Cycle Checkpoint Control Requires CtIP, a Phosphorylation-Dependent Binding Partner of BRCA1 C-Terminal Domains[END_REF]). UBQLN4 (Ubiquilin-4) promotes MRE11

proteasomal degradation and DYNLL1 (Dynein light chain 1) blocks its function via direct interaction, therefore inhibiting HR activation [START_REF] He | DYNLL1 binds to MRE11 to limit DNA end resection in BRCA1-deficient cells[END_REF][START_REF] Jachimowicz | UBQLN4 Represses Homologous Recombination and Is Overexpressed in Aggressive Tumors[END_REF]Jachimowicz, Goergens, et al., 2019). 53BP1 acts in cooperation with its interactor PTIP (PAX transactivation activation domain-interacting protein) and effectors RIF1 and REV7 (Rev7-like terminal deoxycytidyl transferase) [START_REF] Bunting | 53BP1 inhibits homologous recombination in brca1-deficient cells by blocking resection of DNA breaks[END_REF][START_REF] Escribano-Díaz | A Cell Cycle-Dependent Regulatory Circuit Composed of 53BP1-RIF1 and BRCA1-CtIP Controls DNA Repair Pathway Choice[END_REF][START_REF] Feng | RIF1 counteracts BRCA1-mediated end resection during DNA repair[END_REF]Jachimowicz, Goergens, et al., 2019;[START_REF] Munoz | Phospho-epitope binding by the BRCT domains of hPTIP controls multiple aspects of the cellular response to DNA damage[END_REF][START_REF] Wang | PTIP associates with artemis to dictate DNA repair pathway choice[END_REF][START_REF] Xu | REV7 counteracts DNA double-strand break resection and affects PARP inhibition[END_REF][START_REF] Zimmermann | 53BP1 Regulates DSB Repair Using[END_REF]. PTIP directly interacts with Artemis and ensures its retention at the break site (Jachimowicz, Goergens, et al., 2019;[START_REF] Wang | PTIP associates with artemis to dictate DNA repair pathway choice[END_REF]. The mechanism by which the 53BP1-RIF1-REV7 pathway promotes NHEJ was recently identified to be via the Shieldin complex, which consists of REV7, SHLD1, SHLD2, and SHLD3 (Shieldin complex subunit 1-3). This complex is recruited to break sites in 53BP1 and RIF1 dependent way and protects them from extensive resection. The loss of its components leads to PARP inhibition resistance in cancers due to HR reinstatement [START_REF] Findlay | SHLD 2/ FAM 35A co-operates with REV 7 to coordinate DNA double-strand break repair pathway choice[END_REF][START_REF] Ghezraoui | 53BP1 cooperation with the REV7-shieldin complex underpins DNA structure-specific NHEJ[END_REF][START_REF] Gupta | DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity[END_REF]Jachimowicz, Goergens, et al., 2019;[START_REF] Noordermeer | The shieldin complex mediates 53BP1-dependent DNA repair[END_REF][START_REF] Tomida | FAM 35A associates with REV 7 and modulates DNA damage responses of normal and BRCA 1-defective cells[END_REF]. It has also been proposed that it fills in ssDNA ends to enable them for NHEJ [START_REF] Mirman | 53BP1-RIF1-shieldin counteracts DSB resection through CSTand Polα-dependent fill-in[END_REF][START_REF] Scully | DNA double-strand break repair-pathway choice in somatic mammalian cells[END_REF]. REV7 and SHLD3 have been shown

to inhibit CtIP-dependent resection and therefore represses HR [START_REF] Gupta | DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity[END_REF]Jachimowicz, Goergens, et al., 2019;[START_REF] Xu | REV7 counteracts DNA double-strand break resection and affects PARP inhibition[END_REF].

Misregulation of the repair pathway choice leads to severe consequences, including various syndromes and cancer (Jachimowicz, Goergens, et al., 2019;[START_REF] Stewart | The RIDDLE Syndrome Protein Mediates a Ubiquitin-Dependent Signaling Cascade at Sites of DNA Damage[END_REF][START_REF] Terabayashi | Genome instability syndromes caused by impaired DNA repair and aberrant DNA damage responses[END_REF].

Chromatin

DNA organization in eukaryotic cells is organized in chromatin, which is an intricate nucleoprotein structure.

Studies on chromatin began as early as the 1880s when Flemming in his experiments on cellular division discovered an easily-stainable structure that he named 'chromatin' and Miescher, Kossel, and Hoppe- Seylercontinued found that this substance consists of nucleic acid and proteins that were termed 'histones'

and continued in increasing intensity and details ever since (D. E. [START_REF] Olins | Olins-2003-Chromatin-history-our-view-from-the[END_REF][START_REF] Paweletz | Walther Flemming: Pioneer of mitosis research[END_REF]. After many years of elaborate exploration and major discoveries that included identification of DNA as a carrier of inherited information and deciphering of its structure, chromatin research came to a new level in 1960s when the chromosome structure, as well as the role of histone modifications, were defined [START_REF] Allfrey | Acetylation and Methylation of Histones and Their Possible Role in the[END_REF][START_REF] Avery | Studies on the Chemical Nature of the Substance Inducing Transformation of Pneumococcal Types[END_REF][START_REF] Gall | Chromosome Fibers from an Interphase Nucleus[END_REF][START_REF] Olins | Olins-2003-Chromatin-history-our-view-from-the[END_REF][START_REF] Watson | Molecular Structure of Nucleic Acids. A Strucrure for Deoxyribose Nucleic Acid[END_REF]. Another key advance was achieved in the 1970s by identifying and characterizing a nucleosome as a basic chromatin subunit (A.

L. [START_REF] Olins | Spheroid chromatin units (v bodies)[END_REF][START_REF] Olins | Olins-2003-Chromatin-history-our-view-from-the[END_REF][START_REF] Oudet | Electron Microscopic and Biochemical that Chromatin Structure Is a Repeating Unit[END_REF]. Since that time our understanding of both the principles and the importance of chromatin organization has been steadily increasing.

Chromatin structure is extremely complex and includes several levels of organization. Each of these levels represents an additional mean for modulating the use of inherited information as long as its structure is in order. However, they also pose additional obstacles for maintaining it or modifying it in the desired way.

First, DNA is wrapped around histone octamers to form "beads on a string" structure, with H1 histone bound to nucleosome-free parts [START_REF] Allan | The structure of histone H1 and its location in chromatin[END_REF][START_REF] Baldi | Beads on a string-nucleosome array arrangements and folding of the chromatin fiber[END_REF][START_REF] Felsenfeld | Controlling the double helix[END_REF][START_REF] Fyodorov | Emerging roles of linker histones in regulating chromatin structure and function[END_REF][START_REF] Kornberg | Chromatin Structure : A Repeating Unit of Histones and DNA Chromatin structure is based on a repeating unit of eight[END_REF][START_REF] Woodcock | Structural repeating units in chromatin. I. Evidence for their general occurrence[END_REF]. Each nucleosome represents a globular structure with two copies of each histone, H2A, H2B, H3, and H4, and 174 base pair-long DNA is wrapped around it [START_REF] Arents | Topography of the histone octamer surface: Repeating structural motifs utilized in the docking of nucleosomal DNA[END_REF][START_REF] Fyodorov | Emerging roles of linker histones in regulating chromatin structure and function[END_REF][START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 Å resolution[END_REF]. However, first three of the abovementioned histones, as well as a linker histone H1, have several known structural variances, and each of the core histones can be chemically modified, so this structure represents a broad layer of regulation [START_REF] Biterge | Histone variants: Key players of chromatin[END_REF][START_REF] Marzluff | The Human and Mouse Replication-Dependent Histone Genes[END_REF]. The abovementioned H2AX variant is of the utmost interest in relation to DNA repair, but other histone variants play an equally important role in other aspects of cellular homeostasis. For instance, H3 variant CENPA is a hallmark of a centromere and is essential for a kinetochore complex assembly, and H2A.Z and H3.3 are predominantly found in transcriptionally active chromatin whereas macro H2A is linked to a repressed chromatin state [START_REF] Biterge | Histone variants: Key players of chromatin[END_REF][START_REF] Chakravarthy | The histone variant macro-H2A preferentially forms "hybrid nucleosomes[END_REF][START_REF] Rogakou | DNA double-stranded breaks induce histone H2AX phosphorylation on serine 139[END_REF][START_REF] Thakar | H2A.Z and H3.3 histone variants affect nucleosome structure: Biochemical and biophysical studies[END_REF][START_REF] Yoda | Human centromere protein A (CENP-A) can replace histone H3 in nucleosome reconstitution in vitro[END_REF].

Posttranslational modification of histone tails has long been known to play an important role in chromatin state regulation [START_REF] Allfrey | Acetylation and Methylation of Histones and Their Possible Role in the[END_REF][START_REF] Allis | The molecular hallmarks of epigenetic control[END_REF][START_REF] Hebbes | Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken β-globin chromosomal domain[END_REF][START_REF] Jeppesen | The inactive X chromosome in female mammals is distinguished by a lack of histone H4 acetylation, a cytogenetic marker for gene expression[END_REF][START_REF] Kayne | Extremely conserved histone H4 N terminus is dispensable for growth but essential for repressing the silent mating loci in yeast[END_REF]. It has been shown that interaction of histone octamer has an ionic nature and is influenced by the charge, therefore those modifications that affect protein charge either by being charged such as acetylation of lysine or by affecting a residue they bind to. This allows DNA accessibility modulation simply at a level of physical interactions [START_REF] Davey | Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 Å resolution[END_REF][START_REF] Korolev | The polyelectrolyte properties of chromatin[END_REF][START_REF] North | Regulation of the nucleosome unwrapping rate controls DNA accessibility[END_REF][START_REF] Shimko | Preparation of fully synthetic histone H3 reveals that acetyl-lysine 56 facilitates protein binding within nucleosomes[END_REF][START_REF] Simon | Histone fold modifications control nucleosome unwrapping and disassembly[END_REF][START_REF] Zhou | Nucleosome structure and dynamics are coming of age[END_REF]. To add more complexity, each modification can promote or inhibit interaction with multiple proteins, which underlies multiple processes in the nucleus such as the abovementioned H2AX phosphorylation that targets all downstream repair machinery or heterochromatic domain formation with H3K9me3-mediated recruitment of HP1 (Heterochromatin protein 1) proteins. The principles of chromatin modifications influence on the biological outcome were formulated as "histone code hypothesis" which was further expanded into the "epigenetic code" theory. It postulates that epigenetic landscape features, which are created by specialized enzymes ("writers" that introduce modifications and "erasers" that remove them), are recognized by an array of dedicated proteins ("readers") in order to control biological functions [START_REF] Allis | The molecular hallmarks of epigenetic control[END_REF][START_REF] Jenuwein | Translating the histone code[END_REF][START_REF] Strahl | The language of covalent histone modifications[END_REF].

For many years the consensus was that "beads on a string" structures further fold into 30 nm fibers [START_REF] Fyodorov | Emerging roles of linker histones in regulating chromatin structure and function[END_REF][START_REF] Ghirlando | Hydrodynamic Studies on Defined Heterochromatin Fragments Support a 30nm Fiber Having Six Nucleosomes per Turn[END_REF]. However, recent studies using super-resolution microscopy have shown that in vivo nucleosomes do not form a regular fiber but are arranged into heterogeneous clusters of various sizes [START_REF] Fyodorov | Emerging roles of linker histones in regulating chromatin structure and function[END_REF][START_REF] Ou | ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells[END_REF][START_REF] Ricci | Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo[END_REF]. This is in accordance with another recent study where relaxed nucleosome zigzag chains rather than 30 nm fibers were observed in vivo using electron microscopy [START_REF] Fyodorov | Emerging roles of linker histones in regulating chromatin structure and function[END_REF][START_REF] Grigoryev | Hierarchical looping of zigzag nucleosome chains in metaphase chromosomes[END_REF].

Organized or not, chromatin fibers are further looped into topologically associated domains (TADs). TADs represent chromatin regions with a high frequency of interactions as demonstrated by Hi-C experiments [START_REF] Bonev | Organization and function of the 3D genome[END_REF][START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Nora | Spatial partitioning of the regulatory landscape of the X-inactivation centre[END_REF][START_REF] Sexton | Three-dimensional folding and functional organization principles of the Drosophila genome[END_REF]. It has been shown that TADs are correlating with many genomic features such as transcription or chromatin marks, and that promoter-enhancer interactions are mostly occurring within a TAD [START_REF] Bonev | Organization and function of the 3D genome[END_REF][START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Shen | A map of the cis-regulatory sequences in the mouse genome[END_REF]. They are separated by boundaries established by architectural proteins, the most prominent of which are CTCF (CCCTC-binding factor) and Cohesin [START_REF] Bonev | Organization and function of the 3D genome[END_REF][START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. In mammalian cells, TADs have a complex structure containing smaller domains called subTADs [START_REF] Bonev | Organization and function of the 3D genome[END_REF][START_REF] Phillips-Cremins | Architectural protein subclasses shape 3D organization of genomes during lineage commitment[END_REF][START_REF] Wijchers | Cause and Consequence of Tethering a SubTAD to Different Nuclear Compartments[END_REF]. Two chromatin compartments have been specified based on inter-TAD interactions, active A compartment and inactive B compartment [START_REF] Bonev | Organization and function of the 3D genome[END_REF][START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF]. However, it has later been shown that these compartments can be further divided into subtypes [START_REF] Bonev | Organization and function of the 3D genome[END_REF]S. Wang et al., 2016;[START_REF] Wijchers | Cause and Consequence of Tethering a SubTAD to Different Nuclear Compartments[END_REF]. TADs were shown to be predominantly conserved between cell types [START_REF] Bonev | Organization and function of the 3D genome[END_REF][START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Rao | A 3D map of the human genome at kilobase resolution reveals principles of chromatin looping[END_REF]. However, this is not the case regarding compartments, which tend to switch in a cell type-specific manner [START_REF] Bonev | Organization and function of the 3D genome[END_REF][START_REF] Dixon | Chromatin architecture reorganization during stem cell differentiation[END_REF][START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF]. Other well-defined domains, such as lamina-associated domains (LADs), nucleolusassociated domains, pericentromere-associated domains as well as the nucleolus itself and chromocenters can be defined within the nucleus [START_REF] Guelen | Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions[END_REF][START_REF] Németh | Initial genomics of the human nucleolus[END_REF][START_REF] Solovei | How to rule the nucleus: divide et impera[END_REF][START_REF] Wijchers | Characterization and dynamics of pericentromere-associated domains in mice[END_REF]. Finally, at larger scale chromatin is organized into chromosome territories [START_REF] Bonev | Organization and function of the 3D genome[END_REF][START_REF] Lieberman-Aiden | Comprehensive mapping of long-range interactions reveals folding principles of the human genome[END_REF][START_REF] Dc | Delineation of individual human chromosomes in metaphase and interphase cells by in situ suppression hybridization using recombinant DNA libraries[END_REF][START_REF] Pinkel | Fluorescence in situ hybridization with human chromosome-specific libraries: Detection of trisomy 21 and translocations of chromosome 4[END_REF]. Such a complex structure obviously leads to a complicated hierarchy of interactions and interconnections but in the simplest and the most intuitive classification would be discerning active and inactive parts, accessible and not accessible. And this, with a huge simplification, is a division to euchromatin and heterochromatin.

Euchromatin and heterochromatin

Euchromatin is gene-rich, transcriptionally active, and belongs to early replicating parts of a genome. As a contrast, heterochromatin is transcriptionally inactive, gene-poor, and late-replicating [START_REF] Dileep | Single-cell replication profiling to measure stochastic variation in mammalian replication timing[END_REF][START_REF] Hildebrand | Mechanisms and Functions of Chromosome Compartmentalization[END_REF][START_REF] Liu | Establishment and evolution of heterochromatin[END_REF][START_REF] Passarge | Emil Heitz and the concept of heterochromatin: Longitudinal chromosome differentiation was recognized fifty years ago[END_REF][START_REF] Schultz | The Relation of a Dominant Eye Color in Drosophila Melanogaster to the Associated Chromosome Rearrangement[END_REF][START_REF] Solovei | How to rule the nucleus: divide et impera[END_REF][START_REF] Wakimoto | The effects of chromosome rearrangements on the expression of heterochromatic genes in chromosome 2L of Drosophila melanogaster[END_REF]. Nucleosomes within euchromatin carry post-translational histone modifications (histone marks) characteristic for transcription [START_REF] Hildebrand | Mechanisms and Functions of Chromosome Compartmentalization[END_REF][START_REF] Lawrence | Lateral Thinking: How Histone Modifications Regulate Gene Expression[END_REF]. Prominent among these marks are H4K16ac that enhances transcription, H3K4me3 that marks active TSS (transcription start sites), H3K36me3 that is present throughout the hole actively transcribed region, H3K27ac that is a part of active enhancers and directly opposes establishing a repressive H3K27me3 modification, H3K9ac that marks active promoters and many others [START_REF] Akhtar | Chromodomains are protein-RNA interaction modules[END_REF][START_REF] Bannister | Spatial distribution of di-and tri-methyl lysine 36 of histone H3 at active genes[END_REF][START_REF] Bannister | Regulation of chromatin by histone modifications[END_REF][START_REF] Creyghton | Histone H3K27ac separates active from poised enhancers and predicts developmental state[END_REF][START_REF] Karmodiya | H3K9 and H3K14 acetylation cooccur at many gene regulatory elements, while H3K14ac marks a subset of inactive inducible promoters in mouse embryonic stem cells[END_REF][START_REF] Lawrence | Lateral Thinking: How Histone Modifications Regulate Gene Expression[END_REF] EZH2 (Enhancer of zeste homolog 2) [START_REF] Kuzmichev | Histone methyltransferase activity associated with a human multiprotein complex containing the enhancer of zeste protein[END_REF][START_REF] Simon | Occupying Chromatin: Polycomb Mechanisms for Getting to Genomic Targets, Stopping Transcriptional Traffic, and Staying Put[END_REF]. Once marked, facultative heterochromatic regions are recognised and bound by PRC1 or PRC2 complex to maintain transcriptional repression [START_REF] Boyer | Polycomb complexes repress developmental regulators in murine embryonic stem cells[END_REF][START_REF] Bracken | Genome-wide mapping of polycomb target genes unravels their roles in cell fate transitions[END_REF][START_REF] Cao | Role of histone H3 lysine 27 methylation in polycomb-group silencing[END_REF]; T. I. [START_REF] Lee | Control of Developmental Regulators by Polycomb in Human Embryonic Stem Cells[END_REF][START_REF] Wang | Role of histone H2A ubiquitination in Polycomb silencing[END_REF][START_REF] Wiles | H3K27 methylation: a promiscuous repressive chromatin mark[END_REF]. Polycomb complexes play a crucial role in establishing and maintaining expression and silencing patterns in the course of development, and either their loss or malfunction leads to developmental abnormalities or cancer [START_REF] Akasaka | A role for mel-18, a Polycomb group-related vertebrate gene, during the anteroposterior specification of the axial skeleton[END_REF][START_REF] Ben-Porath | An embryonic stem cell-like gene expression signature in poorly differentiated aggressive human tumors[END_REF][START_REF] Bracken | Genome-wide mapping of polycomb target genes unravels their roles in cell fate transitions[END_REF][START_REF] Conway | PRC2 mediated H3K27 methylations in cellular identity and cancer[END_REF][START_REF] Nikoloski | Somatic mutations of the histone methyltransferase gene EZH2 in myelodysplastic syndromes[END_REF][START_REF] Ohm | Stem cell chromatin patterns: An instructive mechanism for DNA hypermethylation?[END_REF][START_REF] Sneeringer | Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas[END_REF].

And finally, LADs are the most repressed part of a genome. They are known to be strictly repressive for transcription, and were proven to be physically compacted [START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Ou | ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells[END_REF][START_REF] Reddy | Transcriptional repression mediated by repositioning of genes to the nuclear lamina[END_REF][START_REF] Solovei | How to rule the nucleus: divide et impera[END_REF]. LADs are enriched for repressive chromatin marks like H3K9me2/3 and H3K27me3 and directly interact with nuclear lamins [START_REF] Guelen | Domain organization of human chromosomes revealed by mapping of nuclear lamina interactions[END_REF][START_REF] Harr | Directed targeting of chromatin to the nuclear lamina is mediated by chromatin state and A-type lamins[END_REF][START_REF] Kind | Single-cell dynamics of genome-nuclear lamina interactions[END_REF][START_REF] Lochs | Lamina Associated Domains and Gene Regulation in Development and Cancer[END_REF][START_REF] Meuleman | Constitutive nuclear lamina-genome interactions are highly conserved and associated with A/T-rich sequence[END_REF][START_REF] Solovei | How to rule the nucleus: divide et impera[END_REF][START_REF] Wu | Identifying novel transcriptional and epigenetic features of nuclear lamina-associated genes[END_REF].

All these levels of the organization are interconnected and influence all processes that take place in the nucleus, including DNA repair.

DNA repair in the chromatin context

It has been shown multiple times by many researchers that chromatin context surrounding a break site has a strong influence over the flow of repair.

Chromatin sensitively responds to DNA damage. DDR activation leads to immediate chromatin relaxation that results from PARP1 activity and chromatin remodelers [START_REF] Burgess | Activation of DNA Damage Response Signaling by Condensed Chromatin[END_REF][START_REF] Clouaire | A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks[END_REF][START_REF] Luijsterburg | PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining[END_REF][START_REF] Sellou | The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage[END_REF][START_REF] Smith | CHD3 and CHD4 recruitment and chromatin remodeling activity at DNA breaks is promoted by early poly(ADP-ribose)-dependent chromatin relaxation[END_REF]. However, this relaxation is only transient, it is followed by an ATM-dependent re-condensation, and this re-condensation is crucial for normal DDR propagation [START_REF] Burgess | Activation of DNA Damage Response Signaling by Condensed Chromatin[END_REF][START_REF] Clouaire | A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks[END_REF]. Even the most prominent DNA damage marker, gH2AX, is itself a chromatin modification, and therefore chromatin is an effector for DDR. Genome-wide studies of chromatin response to double-strand break introduction revealed a plethora of induced chromatin changes, some local and some ranging megabases [START_REF] Clouaire | Comprehensive Mapping of Histone Modifications at DNA Double-Strand Breaks Deciphers Repair Pathway Chromatin Signatures[END_REF][START_REF] Clouaire | A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks[END_REF]. Large-scale modifications are common for all mechanisms of repair include gH2AX foci formation (discussed above) linked with de novo H2AX deposition and the loss of H1 [START_REF] Clouaire | A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks[END_REF][START_REF] Piquet | The Histone Chaperone FACT Coordinates H2A.X-Dependent Signaling and Repair of DNA Damage[END_REF][START_REF] Sellou | The poly(ADP-ribose)-dependent chromatin remodeler Alc1 induces local chromatin relaxation upon DNA damage[END_REF][START_REF] Strickfaden | Poly(ADP-ribosyl)ation-dependent transient chromatin decondensation and histone displacement following laser microirradiation[END_REF]. Conversely, local chromatin changes are repair pathway specific [START_REF] Clouaire | A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks[END_REF]. H3.3 histone variant deposition, were reported to contribute to Ku and XRCC4 recruitment and thus promote NHEJ [START_REF] Clouaire | A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks[END_REF][START_REF] Luijsterburg | PARP1 Links CHD2-Mediated Chromatin Expansion and H3.3 Deposition to DNA Repair by Non-homologous End-Joining[END_REF].

H2A.Z loading also plays a role in the Ku70 deposition albeit it has to be removed later in order for repair the process to proceed [START_REF] Clouaire | A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks[END_REF][START_REF] Gursoy-Yuzugullu | Correction for "Histone chaperone Anp32e removes H2A.Z from DNA double-strand breaks and promotes nucleosome reorganization and DNA repair[END_REF]Y. Xu et al., 2012) Uckelmann & Sixma, 2017). H2AK15 and H4K16 acetylation performed by TIP60 has been shown to promote resection [START_REF] Clouaire | A Snapshot on the Cis Chromatin Response to DNA Double-Strand Breaks[END_REF][START_REF] Jacquet | The TIP60 Complex Regulates Bivalent Chromatin Recognition by 53BP1 through Direct H4K20me Binding and H2AK15 Acetylation[END_REF][START_REF] Tang | Acetylation limits 53BP1 association with damaged chromatin to promote homologous recombination[END_REF].

However, not only is the chromatin environment affected DNA damage, but it can also influence the way this damage would be repaired. It has been proposed that euchromatic regions are more prone to HR [START_REF] Aymard | Transcriptionally active chromatin recruits homologous recombination at DNA double-strand breaks[END_REF][START_REF] Mitrentsi | How to maintain the genome in nuclear space[END_REF]. It is achieved by the affinity of HR promoting proteins, namely BRCA1 and CtIP interactor LEDGF (Lens epithelium-derived growth factor), to active chromatin marks [START_REF] Daugaard | LEDGF (p75) promotes DNA-end resection and homologous recombination[END_REF][START_REF] Mitrentsi | How to maintain the genome in nuclear space[END_REF][START_REF] Tamburini | Localized Histone Acetylation and Deacetylation Triggered by the Homologous Recombination Pathway of Double-Strand DNA Repair[END_REF][START_REF] Wilson | Reading chromatin signatures after DNA double-strand breaks[END_REF]. It has also been shown that proteins involved in R-loops resolution can recruit RAD51 and BRCA2 [START_REF] Cohen | Senataxin resolves RNA:DNA hybrids forming at DNA double-strand breaks to prevent translocations[END_REF][START_REF] D'alessandro | BRCA2 controls DNA:RNA hybrid level at DSBs by mediating RNase H2 recruitment[END_REF][START_REF] Mitrentsi | How to maintain the genome in nuclear space[END_REF]. It has even been suggested that if DSBs are acquired at transcriptionally active regions during G1 when HR cannot be engaged they are held unrepaired until S or G2 phase [START_REF] Aymard | Genome-wide mapping of long-range contacts unveils clustering of DNA double-strand breaks at damaged active genes[END_REF][START_REF] Mitrentsi | How to maintain the genome in nuclear space[END_REF]. Another hypothesis implies the use of RNA as a template for DSB repair [START_REF] Meers | DNA repair by RNA: Templated, or not templated, that is the question[END_REF][START_REF] Mitrentsi | How to maintain the genome in nuclear space[END_REF]. At the same time, heterochromatin has been shown to be not a very HR-friendly environment and rather promotes NHEJ [START_REF] Kalousi | Nuclear compartmentalization of DNA repair[END_REF][START_REF] Pfister | SETD2-Dependent Histone H3K36 Trimethylation Is Required for Homologous Recombination Repair and Genome Stability[END_REF]. Despite its compaction might represent a barrier to repair factor recruitment, it is unclear whether heterochromatin proteins such as HP1 are dismissed or the repair process can proceed without it [START_REF] Kalousi | The nuclear oncogene SET controls DNA repair by KAP1 and HP1 retention to chromatin[END_REF][START_REF] Mitrentsi | How to maintain the genome in nuclear space[END_REF][START_REF] Noon | 53BP1-dependent robust localized KAP-1 phosphorylation is essential for heterochromatic DNA double-strand break repair[END_REF][START_REF] Tsouroula | Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin[END_REF]. Some studies even show an increase of H3K9me3 in close proximity to DSBs [START_REF] Ayrapetov | DNA double-strand breaks promote methylation of histone H3 on lysine 9 and transient formation of repressive chromatin[END_REF][START_REF] Mitrentsi | How to maintain the genome in nuclear space[END_REF]. It was shown that LADs repress HR and a double-strand break that occurs there could be repaired by other pathways, even though more mutagenic, because in this case MMEJ became an alternative for NHEJ, but HR is inhibited in this nuclear compartment. At the same time, breaks around nuclear pores that also reside at the nuclear periphery but are surrounded by transcriptionally active chromatin, are capable of utilizing HR normally [START_REF] Kalousi | Nuclear compartmentalization of DNA repair[END_REF][START_REF] Lemaître | Nuclear position dictates DNA repair pathway choice[END_REF]. It has also been shown that when a break occurs in heterochromatinised chromocenters it can only be repaired by HR at the periphery of a chromocenter, thus escaping a heterochromatic environment [START_REF] Kalousi | Nuclear compartmentalization of DNA repair[END_REF][START_REF] Tsouroula | Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin[END_REF].

However, facultative heterochromatin is relatively poorly investigated in the context of DNA repair.

Bivalency

Another type of chromatin that has never to my knowledge been investigated in the context of DNA repair is bivalent chromatin. Generally, any kind of state that combines active and repressive chromatin marks is bivalent chromatin. To date, these include a simultaneous presence of H3K4me3 and H3K27me3, H3K4me3

and H3K9me3, H3K36me2/3 and H3K9me3, or a combination of H3K9acK14ac, H3K4me3 and H3K27me3 [START_REF] Azuara | Chromatin signatures of pluripotent cell lines[END_REF][START_REF] Bernstein | A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells[END_REF]Harikumar & Meshorer, 2015;[START_REF] Matsumura | H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation[END_REF][START_REF] Mauser | Application of dual reading domains as novel reagents in chromatin biology reveals a new H3K9me3 and H3K36me2/3 bivalent chromatin state[END_REF][START_REF] Roh | The genomic landscape of histone modifications in human T cells[END_REF][START_REF] Vakoc | Histone H3 lysine 9 methylation and HP1γ are associated with transcription elongation through mammalian chromatin[END_REF]. But the first kind of such chromatin state that has been observed (currently termed 'classic' bivalent domains) is a combination of H3K4me3 and H3K27 me3. They were initially found in mouse embryonic stem (ES) cells in culture and for the first several years it was rather common to consider them a culture artifact [START_REF] Azuara | Chromatin signatures of pluripotent cell lines[END_REF][START_REF] Bernstein | A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells[END_REF]Harikumar & Meshorer, 2015). However, later bivalent domains has been observed in other cell types, and even in vivo during development and in cancer [START_REF] Béguelin | EZH2 and BCL6 Cooperate to Assemble CBX8-BCOR Complex to Repress Bivalent Promoters, Mediate Germinal Center Formation and Lymphomagenesis[END_REF][START_REF] Kampilafkos | Epigenetic modifications in cutaneous malignant melanoma: EZH2, H3K4me2, and H3K27me3 immunohistochemical expression is enhanced at the invasion front of the tumor[END_REF][START_REF] Matsumura | H3K4/H3K9me3 Bivalent Chromatin Domains Targeted by Lineage-Specific DNA Methylation Pauses Adipocyte Differentiation[END_REF][START_REF] Minoux | Gene bivalency at Polycomb domains regulates cranial neural crest positional identity[END_REF][START_REF] Sachs | Bivalent Chromatin Marks Developmental Regulatory Genes in the Mouse Embryonic Germline InVivo[END_REF][START_REF] Sin | Poised chromatin and bivalent domains facilitate the mitosis-to-meiosis transition in the male germline[END_REF]. In the course of differentiation, one of the marks is normally lost from bivalent promoters [START_REF] Bernstein | A Bivalent Chromatin Structure Marks Key Developmental Genes in Embryonic Stem Cells[END_REF]. Another question was addressed, whether real bivalency exists, it is only resulted from a heterogeneity of cells in the population or from the close proximity of two differentially marked genomic region. However, the existence of bivalency has later been proven by genome-wide assessing individual bivalent nucleosomes. Sequential immunoprecipitation of single nucleosomes confirmed that both marks can coexist on the same nucleosome, although on different histone H3 tails [START_REF] Sen | Genome-wide positioning of bivalent mononucleosomes[END_REF].

A currently accepted explanation of this phenomenon is based on the observation that many of the bivalently marked regions found in ES cells are promoters of developmental genes. The hypothesis is that such a chromatin state is needed to enable rapid activation or complete inactivation of a respective gene (Harikumar & Meshorer, 2015). However, RNA polymerase II (RNAPII) pausing was not observed on these promoters in accordance with RNAPII association with actively transcribed and not bivalent genes [START_REF] Mantsoki | Dynamics of promoter bivalency and RNAP II pausing in mouse stem and differentiated cells[END_REF]. Additionally, it has also been shown that bivalency is linked to the cell cycle. MLL2

(Myeloid/lymphoid or mixed-lineage leukemia protein 2), as a part HMT (Histone methyltransferases) complex, establishes H3K4me3 at developmental genes and thus real bivalency, and during the rest of the cell cycle genes are robustly repressed [START_REF] Singh | Cell-Cycle Control of Bivalent Epigenetic Domains Regulates the Exit from Pluripotency[END_REF]. According to other studies, exit from pluripotency accrues in a cell cycle-dependent manner, so these two studies go in line [START_REF] Boward | Concise Review: Control of Cell Fate Through Cell Cycle and Pluripotency Networks[END_REF][START_REF] Pauklin | XThe cell-cycle state of stem cells determines cell fate propensity[END_REF].

This chromatin type has not been studied extensively yet and the best model for it are embryonic stem cells where this type of chromatin is rather abundant.

Embryonic stem cells

Stem cells are defined by the ability of both self-renewal and differentiation. There are three classes of stem cells (although the last one is sometimes divided into two). The first one, totipotent stem cells, are capable of differentiation into any type of adult, embryonic, or extraembryonic tissue. The second one, pluripotent stem cells, keeps the potential and can give rise to any adult or embryonic cell type but not some of the extraembryonic tissues. Finally, multipotent stem cells are tissue-specific and usually reside in mouse embryonic stem cells. Unipotent stem cells, that are sometimes considered separately from multipotent ones, can only differentiate on one cell type [START_REF] Alison | Attributes of adult stem cells[END_REF]. Embryonic stem (ES) cells represent pluripotent stem cells derived from a preimplantation blastocyst [START_REF] Martello | The Nature of Embryonic Stem Cells[END_REF]. Mouse ES cells were first obtained by Evans and Kaufman in 1981 and until now stay a very attractive research model and potentially powerful therapeutic tool as well as a fascinating research subject [START_REF] Evans | Establishment in culture of pluripotential cells from mouse embryos[END_REF].

The pluripotent state is maintained by a regulatory network of transcription factors. The core factors are Oct4 (Octamer-binding protein 4), Sox2 (SRY-box 2), and Nanog, and their key role has been proven many times both by demonstrating their importance for stemness and viability and by their capacity to initiate somatic cell reprogramming [START_REF] Kehler | Oct4 is required for primordial germ cell survival[END_REF][START_REF] Li | Deconstructing the pluripotency gene regulatory network[END_REF][START_REF] Mitsui | The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[END_REF][START_REF] Nichols | Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4[END_REF][START_REF] Takahashi | Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors[END_REF]. Oct4 and Sox2 form heterodimers and induce numerous targets including their own genes [START_REF] Chew | Reciprocal Transcriptional Regulation of Pou5f1 and Sox2 via the Oct4/Sox2 Complex in Embryonic Stem Cells[END_REF][START_REF] Li | Deconstructing the pluripotency gene regulatory network[END_REF][START_REF] Rodda | Transcriptional regulation of Nanog by OCT4 and SOX2[END_REF]. However, change of expression levels or interaction partners of Oct4 and Sox2 drive mesendodermal or ectodermal differentiation respectively [START_REF] Avilion | Multipotent cell lineages in early mouse development depend on SOX2 function[END_REF][START_REF] Ferri | Sox2 deficiency causes neurodegeneration and impaired neurogenesis in the adult mouse brain[END_REF][START_REF] Li | Deconstructing the pluripotency gene regulatory network[END_REF][START_REF] Niwa | Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells[END_REF][START_REF] Thomson | Pluripotency factors in embryonic stem cells regulate differentiation into germ layers[END_REF]. Nanog is required for maintenance of pluripotency and appear to be the key LIF/STAT3 (Leukemia inhibitory factor 1, Signal transducer and activator of transcription 3) pathway as its overexpression makes LIF redundant [START_REF] Chambers | Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells[END_REF][START_REF] Li | Deconstructing the pluripotency gene regulatory network[END_REF][START_REF] Mitsui | The homeoprotein nanog is required for maintenance of pluripotency in mouse epiblast and ES cells[END_REF]. Together Oct4, Sox2, and Nanog binding cover the majority of pluripotency regulatory network transcription factors (X. Chen et al., 2008;[START_REF] Li | Deconstructing the pluripotency gene regulatory network[END_REF][START_REF] Loh | The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells[END_REF]. Their synergistic action plays a role in both propagating pluripotency by activating transcription of pluripotency-related genes and preventing differentiation by counter-balancing each other and blocking transcription of developmental regulators [START_REF] Boyer | Core transcriptional regulatory circuitry in human embryonic stem cells[END_REF][START_REF] Loh | The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells[END_REF][START_REF] Young | Control of the Embryonic Stem Cell State[END_REF].

Another curious feature of ES cells should be mentioned. They have a distinct cell cycle profile with a very short G1 phase, which is tightly controlled by the pluripotency regulatory network and, in turn, has been shown to suppress differentiation [START_REF] Coronado | A short G1 phase is an intrinsic determinant of naïve embryonic stem cell pluripotency[END_REF][START_REF] Kareta | Crosstalk between stem cell and cell cycle machineries[END_REF][START_REF] Li | Molecular ties between the cell cycle and differentiation in embryonic stem cells[END_REF][START_REF] Savatier | Analysis of the cell cycle in mouse embryonic stem cells[END_REF]. One of the core network targets, Myc (Myc proto-oncogene protein), enhances proliferation by activating transcription of cell-cycle related genes as well as directly represses differentiation by inhibiting FGF/ERK (Fibroblast growth factor, Extracellular signal-regulated kinase)

signaling pathway [START_REF] Cartwright | LIF/STAT3 controls ES cell selfrenewal and pluripotency by a Myc-dependent mechanism[END_REF][START_REF] Chappell | MYC/MAX control ERK signaling and pluripotency by regulation of dual-specificity phosphatases 2 and 7[END_REF][START_REF] Kareta | Crosstalk between stem cell and cell cycle machineries[END_REF][START_REF] Kim | An Extended Transcriptional Network for Pluripotency of Embryonic Stem Cells[END_REF][START_REF] Loh | The Oct4 and Nanog transcription network regulates pluripotency in mouse embryonic stem cells[END_REF]. A collective action of Myc and core pluripotency factors leads to a compromised G1/S cell cycle checkpoint that contributes to G1 shortening [START_REF] Kanai | Oct3/4 directly regulates expression of E2F3a in mouse embryonic stem cells[END_REF][START_REF] Kareta | Crosstalk between stem cell and cell cycle machineries[END_REF][START_REF] Kim | A Myc Network Accounts for Similarities between Embryonic Stem and Cancer Cell Transcription Programs[END_REF][START_REF] Suvorova | G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21waf1 signaling[END_REF][START_REF] Zhang | PALB2 functionally connects the breast cancer susceptibility proteins BRCA1 and BRCA2[END_REF].

Among multiple regulatory measures than ensure stem cell proficiency for pluripotency and differentiation of them unique chromatin organization is a prominent one.

Chromatin in embryonic stem cells

As previously mentioned, one of the prominent features of stem cells is the enrichment for bivalent domains. However, it is not the only unique feature of ES cell chromatin.

Pluripotent stem cells have less compacted chromatin and higher dynamics of interaction with chromatinassociated proteins and increased turnover of a linker histone H1, which most likely results from overexpression of chromatin remodelers. Even the distribution of chromatin in the nucleus is more even in pluripotent than in somatic cells. These features have been observed both in vitro and in vivo [START_REF] Ahmed | Global chromatin architecture reflects pluripotency and lineage commitment in the early mouse embryo[END_REF][START_REF] Azuara | Chromatin signatures of pluripotent cell lines[END_REF][START_REF] Bouwman | Architectural hallmarks of the pluripotent genome[END_REF][START_REF] Efroni | Global Transcription in Pluripotent Embryonic Stem Cells[END_REF][START_REF] Fussner | Constitutive heterochromatin reorganization during somatic cell reprogramming[END_REF][START_REF] Gaspar-Maia | Open chromatin in pluripotency and reprogramming[END_REF]Meshorer et al., 2006;Meshorer & Misteli, 2006;[START_REF] Otterstrom | Superresolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo[END_REF][START_REF] Ricci | Chromatin fibers are formed by heterogeneous groups of nucleosomes in vivo[END_REF][START_REF] Schlesinger | Open Chromatin, Epigenetic Plasticity, and Nuclear Organization in Pluripotency[END_REF]. As a result, basal transcription is also elevated in pluripotent cells [START_REF] Efroni | Global Transcription in Pluripotent Embryonic Stem Cells[END_REF][START_REF] Kobayashi | Epigenetic regulation of open chromatin in pluripotent stem cells[END_REF][START_REF] Ramalho-Santos | Stemness": Transcriptional profiling of embryonic and adult stem cells[END_REF]. This includes exonic, intronic, and non-genic regions, causing a considerable profusion of nascent RNA as compared to differentiated cells [START_REF] Bouwman | Architectural hallmarks of the pluripotent genome[END_REF][START_REF] Efroni | Global Transcription in Pluripotent Embryonic Stem Cells[END_REF]. Reduced levels of heterochromatic histone marks and increased levels of those associated with active transcription characteristic for ES cells fall in line with these observations [START_REF] Efroni | Global Transcription in Pluripotent Embryonic Stem Cells[END_REF][START_REF] Kobayashi | Epigenetic regulation of open chromatin in pluripotent stem cells[END_REF][START_REF] Lee | Histone Deacetylase Activity Is Required for Embryonic Stem Cell Differentiation[END_REF]Meshorer et al., 2006;[START_REF] Schlesinger | Open Chromatin, Epigenetic Plasticity, and Nuclear Organization in Pluripotency[END_REF][START_REF] Wen | Large histone H3 lysine 9 dimethylated chromatin blocks distinguish differentiated from embryonic stem cells[END_REF]. PRC2 complex plays an important role in maintaining pluripotency as in ES cells it is predominantly bound to promoters of genes related to differentiation [START_REF] Boyer | Polycomb complexes repress developmental regulators in murine embryonic stem cells[END_REF][START_REF] Bracken | Genome-wide mapping of polycomb target genes unravels their roles in cell fate transitions[END_REF][START_REF] Denholtz | Long-range chromatin contacts in embryonic stem cells reveal a role for pluripotency factors and Polycomb proteins in genome organization[END_REF][START_REF] Kobayashi | Epigenetic regulation of open chromatin in pluripotent stem cells[END_REF].

Lower levels of DNA methylation, which is largely associated with repressed chromatin state, were observed in ES cells [START_REF] Lehnertz | Suv39h-Mediated Histone H3 Lysine 9 Methylation Directs DNA Methylation to Major Satellite Repeats at Pericentric Heterochromatin[END_REF][START_REF] Marks | The transcriptional and epigenomic foundations of ground state pluripotency[END_REF][START_REF] Schlesinger | Open Chromatin, Epigenetic Plasticity, and Nuclear Organization in Pluripotency[END_REF]. Finally, one of the prominent features observed both in early embryos and in ES cells is a reactivation of an imprinted X chromosome [START_REF] Mak | Reactivation of the Paternal X Chromosome in Early Mouse Embryos[END_REF][START_REF] Okamoto | Epigenetic Dynamics of Imprinted X Inactivation during Early Mouse Development[END_REF][START_REF] Surani | Genetic and Epigenetic Regulators of Pluripotency[END_REF][START_REF] Wutz | A shift from reversible to irreversible X inactivation is triggered during ES cell differentiation[END_REF].

Unraveling a great number of pluripotent stem cell-specific long-range interactions resulted from genome architecture studies [START_REF] Apostolou | Genome-wide chromatin interactions of the nanog locus in pluripotency, differentiation, and reprogramming[END_REF]De Wit et al., 2013;[START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Kobayashi | Epigenetic regulation of open chromatin in pluripotent stem cells[END_REF][START_REF] Wei | Klf4 organizes long-range chromosomal interactions with the OCT4 locus inreprogramming andpluripotency[END_REF]. Moreover, whereas the genome of differentiated cells is predominantly shaped in 3D around repressed heterochromatic regions, in ES cells the main structure-forming entities are hubs of active transcription dependent on core pluripotency factors (de Wit et al., 2013;[START_REF] Dixon | Topological domains in mammalian genomes identified by analysis of chromatin interactions[END_REF][START_REF] Kobayashi | Epigenetic regulation of open chromatin in pluripotent stem cells[END_REF]. These changes are notable even on a whole-nucleus level. For example, chromocenters, prominent in differentiated cells, show higher numbers and are less distinct in embryonic stem cells [START_REF] Aoto | Nuclear and chromatin reorganization in the MHC-Oct3/4 locus at developmental phases of embryonic stem cell differentiation[END_REF][START_REF] Bouwman | Architectural hallmarks of the pluripotent genome[END_REF][START_REF] Mayer | Common themes and cell type specific variations of higher order chromatin arrangements in the mouse[END_REF][START_REF] Schlesinger | Open Chromatin, Epigenetic Plasticity, and Nuclear Organization in Pluripotency[END_REF][START_REF] Wiblin | Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells[END_REF]. Heterochromatin protein HP1b is almost diffused in ES cells but becomes accumulated at heterochromatic foci in the course of differentiation [START_REF] Mattout | Heterochromatin Protein 1β (HP1β) has distinct functions and distinct nuclear distribution in pluripotent versus differentiated cells[END_REF][START_REF] Schlesinger | Open Chromatin, Epigenetic Plasticity, and Nuclear Organization in Pluripotency[END_REF].

Even interactions with nuclear lamina are massively reorganized between ES and differentiated cells [START_REF] Kobayashi | Epigenetic regulation of open chromatin in pluripotent stem cells[END_REF][START_REF] Peric-Hupkes | Molecular Maps of the Reorganization of Genome-Nuclear Lamina Interactions during Differentiation[END_REF]. Nuclear lamina itself lacks Lamin A and is less organized in pluripotent cells [START_REF] Melcer | Histone modifications and lamin A regulate chromatin protein dynamics in early embryonic stem cell differentiation[END_REF][START_REF] Pagliara | Auxetic nuclei in embryonic stem cells exiting pluripotency[END_REF][START_REF] Schlesinger | Open Chromatin, Epigenetic Plasticity, and Nuclear Organization in Pluripotency[END_REF].

All in all, the evidence point that chromatin structure in pluripotent stem cells is distinct and is tightly interconnected with their undifferentiated state.

DNA repair in embryonic stem cells ES cells represent a fascinating model for us not only for having unique chromatin features but also for their special mechanisms for maintaining genome integrity. Similar to other cell types, they are permanently exposed to DNA damaging agents of both exogenous and endogenous origin. However, due to their rapid proliferation (as discussed above) they also undergo constant replication stress. These factors combined lead to constant activation of DDR [START_REF] Ahuja | A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells[END_REF][START_REF] Juan | Cdk12 is essential for embryonic development and the maintenance of genomic stability[END_REF][START_REF] Nagaria | DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity[END_REF][START_REF] Savatier | Analysis of the cell cycle in mouse embryonic stem cells[END_REF]. However, ES cells are highly tolerant of this stress and show a lower level of spontaneously induced mutations and karyotypic aberrations than somatic cells [START_REF] Brimble | Karyotypic Stability, Genotyping, Differentiation, Feeder-Free Maintenance, and Gene Expression Sampling in Three Human Embryonic Stem Cell Lines Derived Prior to August 9, 2001[END_REF][START_REF] Hong | Protecting genomic integrity in somatic cells and embryonic stem cells[END_REF][START_REF] Nagaria | DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity[END_REF][START_REF] Thomson | Embryonic stem cell lines derived from human blastocysts[END_REF]. Enhanced maintenance of genome integrity appears to be a characteristic of pluripotent cells (Y. [START_REF] Hong | Protecting genomic integrity in somatic cells and embryonic stem cells[END_REF][START_REF] Nagaria | DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity[END_REF]. In order to protect the genome of the whole pool stem cells readily sacrifice themselves by initiating apoptosis or differentiation [START_REF] Aladjem | ES cells do 118 not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage[END_REF][START_REF] Hong | Restoration of an absent G1 arrest and protection from apoptosis in embryonic stem cells after ionizing radiation[END_REF][START_REF] Lin | p53 induces differentiation of mouse embryonic stem cells by suppressing Nanog expression[END_REF][START_REF] Liu | Structural mechanism of the phosphorylation-dependent dimerization of the MDC1 forkhead-associated domain[END_REF][START_REF] Nagaria | DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity[END_REF]. Of note, p53 plays a less role in ES cells, particularly it does not activate G1/S or intra-S checkpoint, and processes related DNA damage response and repair are regulated by a stem-cell specific protein Filia [START_REF] Aladjem | ES cells do 118 not activate p53-dependent stress responses and undergo p53-independent apoptosis in response to DNA damage[END_REF][START_REF] Chuykin | Activation of DNA damage response signaling in mouse embryonic stem cells[END_REF][START_REF] Nagaria | DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity[END_REF][START_REF] Suvorova | G1 checkpoint is compromised in mouse ESCs due to functional uncoupling of p53-p21waf1 signaling[END_REF][START_REF] Vanderlaan | High Dub3 Expression in Mouse ESCs Couples the G1/S Checkpoint to Pluripotency[END_REF][START_REF] Vitale | DNA Damage in Stem Cells[END_REF]B. Zhao et al., 2015). It has also been shown that some known pluripotency markers such as Sall4 (Sal-like protein 4) are directly involved in the process of DNA repair. It gets recruited to DSBs and helps to stabilize MRN complex and enhance ATM activation [START_REF] Xiong | Stemness factor Sall4 is required for DNA damage response in embryonic stem cells[END_REF]. ES cells have enriched levels of DNA repair proteins, including HR and NHEJ factors, and enhanced levels of pro-apoptotic factors that are lost upon differentiation [START_REF] Cooper | Co-regulation of pluripotency and genetic integrity at the genomic level[END_REF]. Together with constant activation of DDR, it is capable of sufficiently safeguarding their genome [START_REF] Ahuja | A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells[END_REF]. Mouse ES cells were shown to have a strong preference towards HR and NHEJ pathway in DSB repair [START_REF] Bañuelos | Mouse but not human embryonic stem cells are deficient in rejoining of ionizing radiation-induced DNA double-strand breaks[END_REF][START_REF] Nagaria | DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity[END_REF][START_REF] Serrano | Homologous recombination conserves DNA sequence integrity throughout the cell cycle in embryonic stem cells[END_REF][START_REF] Tichy | Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks[END_REF]. They have been shown to repair 80% of DSBs by HR and only 20% by NHEJ, whereas in somatic cells the ratio is inverted. Alt-EJ was equally both cases, which means a shifted balance towards the precise repair pathway choice [START_REF] Nagaria | DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity[END_REF][START_REF] Serrano | Homologous recombination conserves DNA sequence integrity throughout the cell cycle in embryonic stem cells[END_REF][START_REF] Tichy | Mouse embryonic stem cells, but not somatic cells, predominantly use homologous recombination to repair double-strand DNA breaks[END_REF]. Therefore, it is of no surprise that knockouts of key HR players usually result in embryonic lethality [START_REF] Lim | A mutation in mouse rad51 results in an early embryonic lethal that is suppressed by a mutation in p53[END_REF][START_REF] Nagaria | DNA double-strand break response in stem cells: Mechanisms to maintain genomic integrity[END_REF][START_REF] Tsuzuki | Targeted disruption of the Rad51 gene leads to lethality in embryonic mice[END_REF][START_REF] Xiao | Conditional gene targeted deletion by Cre recombinase demonstrates the requirement for the double-strand break repair Mre11 protein in murine embryonic stem cells[END_REF][START_REF] Zhu | Targeted disruption of the Nijmegen breakage syndrome gene NBS1 leads to early embryonic lethality in mice[END_REF].

Taken together, the presence of unique mechanisms of replications stress and DNA damage tolerance makes ES cells a fascinating model for investigation.

Genome editing

Artificial genome editing in a variety of species has long been an important task for both medical and research purposes. Medical applications include perspectives of gene editing for correction of diseaserelated mutations, disruption of disease-promoting genes or even introducing novel genes (e.g. for sensitising the immune system to tumour cells). The research application range is even broader: it includes creating knock-outs, knock-ins, and introducing mutations to study the role of particular proteins, processes, create disease models, and more.

Given tempting perspectives of practical use, it is of no surprise that the development of methods that would allow gene editing has been going on for a considerable time. At first, the strategy implied the use of random recombination, either to change the sequence or to allow further use of nucleases (such as Cre) [START_REF] Carroll | Genome editing: Past, present, and future[END_REF][START_REF] Rothstein | One-Step Gene Disruption in Yeast[END_REF][START_REF] Scherer | Replacement of chromosome segments with altered DNA sequences constructed in vitro[END_REF][START_REF] Smithies | Insertion of DNA sequences into the locus by homologous recombination[END_REF][START_REF] Thomas | High frequency targeting of genes to specific sites in the mammalian genome[END_REF]. The discovery of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) allowed a considerable advance if the field. However, the largest step has been done with the discovery of CRISPR-Cas9.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) are parts of bacterial mechanisms of antiviral defence [START_REF] Carroll | Genome engineering with targetable nucleases[END_REF][START_REF] Jansen | Identification of genes that are associated with DNA repeats in prokaryotes[END_REF][START_REF] Sorek | CRISPR-mediated adaptive immune systems in bacteria and archaea[END_REF].

Shortly after their discovery, the system was adopted as a tool of genome editing in other organisms, such as plants, fish, human, mice, and many more [START_REF] Carroll | Genome engineering with targetable nucleases[END_REF][START_REF] Cho | Targeted genome engineering in human cells with the Cas9 RNAguided endonuclease[END_REF][START_REF] Hall | Genome Editing in Mice Using CRISPR/Cas9 Technology[END_REF][START_REF] Hwang | Efficient genome editing in zebrafish using a CRISPR-Cas system[END_REF][START_REF] Jinek | A Programmable Dual-RNA -Guided[END_REF][START_REF] Ma | A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants[END_REF].

The editing system consists of Cas9 protein and a short non-coding RNA that contains two parts, a targetspecific CRISPR RNA (crRNA) and a helper trans-activating RNA (tracrRNA) [START_REF] Karvelis | crRNA and tracrRNA guide Cas9-mediated DNA interference in Streptococcus thermophilus[END_REF][START_REF] Memi | CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations[END_REF]. In the guide RNA, tracrRNA component is responsible for binding Cas9 and crRNA acts in sequence recognition by pairing to the target sequence (also called protospacer) [START_REF] Memi | CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations[END_REF][START_REF] Pattanayak | High-throughput profiling of off-target DNA cleavage reveals RNA-programmed Cas9 nuclease specificity[END_REF]. However, Cas9 protein only performs catalytic reaction if a protospacer-adjacent motif (PAM) is present at the 5' side of the protospacer [START_REF] Memi | CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations[END_REF][START_REF] Núñez | DNA damage and prediction of radiation response in lymphocytes and epidermal skin human cells[END_REF]. Later on single guide RNAs containing both crRNA and tracRNA sequences were designed to simplify the practical use [START_REF] Mali | RNA-guided human genome engineering via Cas9[END_REF][START_REF] Memi | CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations[END_REF]. Promising as it is, this system still requires a thorough investigation, for the mechanism of action to be fully understood. Cas9 applications lie beyond genome editing and also include modulation of transcription, epigenetic modifications, genomic imaging, and lineage tracing (W. [START_REF] Deng | CASFISH: CRISPR/Cas9-mediated in situ labeling of genomic loci in fixed cells[END_REF][START_REF] Gilbert | XCRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes[END_REF][START_REF] Hilton | Epigenome editing by a CRISPR-Cas9-based acetyltransferase activates genes from promoters and enhancers[END_REF][START_REF] Spanjaard | Simultaneous lineage tracing and cell-type identification using CrIsPr-Cas9-induced genetic scars[END_REF][START_REF] Wang | CRISPR/Cas9 in Genome Editing and beyond[END_REF].

It is important for the procedures both in research and in clinical applications is that Cas9-mediated genome editing is achieved by introducing a break in a selected location. All the following events leading to repair of this break, either in a desired or in an undesired way, are carried out by the cellular mechanisms of DNA repair. In order to predict a repair outcome, we need an excellent understanding how these mechanisms work.

Goals

In my project, I intended to investigate two different questions.

First, I wanted to elucidate the influence of the pluripotent state on the fidelity of DSB repair and a pathway choice. It has been previously reported that HR is enhanced in ES cells, which provides the necessary level of genome stability. However, current knowledge is mostly based on either a study of indirect factors, such as proteins involved in certain DNA repair pathways, or studying single loci, which cannot always be extrapolated to the whole genome. Therefore, we found it relevant to perform a study tackling several targets and relying on repair outcomes rather than factors recruiting.

Second, I aimed to investigate the role of a local chromatin structure on a repair outcome and a balance between different repair pathways. Bivalent chromatin was one of the types of particular interest for me as its influence on the process of DNA repair has never been studied yet. Other chromatin types, like euchromatin and facultative heterochromatin, appeared important to study as well. On the one hand, they could serve as a reference to compare behaviour of bivalent chromatin to. On the other hand, existing studies provided incomplete information, as they were based on indirect detection of a pathway use rather than assessing a direct repair outcome.

Therefore, the goals of my work were as following:

1. Compare fidelity of double-strand break repair between different chromatin contexts, and particularly in bivalent chromatin 2. Compare fidelity of double-strand break repair between pluripotent and differentiated cells.

Results

Establishing an experimental system

In my project, I set a goal to follow the process of a single break repair, contrary to many previous studies that were inducing DNA damage globally or at repetitive elements. Those experiments were leading to a large number of simultaneous breaks, which models certain extreme situations cells might face during the lifespan but not physiological conditions. The discovery of the CRISPR-Cas9 system provided an excellent tool for creating DSBs at any selected single locus. Being particularly interested in DSB repair in mouse ES cells I, therefore, needed to establish a mouse ES cell line stably expressing Cas9. As an original plan for the project included an option of time-course experiments, we decided to use a degradable system. Therefore, I cloned a construct that consisted of Cas9 fused to GFP and a mutated FKBP12-derived destabilization domain (DD), and NeoR gene as a selective marker (fig. 12). Fusion to DD is often used for controlled protein function perturbation, as it allows rapid proteasomal degradation in mammalian cells, unless bound by a synthetic ligand Shield 1 [START_REF] Banaszynski | A Rapid, Reversible, and Tunable Method to Regulate Protein Function in Living Cells Using Synthetic Small Molecules[END_REF]. Two selected clones were additionally transfected with the suicidal cassette (kindly provided by Dr. Alexey

Tomilin). This cassette consists of PuroR and thymidine kinase (TK) genes under the control of the minimal Oct4 promoter. Such cassette allows selective elimination of either pluripotent of differentiated cells by applying ganciclovir or puromycine respectively [START_REF] Liskovykh | Derivation, characterization, and stable transfection of induced pluripotent stem cells from fischer344 rats[END_REF]. The functionality of the system was proven by transfecting selected clones with the guide RNA for major satellite repeats previously designed in the lab [START_REF] Tsouroula | Temporal and Spatial Uncoupling of DNA Double Strand Break Repair Pathways within Mammalian Heterochromatin[END_REF]. Bound to this guide RNA, Cas9 is capable of causing hundreds of DSBs per cell, and the amount of DNA damage is enough to allow gH2AX increase detection by Western Blotting (fig. 14). After confirming that the system is functional I tested the kinetics of Cas9 activation and inactivation. The test was performed by FACS analysis of GFP fluorescence, and it showed that both activation and inactivation reach a plateau after 10 hours after Shield1 addition or withdrawal respectively (fig. 14 a,b) Titration of Shield1 allowed me to find the working concentration necessary and sufficient for the full activation (fig. 14 c).

Having the system set up and tested I was able to switch to experiments with single-cutting guide RNAs. Target choice and guide RNA design

The goal of my project was to compare DSB repair in different chromatin contexts. Therefore, targets were selected so that they would represent different chromatin types. I chose several groups of genes depending on their functionality and epigenetic status:

-housekeeping genes (Gapdh and Actin), further colourcoded as pink -pluripotency regulators (Pou5f1, Nanog, and Tfcp2l1), further colourcoded as green -developmental regulators that are bivalent in ES cells (Pax6, Ngn2, Zic1, Dlx2, Zfpm2, Foxa), further colourcoded as yellow -facultative heterochromatic genes (Hoxb1, Tdrd1, and Mc4r), further colourcoded as red -genes belonging to LADs (Sox6, Ptn, and Nrp1), further colourcoded as purple Given that one of the chromatin types of interest was bivalent, and these domains are typically found at promoter regions, most of the guide RNAs were designed within a proximal promoter of a corresponding gene. The only exception was the group of genes located in LADs, as this part was connected to another project running at the lab, and introducing breaks at the intrones was required. Proximal promoters were defined as 1 kb up-and downstream of the TSS according to the BindDB web tool [START_REF] Livyatan | BindDB: An Integrated Database and Webtool Platform for "reverse-ChIP" Epigenomic Analysis[END_REF].

Guide RNAs were designed using the Benchling website (https://www.benchling.com/). guide RNA sequences are provided in Table 1 in the Materials and methods section.

Chromatin status at targeted sites was confirmed by ChIP. I used H3K4me3 as a mark of active chromatin, H3K27me3 as a mark of facultative heterochromatin, and H3K9me2 as a mark for LADs. I also tested H3K9me3 as a marker of constitutive heterochromatin. I performed ChIP in ES cells. 3T3 cells were used as a differentiated control. I could observe an expected chromatin pattern in ES cells. In 3T3 cells only housekeeping genes of all tested were active, and the rest represented facultative heterochromatin (fig. 15).

TIDE analysis. Indel pattern is sequence-specific and is not influenced by a cell type or a chromatin context

To analyse repair fidelity after introducing a break I employed the Tracking indels by decomposition (TIDE) method [START_REF] Brinkman | Easy quantitative assessment of genome editing by sequence trace decomposition[END_REF]. This method is based on PCR and Sanger sequencing and allows calculating the percentage of insertions or deletions (indels) of a particular length out of the total pool of DNA present in the sample. In this method, a fragment around the expected cut is PCRed from gDNA extracted from cells transfected with the guide RNA and from non-transfected (control) cells. PCR products are then sent for Sanger sequencing. If a break was introduced by Cas9 and repaired with indels, a corresponding shift of the Actin and Hoxb1 guide RNAs are taken as representative example.

However, in some examples, notably pg418 guide targeting Pou5f1, pg450 guide targeting Hoxb1, and pgo3 guide targeting Nrp1, a considerable contribution of +1 insertion was observed in 3T3 but not ES cells (fig.

18 a). However, the number of guide RNAs manifesting such changes was low, so I think we can consider them as an exception, which also fits the general idea in the field. At the same time, no link could be made between indel patterns of two guide RNAs for the same gene even in the same cell type (fig. 18 b), despite in some cases distance between their cutting sites was rather short. This observation also fits the hypothesis of the indel pattern being mostly dictated by the surrounding sequence. The size of deletions has been largely debated in the context of the balance between error-prone repair pathways, NHEJ and MMEJ. While it remains under debate which of these pathways could be the source of relatively small indels, there is no doubt that deletions larger than 10 b.p. are generated by MMEJ. insertions can be seen for one but not another guide RNA for the same locus on an example of Zic1.

Nevertheless, it was a general trend among most of the guide RNAs that the +1 insertion level was slightly reduced in ES cells and a proportion of deletions was increased as compared to 3T3 cells (fig. 20). This may result from a generally more open chromatin state allowing to shift a balance towards MMEJ rather than NHEJ or from ES cells highly relying on resection-dependent methods. This hypothesis would require additional experiments to prove this. However, these changes related to exact proportions of indels rather than overall pattern, which stayed constant between cell types. All in all, experiments I have performed using multiple guide RNAs to introduce breaks in a variety of loci in 3T3 and ES cells lead to the conclusion that indel pattern is predominantly defined by the sequence surrounding the break site and the influence of a cell type and a chromatin context is minor. This fits the concept currently accepted in the field and can be considered another evidence supporting it.

3T3 cells have a higher rate of error-prone repair in housekeeping genes but not in pluripotency regulators

Assuming that the total percentage of mutated sequences gives us the means to estimate the overall employment of error-prone repair, I compared this parameter in 3T3 and ES cells for some of the target genes (fig. 21). Interestingly, we observed higher rates of error-prone repair than it was reported in the literature. The proportion of mutated sequences was going over 20% in some cases, which would mean at least 30% of breaks repaired by NHEJ of MMEJ, considering that neither induction nor transfection was absolute, and efficiency of both processes can be considered close to 80% judging from previous tests. Housekeeping genes demonstrated a result that could be expected from literature: levels of error-prone repair were significantly higher in differentiated cells than in ES cells. Surprisingly, when I looked at pluripotency regulators the picture changed dramatically, and no difference between cell types could be observed. This observation was rather surprising, considering that pluripotency regulators are transcriptionally inactivate and heterochromatinised in 3T3 cells, which should lead to reduced use of HR and therefore increased mutagenesis according to literature.

One possible explanation for that discrepancy was the influence of transcriptional regulators on the cell cycle. It is known that the cell cycle of mouse ES cells is dramatically different from the cell cycle of differentiated cells, including 3T3 (fig. 22 a). ES cells are characterised by a short G1 phase and an absence of G1-S checkpoint. As a consequence, a larger proportion of cells in a population is in S and G2 phases of the cell cycle, hence HR-proficient. It was therefore important to rule out the possibility that mutations in pluripotency regulators lead to changes in the cycle that would prevent ES cells from employing error-free repair. I performed a cell cycle analysis after transfecting cells with guide RNAs targeting Gapdh as a housekeeping gene or Nanog as a pluripotency regulator (fig. 22 b,c). The cell cycle was not affected after targeting Nanog as compared to targeting Gapdh at the usual collection time (48h), as well as at an earlier time point (24h). Moreover, cell cycle distribution in both cases matched those of non-treated cells.

Therefore, we could conclude that an observed effect was not an artifact caused by a cell cycle perturbation. As transcription levels can be different from mRNA rates, which depend not only on transcription but also on mRNA degradation, I performed nascent transcript qPCR. However, transcription levels were not necessarily higher for pluripotency regulators than for housekeeping genes as well (fig. 23 b)

A technical problem we were facing was our inability to detect HR repair outcomes using TIDE, as it is indistinguishable from uncut alleles for methods that involve analysis of the sequence. Therefore we were not able to say whether differences we observed resulted from differential Cas9 binding, HR involvement, or even transfection efficiency variability between different replicates. Therefore, we needed to modify our experimental approach.

HR-TIDE: improving methodological procedures in order to detect HR

We decided to modify the TIDE method to allow the detection of HR outcomes (fig. 24). This modified TIDE (HR-TIDE) implied an addition of templates for HR, that contended a 9 b.p. insert (to avoid a frameshift when the cut was introduced in an exon) located at the place of a break and 500 b.p.-long homology arms at both sides of it. So as by using HR templates we were taking into account all repair pathways, we assumed that the percentage of perturbed sequences represented total cutting efficiency. We could see that it was sometimes varying between cell types (which could be explained both by differential Cas9 binding caused by different chromatin context and by transfection efficiency fluctuations) and even between different replicates (definitely resulting from transfection variations).

Overall cutting efficiency is slightly higher in 3T3 than in ES cells

However, it was interesting to evaluate whether there is an actual difference in Cas9-mediated cleavage between cell types. To this end, I looked at the whole array of results to find out whether any trend could be observed from a comparison between two cell types. In this analysis, I considered cutting efficiency for each guide RNA, calculated as an average from all biological replicates, as a single measurement. This allowed me to assess the break introduction as a parameter of interest, where cutting efficiencies for genome locations recognised by different guide RNAs became single measurements of this parameter.

I observed that the median value in ES cells was close to 34%, whereas in 3T3 cells it was around 43% (fig. 26). The increase of the break introduction efficiency in 3T3 cells was statistically significant (p=0.0065, twotailed Mann-Whitney U-criteria).

In order to check whether this held true when we compare results in two cell types for the same guide RNA, I found a ratio between cutting efficiency in 3T3 and ES cells and considered this as an experimental array.

The median of this array was 1.24, which shows an increase at the level of individual guide RNAs. Therefore Cas9-mediated DSB introduction is slightly efficient in 3T3 cells as compared to ES cells. Most likely, this difference resulted from differential transfection efficiency. The efficiency of Cas9-mediated break introduction in heterochromatin as compared to euchromatin is broadly discussed. It has been previously shown that constitutive heterochromatin considerably reduces break introduction by Cas9 [START_REF] Kallimasioti-Pazi | Heterochromatin delays CRISPR-Cas9 mutagenesis but does not influence repair outcome. Heterochromatin Delays CRISPR-Cas9 Mutagenesis but Does Not Influence the Outcome of Mutagenic DNA Repair[END_REF]. There is also evidence that facultative heterochromatin, and particularly the presence of H3K27me3 has a similar effect [START_REF] Schep | Impact of chromatin context on Cas9induced DNA double-strand break repair pathway balance[END_REF] By analysing HR efficiency in the same way as the cutting efficiency, I observed that the median value in ES cells was close to 11%, whereas in 3T3 cells it was around 2% (fig. 27). This increase was statistically significant (p=0.0024, two-tailed Mann-Whitney U-criteria). Interestingly, taking advantage of an opportunity to directly measure a proportion of HR in several different locations we could see again that despite its higher levels in ES as compared to differentiated cells, it still does not match previous evaluations, and the vast majority of breaks is repaired by error-prone methods. After assessing the ratio between HR level in ES and 3T3 cells I found that the median ratio was 5.3, which

shows an increase at the level of individual guide RNAs. Therefore ES cells demonstrate a five-fold increase of HR employment as compared to cycling differentiated cells. This may have resulted from surrounding sequence or chromatin features. Using the same strategy as before, I compared arrays of HR proportions from ES and 3T3 cells for each of these target groups (fig. 29).

In both groups the median of HR utilisation was higher in ES than in 3T3 cells (27% versus 9% for housekeeping genes and 18% versus 1% for pluripotency). However, statistical analysis using Mann-Whitney criteria showed the significance of the increase for pluripotency regulators (p=0.0214) but not housekeeping genes. This could have been caused either by a lower number of guide RNAs analysed for Thus, we can claim with confidence that active genes in ES cells show considerably higher HR rates than they do in differentiated cells, independently of their activation status in the latter case.

I also plotted all investigated groups on the same graph for each of analysed cell types. Despite the inability to do statistical analysis due to the lower number of targets per group, I could notice as a trend that for each cell type those groups of targets, which are active in this cell type, had elevated HR levels as compared to groups of inactive genes (fig. 30). This goes in line with other published data demonstrating that transcriptionally active chromatin is an HR-permissive environment. However, adding more targets to each group to enable proper statistical analysis is needed to secure this conclusion.

Bivalent and inactive genes

Looking at other chromatin types of interest (bivalent domains, facultative heterochromatin, and LADs) we observed a completely different picture.

Pairwise comparison for each guide RNA would show a significant increase for half of target sites analysed in both bivalent domains and facultative heterochromatin (fig. 31 a and b respectively) so no clear conclusion could be drawn on whether HR is more actively employed by ES cells to repair these loci.

Moreover, one gene in each of these two arrays can potentially be exceptional. Hoxb1 demonstrated surprisingly high HR rates in both target sites and both cell types analysed. This could potentially result from differences of Hox locus organisation compared to other genomic regions. However, this assumption would require to assess more Hox genes to confirm it. Considering bivalent regions and facultative heterochromatic targets as independent arrays (fig. 31 c) we found no significant difference between analysed cell types. Moreover, medians of an array of one cell type lied within a confident interval of another cell type for both chromatin environment, and the increase of HR utilisation in ES cells was approximately two-fold (close to 2 and 2.16 respectively). This led us to a

Exceptions

It can be noticed that in ES cells HR in bivalent targets seems to have high variability (fig 31 c). This may result from one of the selected targets, Pax6, demonstrating very low HR levels. It is possible that this locus is an exception due to its DNA sequence or another factor, but assessing more bivalent targets would be necessary to check it.

The same problem can be observed for the group of facultative heterochromatic targets. HR level in this group goes surprisingly high in ES cells (fig. 31 c) and demonstrate a rather prominent variability. It is caused by results from two guide RNAs for the same gene, Hoxb1. However, the Hox cluster is known to have a rather distinct chromatin structure and is sometimes even thought to represent a separate chromatin type [START_REF] Aaronson | Systematic identification of gene family regulators in mouse and human embryonic stem cells[END_REF]. Therefore, it would help to increase the number of heterochromatinised targets as well as to add some more Hox genes into analysis to check whether it stands the same in the context of DNA repair.

LADs

We could observe no significant increase in HR employment for any of analysed loci within LADs (fig. 32). However, it was not completely blocked in any of the targets. This observation does not fit results previously obtained in the lab. However, such a discrepancy could be explained if genes in question are located at the This assumption requires further experimental investigation. However, the fact that two of the genes of interest were reported as bivalent in literature can be considered indirect proof for it.

The cell cycle is partially responsible for the low HR rate in 3T3. Cell cycle block causes an increase in HR utilisation but cannot fully explain differences between cell types

As it was noted before, mouse ES cells are characterised by a specific cell cycle distribution, with a very short G1 phase. As a consequence, only 20% of cells in a population are in this phase at any time point, and the rest of the cells is in S/G2 phases, therefore 80% of cells are HR-proficient. In differentiated cells, these ratios are different, as G1 is longer. 60% of cells in a population are in this state and therefore cannot perform HR. This could potentially be the cause of different ratios of HR in 3T3 and ES cells. To test this hypothesis we first decided to enrich a population of 3T3 cells for G2 cells. To this end, we blocked cells in the G2 phase with RO inhibitor and analysed changes in HR utilisation. We could indeed detect an increase of HR ratios comparable or even larger than those observed I ES cells (fig. 33). However, from previous observations we knew that the degree of HR enhancement varies between different chromatin contexts, and would the cell cycle be the only explanation, it would cause a similar shift in all cases. To further test the degree to which perturbed cell cycle is responsible for higher HR rates in ES cells, we needed to bring it to the state close to those of differentiated cells. This can be achieved by culturing ES cells in a serum-free medium with an addition of MEK and GSK3b inhibitors (so-called 2i medium) [START_REF] Ying | The ground state of embryonic stem cell self-renewal[END_REF]. It has been demonstrated that in this medium a G1-S checkpoint is restored in ES cells and the length of the G1 phase is increased, and 40% of a cell population is in this phase [START_REF] Ter Huurne | Distinct Cell-Cycle Control in Two Different States of Mouse Pluripotency[END_REF].

We could also observe these changes (fig. 34). We performed transfections of some selected guide RNAs in these culture conditions and analysed HR rates.

For most of the analysed target sites, we could detect no difference in HR rates between two culture mediums (fig. 35), although in a few cases we could see a slight decrease or increase. When results from all analysed target sites were combined for statistical analysis, no significant difference was observed. Also, when a ratio between HR levels in serum-containing medium and 2i medium was calculated for each guide RNA, the median ratio amounted to a 1.16-fold increase, which is lower than differences observed between 3T3 and ES cells. an increased level of proteins involved in DNA repair and particularly in HR that was previously reported.

However, a precise answer to this question would require further investigation.

Discussion

In trimethylation and the use of NHEJ in either of the model cell types. However, a small number of targets does not allow us to draw such broad conclusions.

In the same study, a template-dependent repair has been tackled. It was observed that the chromatin environment influences it the same way as it does for NHEJ. However, the pathway chosen for that investigation was single-strand templated repair, so we could not directly compare it to our results as we were interested in HR. We could see that HR utilization was influenced both by a chromatin type and a cell type. Heterochromatic marks appeared to dominate over euchromatic in control of a repair pathway choice and to be repressive for HR. I found that cell cycle characteristics are one of the factors leading to the abovementioned differences between pluripotent and differentiated cells but they are unlikely to be the only factor involved. Therefore, further experiments to dissect this issue might be necessary. Indel patterns between different cell types bore strong similarity for every guide RNA, whereas no resemblance could be detected for two different guide RNAs even in a case of a short distance between break sites. This goes in line with previously published data indicating that DNA sequence around the break site, particularly a few nucleotides surrounding it, and a presence of microhomologies define specific indels found after a break repair.

However, an overall proportion of larger deletions, presumably associated with MMEJ, appeared to be higher in ES cells than in 3T3 cells. This is likely to indicate a shift in a balance between NHEJ and MMEJ.

One possible explanation for this effect could be a more open chromatin organisation in ES cells, considering that such a state is generally reported to be prone to HR and at the same time MMEJ being also resection-dependent. This shift of balance could also be a consequence of an increased level of some proteins simultaneously involved in both HR and MMEJ repair pathways, such as CtIP. Nevertheless, this would require additional experiments to prove.

ES cells use HR more actively than differentiated cells

Focusing more on the balance between error-free and error-prone repair pathways I performed HR-TIDE experiments and analyzed a proportion of sequences that incorporated a provided template for HR.

Despite numbers varying for individual guide RNAs, analysis on the whole array revealed that HR is significantly higher used across the genome in ES cells as compared to 3T3 cells. This corresponds to the current paradigm in the field. However, unlike some other cases, in my setup, I could still see a vast contribution of error-prone pathways in the repair process. Therefore, my results support the hypothesis of HR being more actively employed in ES cells but do not confirm the degree of its mobilization previously claimed. This is likely to result from the difference between experimental setups. Studies leading to conclusions that HR is utilised to repair up to 80% of breaks in ES cells were performed on one single break on a transcriptionally active gene, and I checked several target sites across the genome, in both active and inactive states. Another possible explanation could be the use of Cas9 to introduce breaks in my experiments, as it has previously been suggested that the presence of Cas9 at the DNA after a break was introduced might potentially influence the pathway choice.

HR is enhanced in transcriptionally active genes. It is more prominent in ES than in differentiated cells

A comparison between transcriptionally active regions and other types of chromatin led me to the conclusion that the euchromatic environment is promoting HR. Despite I cannot prove this observation statistically due to a low number of loci explored for each group, it is supported by several other studies, which makes it likely to be correct. Nevertheless, it would be preferable to increase the number of targets to enable a solid conclusion. Therefore, finding that pluripotency regulators demonstrate significantly higher levels of HR in ES than 3T3 cells is not surprising. However, I could also see a threefold increase for housekeeping genes, which supports the previous conclusion that HR levels are generally elevated in pluripotent cells.

Despite both conclusions fit within currently existing views, I think they add considerably to previously developed theories as to my knowledge it is the first time when HR contribution is assessed directly at the level of sequence and not based on the recruitment of different factors, which could also be involved into some other processes. Moreover, it disconfirms a recent study by Janssen et al. that claimed no influence of the chromatin state on the process of HR and therefore sheds light on a still controversial topic [START_REF] Janssen | The Chromatin Structure of CRISPR-Cas9 Target DNA Controls the Balance between Mutagenic and Homology-Directed Gene-Editing Events[END_REF].

HR levels are low in bivalent domains and facultative heterochromatin

Of different types of heterochromatin constitutive heterochromatin is the most studied and it has been shown that DSBs in such an environment are less likely to be repaired by HR. However, facultative heterochromatin has been less studied. We assessed several loci marked with H3K27me3 and found that HR levels, in this case, were low, and more interestingly, they were not significantly increased in ES cells as compared to 3T3 cells. Therefore, we can conclude that facultative heterochromatin represents an environment predominantly repressive for HR.

As euchromatin represents an HR-promoting environment and heterochromatin is repressive to it, it was especially interesting for us to see what results we might observe in the case of bivalent domains, where marks of both are combined. Interestingly, we could see that HR levels stay low and there is no significant difference between cell types. Therefore, the same outcome is observed for bivalent domains behave as for facultative heterochromatin. In other words, in regulating the process of DSB repair repressive chromatin marks appear to be a stronger driver than permissive ones. It is an interesting observation as to our knowledge no one has yet studied the influence of this type of chromatin on the process of DNA repair.

Influence of the cell cycle on the enhanced HR utilization in ES cells

As a cell cycle is known to strongly influence the process and it is also changed in ES cells as compared to differentiated cells, we decided to check to what extent it contributes to the difference in repair pathway choice we see between cell types. For this we blocked 3T3 cells in the G2 phase to check if increasing a proportion of HR-proficient cells in the population would change the balance between repair pathways. It is hardly surprising that we found it to lead to an increase in the proportion of HR products. Therefore, in consonance with existing knowledge, we could see that blocking cells in a cell cycle stage permissive for HR would lead to increase utilisation of this pathway up to levels similar or even exceeding observed in ES cells.

Another apparent question was whether modifying the cell cycle of ES cells to resemble one of the differentiated cells would cause the opposite effect. Fortunately, we had a way to do it, as it has been reported that in 2i medium ES cells acquire a cell cycle that resembles one of the differentiated cells, and what was of particular interest for us, in such culture conditions larger proportion of cells in a population stays in G1 phase. After introducing DSBs using several selected guide RNAs we only found a moderate drop in HR efficiency, which was not statistically significant. However, it has to be taken into account that the share of cells in the G1 phase is smaller in ES cells even in 2i than in 3T3 cells.

All in all, we came to the conclusion that it is very likely that a distinct cell cycle that is known to be characteristic for ES cells is likely to be one of the mechanisms to ensure higher genome stability.

Nevertheless, it is highly doubtful it is the only underlying mechanism, and further experiments would be required to uncover other ways utilised by ES cells to ensure their genome is preserved through generations.

Perspectives

It has to be noted that I performed all experiments in my project on a relatively small number of targets, which limits our ability to generalise conclusions. Therefore, my perspectives could be divided into shortterm, aiming to prove established results without the addition of a large number of other target sites or a major change in the experimental setup, and long-term, aiming to broaden our observations to a genomewide level.

I would like to start discussing the suggested experiments with short-term perspectives. First, considering the heterogeneity of results for bivalent domains and facultative heterochromatin target groups I think adding several more targets for each group to ensure that none of the results for selected targets was impaired by any factor other than chromatin structure (in other words, that we do not deal with any target representing an exception from the general trend) and reduce the influence of outliers. Adding 3 or 4 more targets should be sufficient to complement already performed HR-TIDE-based experiments.

Considering the fact that most of the time using two guide RNAs designed for the same region leads to very similar results, it might be profitable to design one guide RNA per each selected target. Choosing some additional targets within the Hox cluster would also allow evaluating the hypothesis of exceptional DSB repair pathway preference (and therefore indirectly confirm an exceptional chromatin organisation in this part of a genome). Considering the differences observed between publications it would be more tenable to perform ChIP-qPCR for all newly selected targets to confirm their chromatin status.

Second, adding several more guide RNAs for housekeeping genes would enable us to perform a proper statistical analysis to compare different target groups within the same cell type. It ought to be remarked that the target group of LADs was excluded from all comparisons with other chromatin types due to a small size of the group. Therefore, it also requires enlargement. However, considering highly heterogenic results observed for this target group it is also necessary to perform FISH to prove that all of the targeted regions indeed belong to LADs in the utilised cell line.

In my analysis, I have set the NHEJ/MMEJ border voluntary. To stratify this question as well as to prove that +9 incorporation is indeed facilitated by HR, it would be necessary to perform TIDE or HR-TIDE experiments while knocking down or chemically inhibiting key players of investigated pathways, such as DNA-PKcs, DNA Ligase IV, or XRCC4 for NHEJ, Polq, DNA Ligase III, or XRCC1 for MMEJ, and Rad51 or BRCA1 for HR.

According to the obtained results, we could formulate a hypothesis that in case of bivalency, i.e.

coexistence between euchromatic and heterochromatic marks DSB pathway balance is kept similar to those of facultative heterochromatin. However, this assumption requires direct confirmation. In order to achieve it, we plan to remove H3K27me3 from bivalent genes by recruitment of histone demethylases such as JMJD3 or UTX to target sites by fusing them to dCas9 (a catalytically inactive form of Cas9) prior to performing HR-TIDE experiments. Demethylases recruitment can alternatively be substituted by chemical inhibition or knock-down of PRC2 complex subunits. However, it would cause genome-wide perturbations, therefore, targeted demethylation is preferable. On the other hand, targeting PRC2 complex to transcriptionally active targets to form bivalent domains de novo would complement proposed experiments in order to prove a proposed theory. However, if this strategy is chosen, it would be important to confirm that bivalent domain formation and not replacement of euchromatic marks by heterochromatic follows such a recruitment. Therefore, a confirmation by ChIP-qPCR would be required.

We could conclude from our results that HR rates are higher in actively transcribed regions marked by H3K4me3. However, we cannot draw a line between roles transcriptional activity and chromatin status.

Performing HR-TIDE experiments after chemical transcription inhibition would allow us to dissect between these two factors. Triptolide use would be preferable compared to other commonly used compounds, such as α-amanitin, actinomycin D, DRB, or flavopiridol, due to the fact that it inhibits transcription initiation, while other inhibitors block elongation. Considering that selected targets lie within proximal promoter regions, an attitude that blocks transcription inhibition should preferentially be used.

Potential downsides of such an approach are massive perturbation caused in a cell by a prolonged transcription block. One cannot exclude that effects caused by such a block might mask analyzed influence on DNA repair.

Regarding long-term perspectives, I would like to mention a potential practical application of the developed approach. In my opinion, it could be used for predicting knock-in efficiency. Analysing obtained results I have noticed that in some cases HR efficiency differs from what can be expected from the chromatin environment surrounding the introduced break. Knocking-in large fragments, such as fluorescent reporters, is often necessary for research. However, the introduction of large fragments often results in reduced efficiency compared to smaller ones. Therefore, I think that prior testing of designed homology arms with smaller insert might be a good strategy to predict their efficiency before engaging in a large experiment to enable the optimal setup. To allow this, homology arms should be cloned with a small insert containing digestion sites that would enable further replacement by a larger insert of interest.

This way, knock-in efficiency could be tested by HR-TIDE prior to the real experiment. To verify that HR-TIDE results are a good predictor for a larger fragment, it would be good to replace an insert in several of previously used templates with mCherry and evaluate knock-in efficiency by FACS to test whether it correlates with HR proportion observed in HR-TIDE experiments.

Finally, all experiments performed in the course of my doctoral project can be used as a basis to design a genome-wide test of HR utilization. Selecting one or two hundreds of well-cutting guide RNAs targeting different genome regions based on available data and cloning them together with corresponding HR templates into a viral library based on an integration-deficient lentiviral vector could be a solution for such a goal. This library should be used for infecting cells of interest and further submitted to NGS analysis. Such an experiment would provide a map of HR utilisation efficiency and allow assessment of its dependence on a chromatin state as well as other factors. It would also help to select a group of targets for further dissection of mechanisms underlying observed correlations.

Materials and methods

Cell culture

Mouse ES cells were cultured on a feeder layer of mitotically inactive MEFs, growing conditions were set to 37°C, 5% CO2. Cells were passaged every 2-3 days and kept in culture for no more than 10 passages.

Passaging involved trypsinisation for 5 minutes at 37°C, trypsin inactivation with at least equal volume of medium, achieving a state of single-cell suspension, separation from feeders by gravity (after 10 minutes incubation in a 15 ml Falcon tube, top fraction was used for plating) and plating on a fresh dish at 1:10 to 1:20 rate depending on the desired final confluency. Suicidal cassette: for all following experiments cells were treated with puromycine for 2 days starting 24h after thawing.

For experiments in serum-free conditions 2i medium was used. Transfection mix 2 contained 12,5 ml of Lipofectamine 2000, and 250 ml of OptiMEM medium.

Transfection mixes 1 and 2 were incubated for 5 min at room temperature, then mixed and incubated for 10 more min at room temperature. For transfection, cells were centrifuged for 3 min at 1200 rpm, and the supernatant removed. Resulting pellets were carefully disloged, and then gently resuspended in corresponding transfection mixes. Transfection was performed with 15 minutes of incubation in suspension at 37°C. In order to prevent a pellet formation, tubes were gently shaken every 5 min. Cells were plated on 6-well plates pre-coated with gelatine with addition of 0,5 ml of OptiMEM per well for 4 hours, than medium was replaced by standard ES cell culture medium supplemented with Shield1.

For transfections of cells cultured in 2i medium 1*10^6 cells was taken per sample and transfected with total of 2 mg of DNA. Reverse transfection was performed with reducing incubation time to 7 minutes.

Cells were plated in 2i medium supplemented with 2% serum.

Growing conditions for 3T3 cells were set to 37°C, 5% CO2. Cells were passaged every 3-4 days and kept in culture for no more than 8 passages.

3T3 cell medium composition:

High glucose DMEM 10% NCS 40mg/ml Gentamycin Transfection of 3T3 cells was performed using Lipofectamine 2000. Cells were plated at a confluency of 1*10 5 cells per sample on a well of a 6-well plate 18 h prior transfection. On the day of transfection, medium in each well was replaced by 1 ml of a fresh 3T3 cell medium and transfection mixes were prepared. Transfection mix 1 contained total of 2 mg of DNA (when transfecting for HR-TIDE experiments, HR template and a guide RNA were co-transfected at a 3:2 ratio, for template only or guide samples a corresponding plasmid was replaced by the same amount of the empty vector) and 150 ml of OptiMEM medium. Transfection mix 2 contained 10 ml of Lipofectamine 2000 and 150 ml of OptiMEM medium.

Transfection mixes 1 and 2 were incubated for 5 min at room temperature, then mixed and incubated for 10 more min. Next, each transfection mix was added to a corresponding well.

Cell cycle analysis

Cell cycle analysis was performed using Click-iT EdU incorporation kit (Invitrogen) according to a slightly modified manufacturer's protocol. Cell culture medium was changed to fresh medium pre-warmed to 37°C and supplemented with EdU at a final concentration of 50 mM. After 30 min, cells were harvested by trypsinisation, achieving a state of single-cell suspension, washed with 1% BSA in PBS, and fixed with a 100 ml of a provided fixative solution (Component D) for 15 min at room temperature. Then cells were washed with 1% BSA in PBS and incubated with a 100 ml of a provided permeabilization and wash reagent (prepared according to manufacturer's protocol) for 15 min at room temperature. Next, cells were washed with 1% BSA in PBS and a total 200 ml a reaction cocktail was added per sample, followed by a 30 min incubation. Then samples were washed with 1,2 ml per sample of a permeabilization and wash reagent and pelleted, and supernatant was removed. Resulting pellets were resuspended in 200 ml of a permeabilization and wash reagent, supplemented by RNaseA at a final concentration of 4 mg/ml, and PI at a final concentration of 4 mg/ml. Samples were further analysed by FACS.

Reaction cocktail composition (per sample):

175,2 ml of PBS 4 ml of CuSO4 (Component F) 2 ml of Fluorescent dye azide (prepared according to manufacturer's protocol) 20 ml of Reaction Buffer Additive (prepared according to manufacturer's protocol)

Western blotting

Cells were harvested by trypsinisation and lysed in RIPA buffer containing HEPES pH7.6 50mM, Sodium dexicholate 0.5%, NP40 1%, EDTA 1mM, LiCl 0.5M. Quantification was performed with Bradford assay according to manufacturer's protocol. Samples were mixed with NuPAGE lysing buffer and pre-heated for 20 minutes. Samples were loaded on pre-cast 4-12% gradient gels for migration. Wet transfer was performed on nitrocellulose membrane at 400 mA for 90 minute. Membrane was blocked in 5% non-fat dry milk in PBS for 1 hour at room temperature. Primary antibodies were diluted in 3% non-fat dry milk in PBS-0.1% Tween20 and incubated at 4°C overnight. Primary antibodies used were gH2AX (Abcam ab2893) diluted 1:1000, tubulin (Sigma T9026) diluted 1:10000 and GFP (Santa Cruz sc-9,996 (clone B-2))

diluted 1:500. Membranes were washed with PBS-0.1% Tween20 and incubated with secondary antibodies for 1 hour at room temperature. After incubations were washed with PBS-0.1% Tween20 and developed using ECL reagent.

RIPA buffer composition:

Pou5f1 Pg266

CCCTCCTCCTAATCCCGTCT TTCTAGTCCACACTGCGTCG

Gapdh Pg313

GAGGAGTCCTTGGAGTGTGC TGCTGAGTCACTTGGAGCAG

Ngn2 Pg316

CTCTCTCACAACGTGCCTCC GGTGAGCGCCCAGATGTAAT

Tfcp2l1 Pg360

ATGTCACACGAGCCCAGTTT GCTAGCGAAATCCCCACAGA

Hoxb1 Pg361

AGACAGTGTCACACGTAGGC GATGCAAAGGTTGCGGTCTG

Pou5f1 Pg418

CCCTCCTCCTAATCCCGTCT TTCTAGTCCACACTGCGTCG

Actin Pg438

CGTAGCGTCTGGTTCCCAAT AGGTGCGTGTCCTTCTAAGC

Gapdh Pg439

TGGAACTCACCCGTTCACAC GCAGGGCATCCTGACCTATG

Nanog Pg442

TTGCGTTAAAAAGCCGCACT GAGCTTCAGACCTTGGCTCC

Nanog Pg443

TTGCGTTAAAAAGCCGCACT GAGCTTCAGACCTTGGCTCC

Pax6 Pg445

AAGCAGCCGCACTTAGTCAA TAGTGGCTTCTTTCACCGCC

Pax6 pG446

AGGGAGAGGGAGCATCCAAT GGGAACACACCAACTTTCGC

Zic1 pG447

GCACGACTTTTTGGGGTTGG TGGCAGCCCTGTTAGTCAAA

Zic1 pG448

GCACGACTTTTTGGGGTTGG TGGCAGCCCTGTTAGTCAAA

Ngn2 pG449

CTCTCTCACAACGTGCCTCC GGTGAGCGCCCAGATGTAAT

Hoxb1 pG450

CTCTTGCCCTCCTGGACTTG TCCATGTAGAGGCCGAAGGA

Tdrd1 pgl1

GAGGTGGGGCATAACGACTT CAGGAGGGGTTGCACGTTTA

Tdrd1 pgl2

GAGGTGGGGCATAACGACTT CAGGAGGGGTTGCACGTTTA

Mc4r pgl3

TCGATGACGGCGTTACACAT TGAGCCTTCCGTCATTCAGG

Mc4r pgl4

TCGATGACGGCGTTACACAT TGAGCCTTCCGTCATTCAGG

Sox6 pgo1

CGTACTGCACCTCAGTGTGA CTATAGTGTGCGTGAGGCGA

Ptn pgo2

CACCACACCACTTAGCCCAT CAGGGAGGTGACAGAAACCC

Nrp1 Pgo3

  I do not know how to express what I think and what I feel, because nothing I can say would be enough, and words fall short. I can only hope you will understand what is behind them.

C

  'est pourquoi il est important d'étudier le processus de réparation de l'ADN, en particulier les cassures des doubles brins. De plus en plus de preuves appuient l'hypothèse selon laquelle l'organisation de la chromatine joue un rôle notable dans le choix de la voie de réparation. Il a été démontré que l'organisation locale de la chromatine autour du site d'une cassure double brin de l'ADN (Double Strand Break soit DSB en anglais) peut influencer le résultat de la réparation. Par exemple, la présence des marques d'histones telles que la triméthylation de la lysine 36 de l'histone H3 (H3K36me3) et l'acétylation d'une lysine 16 de l'histone H4 (H4K16ac), signe d'une chromatine transcriptionnellement active, ont été observées pour promouvoir une réparatoin par le mécanisme de recombinaison homologue (Homologous Recombination soit HR en anglais). De plus, il a récemment été démontré que dans le cas de DSBs induites dans des régions transcrtiptionnellement actives en G1, où la HR n'est pas possible en raison de l'absence d'une chromatide soeur, la réparation peut être différée à la phase G2 où la HR est activée. D'autre part, il a été démontré que la présence de marques d'histones corrélées avec de la chromatine répressives, telles que la mono-et diméthylation de la lysine 20 de l'histone H4 (H4K20me1 et H4K20me2), favorise le choix de l'assemblage non homologue des extrémités (Non Homologous End Joining soit NHEJ en anglais) comme voie de réparation principale. Au sein de notre laboratoires, il a été montrer que dans les domaines associés aux lamines (Lamins Associated Domains soit LADs en anglais) ou la chromatine fortement réprimées, la réparation par HR est inhibée favorisant l'utilisation du mécanisme alternatif de jonction des extrémités (alternativ End Joining soit alt-EJ an anglais) potentiellement délétère. Il a également été observé en laboratoire que dans l'hétérochromatine péricentrique, le mécanisme de HR est inhibé car il est potentiellement délétère, lors de translocations de séquences pouvant conduire à des ruptures de chromosomes. La réparation de la DSB dans l'hétérochromatine, surtout constitutive, est beaucoup mieux étudiée que dans l'euchromatine ou l'hétérochromatine facultative. Il a été démontré que l'euchromatine est plus sensible aux dommages causés par l'ADN et qu'elle favorise généralement les RH, mais dans l'ensemble, elle nécessite des études plus détaillées. L'un des types de chromatine, relativement abondant dans les cellules souches, qui n'a encore jamais été étudié dans le domaine de la réparation de l'ADN est la chromatine dite bivalente. Domaines bivalents est un nom pour les tronçons de chromatine marqués à la fois par des modifications d'histones permissives et répressives. Les premières observations de domaines bivalents concernent la combinaison des marques d'histone H3K4me3 et H3K27me3. Par la suite, les marques H3K4me3 et H3K9me3 ou H3K36me2/3 et H3K9me3 contenant des domaines bivalents furent trouvés respectivement dans des cellules souches mésenchymateuses humaines et des cellules préadipocytes et HEK293. Pendant quelques années après leur découverte, ces marques ont été considérées comme un artefact de culture cellulaire par de nombreux scientifiques. Cependant, au cours des dernières années, ils ont été observés in vivo par plusieurs groupes, à la fois pendant le développement normal et dans lors de cancer.

Fig. 1

 1 Fig.1Types of DNA lesions, their sources and repair pathways. Adapted from[START_REF] Chatterjee | Mechanisms of DNA Damage, Repair and Mutagenesis[END_REF]

Fig. 2

 2 Fig. 2 Schematic representation of early steps of DDR activation. DSBs are sensed by MRN complex, which binds to free DNA ends and promotes ATM activation. This leads to multiple DNA repair proteins activation, including Mdc1, 53BP, CtIP and BRCA1 and promotes DNA repair. Intra-S and G2-M checkpoints and a cell cycle arrest are activated by phosphorylation of checkpoint kinases Chk1 and Chk2, and p53. Adapted from[START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF]

Fig. 3

 3 Fig. 3 Roles of PI3KKs in the maintenance of genome stability. Adapted from[START_REF] Blackford | ATM, ATR, and DNA-PK: The Trinity at the Heart of the DNA Damage Response[END_REF]

Fig. 3

 3 Fig. 3

Fig. 4

 4 Fig. 4 Consequences of DNA damage-induced Chk1 and Chk2 mediated cell cycle checkpoint activation. Together, they phosphorilate multiple targets, including Cdc25Aand Cdc25C, resulting in DNA repair activation, and finally cell cycle arrest or apoptosis. Adapted from(Bartek & Lukas, 2003).

Fig. 5

 5 Fig. 5 Schematic representation of cNHEJ pathway. It is initiated by Ku70/Ku80 binding to free DNA ends and a subsequent DNAPKcs recruitment and an active DNA-PK complex formation. DNA-PK mediates tethering of broken ends, autophosphorylates its own subunits and phosphorylates other targets including Artemis, PNKP, XRCC4, XLF, and DNA ligase IV. If necessary, end-processing is performed by PNKP, Artemis, WRN, APLF, Pol m/l, and others. DNA Ligase IV, XRCC4, and XLF perform ligation. Finally, Ku heterodimers are removed, presumably by proteasomal degradation. Adapted from[START_REF] Fell | The Ku heterodimer : Function in DNA repair and beyond[END_REF].

Fig. 6

 6 Fig. 6 Schematic representation of HR pathway. DSB is sensed by MRN complex, which recruits CtIP. Mre11 initiates resection, which is further conducted by EXO1, BLM, and DNA2, and Ku heterodimers are displaced from ssDNA.

Fig. 7

 7 Fig. 7 Schematic representation of MMEJ pathway. After PARP1-mediated break recognition, resection is performed by Mre11, aided by CtIP. ssDNA is protected by RPA until gaps are filled by Polq. Ligation is carried out by Lig III and XRCC1. Adapted from (Wright et al., 2018).

Fig. 8

 8 Fig. 8 Schematic representation of SSA pathway. After PARP1-mediated break recognition, initial resection is performed by Mre11, aided by CtIP. It is further extended by EXO1 and DNA2. ssDNA is protected by RPA, and Rad52mediates annealing. ERCC1/XPF complex removes 3' overhangs. Adapted from[START_REF] Wright | Homologous recombination and the repair of DNA double-strand breaks[END_REF]

Fig. 9

 9 Fig.9DSB repair pathway choice. Adapted from(Jachimowicz, Goergens, et al., 2019)

Fig. 9

 9 Fig.9Levels of chromatin organization. Adapted from[START_REF] Fyodorov | Emerging roles of linker histones in regulating chromatin structure and function[END_REF]

Fig. 10

 10 Fig. 10 Chromatin environment influence over DNA repair pathway choice. Adapted from[START_REF] Kalousi | Nuclear compartmentalization of DNA repair[END_REF]

Fig. 11

 11 Fig.11Schematic representation of CRISPR-Cas0 system mechanism of action. Adapted from[START_REF] Memi | CRISPR/Cas9 gene-editing: Research technologies, clinical applications and ethical considerations[END_REF]

Fig. 12 A

 12 Fig. 12 A scheme of the construct used to create stable cell lines

Fig. 13

 13 Fig. 13 a) Typical FACS results for several analysed clones; b) FACS results for clones selected for analysis

  Fig. 14 Confirming Cas9 functionality by WB: gH2AX levels are increased after transfecting with a guide RNA for major satellites.

Fig. 14

 14 Fig. 14 a) Cas9-GFP stabilisation after Shield1 addition for both selected clones; b) Cas9-GFP degradation after washing out Shield1; c) Titration curve for Shield1 for both selected clones. All results are obtained by measuring GFP fluorescence by FACS analysis

Fig. 15

 15 Fig. 15 ChIP-qPCR results for selected targets. Results for 3T3 cells are shown in blue and for ES cells in orange. Error bars represent s.d. from 3 technical replicates.

  . As a result of a merge of sequences with different repair outcomes, superposed nucleotide picks appear on a chromatogram. The original sequence and height of individual peaks available from the control sequence allow TIDE software to decompose the signal and return ratios of different indels (fig.16). This technique gave us an opportunity to estimate both total employment of error-prone repair and an indel pattern for each condition.

Fig. 16 A

 16 Fig.16A scheme of TIDE experiment. Adapted from[START_REF] Brinkman | Easy quantitative assessment of genome editing by sequence trace decomposition[END_REF]

Fig. 17

 17 Fig. 17 Reproducibility of indel patterns. a) 3 biological replicates of transfecting the same guide RNA on the example of a guide RNA targeting Hoxb1 in 3T3 cells; b) Indel pattern comparison between 3T3 (blue) and ES (orange) cells.

  Fig. 18 TIDE results: indel pattern. a) guide RNAs targeting Pou5f1 and Nrp1. +1 insertion can be seen in 3T3 but not in ES cells; b) Comparison of indel patterns of two guide RNAs targeting the same locus in the same cell type.

Fig. 19 .

 19 Fig. 19. Indel size assessment. a) Large deletions are notable in both cell types on an example of Mc4r; b) large

Fig. 20

 20 Fig. 20 Indel size assessment. Larger insertions are more prominent in ES than in 3T3 cells

  Fig. 21 TIDE results comparison between cell types. A rate of impaired sequences is significantly higher in analysed housekeeping genes (Gapdh and Actin). No significant difference can be seen for analysed pluripotency regulators (Pou5f1, Nanog, Tfcp2l1). Results for 3T3 ells are shown in blue, and for ES cells in orange. * p<-0.5, **p<0.01. n>3

Fig. 22

 22 Fig. 22 Quantification of a cell cycle distribution of a) non-transfected ES and 3T3 cells; b) ES cells transfected withguide RNAs for Nanog or Gapdh 24 h or 48 h post transfection (error bars represent s.d., n=3) c) representative FACS plots for ES cells transfected with Nanog and Gapdh 24 hours post transfection.

Fig. 23

 23 Fig. 23 a) RT-qPCR (enrichment shown in logarithmic scale) and b) RT-qPCR for nascent transcripts in ES cells.

Fig. 24

 24 Fig. 24 Scheme of HR-TIDE approach

Fig. 25 A

 25 Fig. 25 A representative graph returned by TIDE software after co-transfecting guide RNA for Oct4 and a corresponding HR template.

Fig. 26

 26 Fig. 26 Comparison of cutting efficiency in a) 3T3 and ES cells, b) euchromatin and H3K27me3-marked regions in ES cells and c) euchromatin and facultative heterochromatin in ES cells.

  . Due to the number of analysed guide RNAs, I could perform such a comparison only in ES but not 3T3 cells. any reduction while considering either in cutting efficiency, and the difference was not significant while comparing either all H3K27me3-marked targets (bivalent and facultative heterochromatin combined, fig. 26 b) or facultative heterochromatin only (fig. 26 c)HR rates are higher in ES than in 3T3 cellsIn order to eliminate the abovementioned sources of variation, I chose to use the cutting efficiency as a normalisation factor for HR and error-prone pathways (NHEJ and MMEJ) outcome values and a further look at their proportions.

Fig. 27

 27 Fig. 27 Comparison between HR efficiencies in 3T3 and ES cells.

  HR employment after break induction with the same guide RNA between different cell types showed a significant increase in ES cells compared to 3T3 cells for housekeeping genes and pluripotency regulators (fig.28) with one exception. Also, one guide for each target group showed abnormally low HR in ES cells.

Fig. 28

 28 Fig.28Comparison of HR use between cell types on exampl of single guide RNAs. HR utilisation is higher in ES than in 3T3 cells for both housekeeping genes and pluripotency regulators.

  by higher variability in this group. In any case, analysing more targets for this group might solve this question.

Fig. 29

 29 Fig. 29 Comparison of arrays of guide RNAs targeting housekeeping genes or pluripotency regulators between cell lines.

  Fig. 30 Comparison betwee different target groups in the same cell type.

  Fig. 31 Comparison of a) bivalent and b) heterochromatic targets between two cell lines on single guide RNAs and c) on combined arrays. There is no significant difference in HR efficiency in different cell types.

  terms of DNA repair bivalent chromatin predominantly behaves similarly to facultative heterochromatin and that repressive chromatin marks tend to play a decisive role in a repair pathway choice.

Fig. 32

 32 Fig. 32 Comparison of targets located in LADs targets between two cell types on single guide RNAs. Different targets behave in a different way, making it impossible to draw a conclusion based on such a small number of targets.

Fig. 33

 33 Fig. 33 HR proportion grows considerably after cell cycle block with RO inhibitor.

  Fig. 34 Cell cycle distribution difference between cells cultured in the presence of serum or in serum-free 2i medium

Fig. 35

 35 Fig.35Comparison of HR proportion in two culture mediums shows that it is not consistent between different targets and no conclusion can be drawn.

  my project, I investigated the role of sequence and chromatin context in the process of DNA doublestrand break repair and the influence of the cell type on it. Cell types in the focus were mouse ES cells and 3T3 cells. ES cells were selected as a model because they were reported to have unique pluripotencyspecific features of DNA repair, and because of the abundance of bivalent chromatin. 3T3 cells were used as a model of differentiated yet cycling cells as a line stably expressing Cas9 was previously acquired in the team. Targets for a break induction were selected at euchromatic, bivalent regions, facultative heterochromatin, and LADs. The first three target types were dictated by our wish to compare bivalent regions to chromatin types a mix of which they represent. I performed TIDE and HR-TIDE experiments for all selected targets in both cell types, in normal conditions or with cell cycle perturbations. I could confirm existing hypothesis of error-prone repair outcome being greatly dependent on the sequence around the break site, and having variations depending on a cell type.The tendency seems to be in accordance with previous observations that euchromatin seems to shift the balance between repair pathway towards HR, as compared to transcriptionally inactive regions such as facultative heterochromatin and bivalent domains, although due to the small number of investigated targets I cannot claim statistical significance. However, these observations come into contradiction with the results of a study bySchep et al. that was recently published on BioRxiv[START_REF] Schep | Impact of chromatin context on Cas9induced DNA double-strand break repair pathway balance[END_REF]. They addressed the question of the role of the chromatin environment in repair pathway choice by inserting a short barcoded reporter cassette with a well-characterised Cas9 target site into multiple genomic locations in a human immortalised cancer cell line. Analysing thousands of target sites, they observed a negative correlation between the presence of H3K27me3 chromatin mark and NHEJ employment and could also see that the euchromatic environment promotes the use of NHEJ. In our investigation we could see prominent recruitment of NHEJ in euchromatic targets in 3T3 but not in ES cells. However, somatic 3T3 cells seem to be a more proper line to compare to another differentiated cell line, such as K562 utilised by Schep et al. At the same time, we did not see the anticorrelation between H3K27

Standard

  cell lines, antibiotics selection begun 24 h post transfection and continued for 2 days with Puromycin (at a final concentration of 2mM) or for 10 days with G418 (at a final concentration of 400 mM).

  transfection, reverse transfection protocol using Lipofectamine 2000 was applied. Cells were pre-treated with Shield1 (at a final concentration of 1:2000) overnight prior transfection. For the transfection, cells were trypsinised and separated from feeders by gravity in 15 ml Falcon tubes for 15 minutes. ES cell containing supernatant was separated from a pellet of feeders and transferred into fresh Falcon tubes. 0.5*10 6 cells per sample were taken if transfecting cells cultured in the standard ES cell medium. In the time of incubation, transfection mixes were prepared. Transfection mix 1 contained total of 5 mg of DNA (when transfecting for HR-TIDE experiments, HR template and a guide RNA were cotransfected at a 3:2 ratio, for template only or guide samples a corresponding plasmid was replaced by the same amount of the empty vector), 10 ml of Lipofectamine 2000, and 250 ml of OptiMEM medium.

  Cells were rinsed with PNS and fixed with 1% PFA in PBS for 15 minutes at 37°C. Cells were washed twice with ice-cold PBS and collected by scraping on ice in PBS with PIC and PhoSTOP. Scraped cells were pulled down at 4°C for 5 minutes at 1200 rpm. Starting from scraping, all samples and buffers were kept on ice, except for SDS-containing sonication buffer. After removing supernatant cells were incubated for 10 minutes of ice in Lysis buffer 1 and centrifuged at 4°C for 2 minutes at 6000 rpm. After removing supernatant cells were incubated for 10 minutes in Lysis buffer 2 and centrifuged at 4°C for 2 minutes at 6000 rpm. Supernatant was removed and after addition of Sonication buffer cells were transferred into Covaris sonication tubes and sonicated for 15 minutes. 50 ml of Protein G Dynabeads was used for each sample. Beads were washed 3 times with 5 mg/ml BSA in PBS, blocked in 5 mg/ml BSA in PBS for 30 minutes at 4°C on a rotating wheel and coated with antibodies for 4 hours. Immunoprecipitation was performed with 20 mg of sonication materiel. Samples were diluted with ChIP dilution buffer and immunoprecipitated overnight at 4°C. Samples were washed twice with SDS wash buffer, once in high salt buffer and once in TE buffer. Samples were eluted with Elution buffer and decrosslinked overnight at were collected 48h post transfection.gDNA extraction was performed by Machry-Nagel NucleoSpin tissue kit according to manufacturer's protocol.PCR amplification was performed using Invitrogen Taq Polymerase.PCR cycle:Stage 1 (repeated once): 95°C, 5 min Stage 2 (repeated 35 cycles): 95°C, 30 sec; 60°C, 30 sec; 72°C, 1 min 30 sec Stage 3 (repeated once): 72°C, 10 min; 10°C, indefinitely PCR cleanup was performed using Beckman AMPure XP kit according to manufacturer's protocol.

  Ces derniers sont souvent observés au niveau des régulateurs du développement et sont impliqués dans la différenciation rapide des cellules ESC. Ainsi, interfère avec le cycle cellulaire, augmentant la proportion de cellules dans la phase G1.Les conséquences de cette perturbation pourrait être une augmentation de l'utilisation de la NHEJ, Cependant nous avons pu écarter cette hypothèse en démontrant qu'il n'y a aucun changement dans les profils de cycle cellulaire des ESCs dans toutes les conditions.Nous avons également remarqué que bien que l'efficacité globale de TIDE dépend du type de cellule, le schéma des insertions et des suppressions (indels) est assez similaire entre les cellules ESC et 3T3 et dépend des ARN guides utilisé pour cibler la protéine Cas9. Comme les délétions inférieures à 10nt sont considérées comme des produits de NHEJ et de plus de 10nt de AltEJ, nos résultats suggèrent que

	observé que les domaines bivalents présentent des niveaux intermédiaires de HR, soutenant l'idée qu'ils Comme perspectives d'avenir, plusieurs expériences peuvent être planifiées. En tirant parti du système Introduction
	représentent un état intermédiaire entre la chromatine active et la chromatine refoulée. En ce qui Cas9 inductible et dégradable (DD/shield1), il est possible d'effectuer des expériences ChIP pour surveiller
	nous avons choisi d'induire des DSB sur plusieurs gènes bivalemment marqués. Ces gènes (Pax6, Zic1 et concerne les gènes situés dans les LAD, nous avons observé une certaine variabilité, certains gènes étant la cinétique d'apparition et de disparition de plusieurs facteurs de réparation de l'ADN et des voies de DNA damage and repair
	Ngn2) sont exprimés à des niveaux très faibles dans les ESCs à des niveaux élevés dans certains types de totalement réprimés et d'autres présentant une compétence plus élevée en matière de ressources signalisation appelées DDR (Damage Response Repair) (H2AX et 53BP1) à différents moments après ajout DNA is constantly assaulted by various endogenous and exogenous damaging agents. Timely and faultless
	cellules différenciées et régulent le développement neuronal. Ces loci ont été comparés à des gènes humaines, ce qui pourrait s'expliquer par leur emplacement à l'intérieur ou à la limite de domaines. et retrait de shield1. Comme ci-dessus, ces expériences doivent être effectuées dans des cellules ES et des correction of acquired damage is necessary for the health and normal survival of an organism. Failure to
	considérés comme des marqueurs de pluripotence (Nanog, Pou5f1, Tfcp2l1, Zfp42) qui s'expriment Voyant des différences dans la fréquence des HR, nous avons réalisé que le cycle cellulaire diffère entre cellules 3T3. Pour corréler l'induction et la réparation des DSB réelles avec l'augmentation de la DDR et properly repair mutilated DNA can lead to severe consequences such as mutagenesis, ageing, and cancer.
	fortement dans les mESCs et sont éteint pendant la différenciation. Nous avons également choisi des les cellules ES et 3T3, la première ayant un G1 beaucoup plus court et donc un plus grand pourcentage de l'arrêt de l'induction de ces DSB, la technique de LM-PCR, permettant de quantifier précisément le
	gènes domestiques (Actb, Gapdh) qui sont exprimés en permanence, et des gènes que l'on rapporte cellules capables de ce mode de réparation. Pour répondre à cette question, nous avons d'abord décidé nombre de cassures ADN, doit être effectuée en même temps après l'ajout et le retrait du shield1 dans
	comme totalement réprimés en présence des marques d'histones H3K27me3 dans les cellules ES (Hoxb1, de bloquer les cellules 3T3 en phase G2 du cycle cellulaire à l'aide d'un inhibiteur RO. Conformément aux toutes cassures et types de cellules.
	rapports précédents, nous avons pu constater une augmentation considérable du pourcentage des Cependant, pour obtenir une image plus large de l'influence de la chromatine sur le choix de la voie de
	ressources humaines. Cependant, nous avions besoin d'une preuve complémentaire que l'extension de la réparation de l'ADN, il serait avantageux d'effectuer une expérience à plus grande échelle. L'optimisation
	phase G1 des cellules ES entraînerait une baisse de l'efficacité RH. Afin de modéliser une telle situation de la conception de séquences homologue pour le HR-TIDE, d'une part, et la disponibilité de données sur
	sans engager les cellules dans une démarche de différenciation, nous avons décidé d'utiliser un milieu de l'efficacité de l'édition du génome de différents guides à l'échelle du génome, d'autre part, permettent de
	culture cellulaire appelé « 2i medium ». Le 2i pour la culture des cellules ES est exempt de sérum et combiner ces deux approches. Pour ce faire, une bibliothèque contenant des ARN guides et des
	et les cellules 3T3 qui expriment de façon stable Cas9 ont été co-transfectées avec un guide ARN et la comprend un cocktail de deux inhibiteurs (inhibiteur MEK et inhibiteur GSK-3, qui bloquent séquences homologues correspondantes pourrait être clonée dans des vecteurs viraux non intégratifs et
	séquence homologue donneuse. Ensuite, le locus a été amplifié par PCR et soumis à une analyse TIDE. La respectivement les voies de signalisation MEK/ERK et Wnt/-Catenin). Cependant, nous n'avons pas pu utilisée pour l'infection de cellules ES ou cellules différenciées, suivit d'une analyse des profils de
	fréquence d'insertion de 9 pb à chaque emplacement génomique indiquait la fréquence des HR. En observer une baisse significative de l'efficacité des ressources humaines dans les gènes actifs, ce qui réparation à l'aide de techniques NGS.
	utilisant la méthode TIDE ainsi modifiée (que nous avons appelée HR-TIDE), nous avons pu obtenir une indique que les différences de cycle cellulaire entre les types cellulaires ne peuvent être à elles seules
	comparaison de l'efficacité HR entre différents types de chromatine, ainsi qu'entre les cellules 3T3 et ES. responsables des différences dans le choix d'une voie de réparation. Dans le même temps, nous avons
	Nous avons pu constater que la fréquence HR dans les cellules 3T3 était généralement faible et ne remarqué une diminution de l'utilisation du mécanisme de HR dans les gènes inactifs, ce qui correspond
	dépassait jamais 20 %. Nous avons pu observer une plus grande variabilité dans les cellules ES, allant au fait que la bivalence est perdue dans les conditions 2i.
	d'aussi peu que 3 % chez certains inactifs jusqu'à 40 % chez les gènes actifs. En général, en utilisant HR-Dans l'ensemble, nous avons établi un système inductible et dégradable pour évaluer la réparation des
	TIDE, nous n'avons pas pu confirmer les rapports précédents selon lesquels 80 % des ORD sont réparés à DSB et nous avons proposé plusieurs essais pour étudier le choix de la voie de réparation de l'ADN. Nos
	l'aide de HR, bien qu'ils apportent une plus grande contribution que dans les cellules différenciées, ce qui données ont mis en lumière le rôle de la chromatine bivalente et de l'hétérochromatine facultative dans
	correspond aux idées actuelles sur le terrain. le processus de choix de la voie de réparation de l'ADN. En même temps, nous avons proposé et optimisé
	En comparant l'efficacité de la HR entre différents contextes chromatiniens, nous avons pu confirmer une méthode simple et rapide d'accès à la compétence HR d'un lieu particulier dans un contexte
	l'observation que les régions de chromatine activent transcriptionnellement sont plutôt promotrice du m, cellulaire, qui peut être d'une utilité pratique dans la conception de mutants knock-in utilisant le système
	alors que l'hétérochromatine facultative représente un environnement répressif. Nous avons également CRISPR-Cas9.

Le but de mon projet était d'étudier la cinétique et les mécanismes de réparation du DSB et sa relation avec le contexte chromatinien dans lequel des DSB sont induites. À cette fin, j'ai utilisé le systeme CRISPR-Cas9 pour induire des DSB dans divers contextes chromatiniens dans des ESC ou des cellules différenciées. J'ai généré des lignées cellulaires mES stables exprimant une protéine Cas9 fusionnées à une GFP et à un domaine dégron (DD). En présence de la drogue appelée Shield1, le domaine dégron est masqué, stabilisant l'expression de la protéine Cas9 dans les cellules. J'ai observé que entre 8 et 10h après de la drogue shield, la protéine Cas9GFP atteint son niveau maximum d'expression. De plus, 10h après le retrait de la drogue shield1, le niveau d'expression de la protéine Cas9GFP retombe à un niveau basal. Pour induire des DSB dans les différents contextes chromatiniens, j'utilise des plasmides ou des ARN guides transcrits in vitro qui ciblent des sites génomiques comportant différentes modifications chromatiniennes. Nous avons choisi d'étudier des domaines bivalents qui n'ont jamais été étudiés auparavant en termes de réparation de l'ADN. Tdrd1, Mc4r). Nous avons également cherché à inclure dans la comparaison certains gènes appartenant aux LADs dans les cellules ES (Sox6, Ptn, Nrp1). À cette fin, nous avons conçu deux guides d'ARN par gène pour introduire les DSB dans leurs promoteurs. Nous avons utilisé la lignée cellulaire « Cas9-expressing NIH 3T3 » précédemment établie en laboratoire pour obtenir une comparaison avec un type cellulaire différencié. Pour mesurer efficacement la NHEJ ainsi que la fidélité de réparation des cassures induites dans les contextes chromatiniens décrits ci-dessus dans les cellules mESCs et NIH3T3, nous avons utilisé une méthode appelée TIDE (Tracking of Indels by Decomposition). TIDE est une méthode basée sur le séquençage qui nous permet de quantifier un pourcentage de séquences mal réparées dans la population, ainsi qu'un pourcentage d'insertion ou de suppression de séquences particulières. Les résultats de l'étude TIDE ont montré que les ESC utilisent la NHEJ ou d'autres voies sujettes aux erreurs plus que prévu dans la littérature. Fait intéressant, nous avons également observé que la fréquence relative des réparations erronées varie selon le type de gènes où la cassure a été introduite. Les ESC utilisent moins les voies de réparation sujettes aux erreurs que les cellules 3T3 pour les gènes de ménage et les régulateurs du développement. Cependant, nous avons pu observer l'inverse pour les gènes marqueurs de pluripotence, suggèrant que la structure chromatinienne ainsi que les niveaux de transcription influencent tous les deux l'efficacité de TIDE. Ce résultat s'explique par la possibilité qu'en perturbant un promoteur d'un régulateur de pluripotence, on l'équilibre NHEJ/AltEJ ne dépend pas directement de la structure chromatiniennes ou du type cellulaire.

Malheureusement, la méthode TIDE ne nous a pas permis de distinguer l'ADN non coupé par Cas9 ou réparé à l'aide de HR. Pour mesurer l'efficacité des RH, nous avons dû modifier la méthode en tirant parti de la sensibilité de TIDE pour mesurer l'efficacité des petites insertions après une induction de DSB. À cette fin, pour chaque ARN guide utilisés, nous avons conçu une séquence donneuse pour la HR spécifique au locus ciblé contenant une homologie de 1000bp entourant une séquence ADN unique de 9bp. Les ESC

  Altogether, despite certain similarities in the experimental setup, we mostly addressed different questions from those of Schep et al. Our studies could therefore be considered as complementary for clarification of the role of chromatin features in defining DSB repair strategy.Sequence predominantly defines the DSB repair outcome. Chromatin and cell type might have no more than moderate influence on the DSB repair outcome After analysing indel patterns for all target sites (26 guide RNAs) designed to induce DSBs in different chromatin contexts in two different cell types I could see no correlation with any of these factors. All types of repair outcomes, including small insertions and deletions, thought to be resulting from NHEJ employment, and larger deletions normally connected to MMEJ could be observed in all chromatin types.

Table 2 .

 2 Primers used for HR-TIDE

	Gene	Guide RNA	Forward primer	Reverse primer
		number
	Actin	Pg255	CGTAGCGTCTGGTTCCCAAT	AGGTGCGTGTCCTTCTAAGC

easily accessible, which is largely promoted by histone acetylation[START_REF] Bassett | The folding and unfolding of eukaryotic chromatin[END_REF][START_REF] Görisch | Histone acetylation increases chromatin accessibility[END_REF][START_REF] Lawrence | Lateral Thinking: How Histone Modifications Regulate Gene Expression[END_REF][START_REF] Otterstrom | Superresolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo[END_REF][START_REF] Shogren-Knaak | Histone H4-K16 acetylation controls chromatin structure and protein interactions[END_REF]. As long as euchromatin is in focus, the matter seems to be relatively straightforward. But heterochromatin comes to the spotlight, no simplicity is left. Heterochromatin is a very complex and complicated concept, as there is no such thing as just heterochromatin. Heterochromatin is often claimed to be compacted but not all data support this theory, and it is possible that heterochromatic regions are inaccessible, not due to physical compaction but rather an environment created by associated proteins[START_REF] Boettiger | Super-resolution imaging reveals distinct chromatin folding for different epigenetic states[END_REF][START_REF] Hildebrand | Mechanisms and Functions of Chromosome Compartmentalization[END_REF][START_REF] Liu | Establishment and evolution of heterochromatin[END_REF][START_REF] Otterstrom | Superresolution microscopy reveals how histone tail acetylation affects DNA compaction within nucleosomes in vivo[END_REF][START_REF] Ou | ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells[END_REF][START_REF] Xu | Super-Resolution Imaging of Higher-Order Chromatin Structures at Different Epigenomic States in Single Mammalian Cells[END_REF]. There are two distinct heterochromatin types, constitutive and facultative, LADs are considered separately as they are distinct from both of them(Brown, 1966;[START_REF] Fu | Functions of p53 in pluripotent stem cells[END_REF][START_REF] Hildebrand | Mechanisms and Functions of Chromosome Compartmentalization[END_REF][START_REF] Nagano | Single-cell Hi-C reveals cell-to-cell variability in chromosome structure[END_REF].Constitutive heterochromatin, marked with H3K9me3, is predominantly formed around genomic regions that have to be strictly inhibited, such as repetitive elements such as satellite DNA, ribosomal DNA, and transposable elements, elements of viral origin or inactivated X chromosome[START_REF] Litt | Correlation between histone lysine methylation and developmental changes at the chicken β-globin locus[END_REF][START_REF] Liu | Establishment and evolution of heterochromatin[END_REF][START_REF] Nakayama | Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly[END_REF][START_REF] Noma | Transitions in distinct histone H3 methylation patterns at the heterochromatin domain boundaries[END_REF][START_REF] Savitsky | Heterochromatin Protein 1 Is Involved in Control of Telomere Elongation in Drosophila melanogaster[END_REF][START_REF] Whitehead | Neurones of the Male and Female , and the Behaviour of the Nucleolar Satellite during[END_REF].H3K9me3 promotes recruitment of HP1, which silences transcription and ensures formation and maintenance of heterochromatic domains[START_REF] Bannister | Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain[END_REF][START_REF] Lachner | Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins[END_REF][START_REF] Lehnertz | Suv39h-Mediated Histone H3 Lysine 9 Methylation Directs DNA Methylation to Major Satellite Repeats at Pericentric Heterochromatin[END_REF][START_REF] Peters | Partitioning and Plasticity of Repressive Histone Methylation States in Mammalian Chromatin[END_REF][START_REF] Schotta | A silencing pathway to induce H3-K9 and H4-K20 trimethylation at constitutive heterochromatin[END_REF][START_REF] Zeng | Specific loss of histone H3 lysine 9 trimethylation and HP1γ/cohesin binding at D4Z4 repeats is associated with facioscapulohumeral dystrophy (FSHD)[END_REF]. Constitutive heterochromatin plays an important role in regulating genome stability by protecting telomeres and pericentromeres from inappropriate treatment by DNA repair machinery, recombination, and chromosomal rearrangements and by preventing transposable elements activation[START_REF] Eberhart | Epigenetics of eu-and heterochromatin in inverted and conventional nuclei from mouse retina[END_REF][START_REF] Fu | Functions of p53 in pluripotent stem cells[END_REF][START_REF] Peng | H3K9 methylation and RNA interference regulate nucleolar organization and repeated DNA stability[END_REF][START_REF] Kunkel | Evolving Views of DNA Replication (In) Fidelity[END_REF][START_REF] Savitsky | Heterochromatin Protein 1 Is Involved in Control of Telomere Elongation in Drosophila melanogaster[END_REF][START_REF] Sentmanat | Ectopic assembly of heterochromatin in Drosophila melanogaster triggered by transposable elements[END_REF].On the contrary, facultative heterochromatin is formed in those parts of a genome that might be transcribed, but have to be inactivated in a particular cell type[START_REF] Hübner | Remodeling of nuclear landscapes during human myelopoietic cell differentiation maintains co-aligned active and inactive nuclear compartments[END_REF][START_REF] Lewis | A gene complex controlling segmentation in Drosophila[END_REF][START_REF] Liu | Establishment and evolution of heterochromatin[END_REF][START_REF] Ou | ChromEMT: Visualizing 3D chromatin structure and compaction in interphase and mitotic cells[END_REF][START_REF] Solovei | How to rule the nucleus: divide et impera[END_REF][START_REF] Zhu | Genome-wide chromatin state transitions associated with developmental and environmental cues[END_REF]. A hallmark of facultative heterochromatin is H3K27me3 histone modification, which is established and maintained by Polycomb repressive complexes, PRC1 and PRC2 complexes[START_REF] Boyer | Polycomb complexes repress developmental regulators in murine embryonic stem cells[END_REF][START_REF] Bracken | Genome-wide mapping of polycomb target genes unravels their roles in cell fate transitions[END_REF][START_REF] Czermin | Drosophila enhancer of Zeste/ESC complexes have a histone H3 methyltransferase activity that marks chromosomal Polycomb sites[END_REF][START_REF] Kuzmichev | Histone methyltransferase activity associated with a human multiprotein complex containing the enhancer of zeste protein[END_REF][START_REF] Liu | Establishment and evolution of heterochromatin[END_REF][START_REF] Müller | Histone methyltransferase activity of a Drosophila Polycomb group repressor complex[END_REF][START_REF] Simon | Occupying Chromatin: Polycomb Mechanisms for Getting to Genomic Targets, Stopping Transcriptional Traffic, and Staying Put[END_REF][START_REF] Wiles | H3K27 methylation: a promiscuous repressive chromatin mark[END_REF]. The only methyltransferase that is capable of creating this mark is a PRC2 catalytic subunit

Acknowledgements

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