My Father: Late Mr Jacob Charles Philip Prasad My Mother: Esther Rani Charles My Brother: Charles Jacob Harrys Kishore My Guide: My Lord and Saviour Jesus Christ that there is a long way to go in order to emulate his ideas but the one thing I can never forget is his commitment to excellence in quality research. A characteristic trait of his that I would like to make my own is his never ending hunger and passion for science and it has been the one feature that I cherish and would like to thank him for being patient in me and training me to be what I am today. I thank Dr Stefan Dimitrov for being such an inspiring person and more importantly for being my guide in the truest sense.

My thanks to Dr Dimitar Anguelov, my thesis co-guide, for teaching me the essentials of chromatin biology and biochemistry. His guidance in designing experiments and executing them is something that I will carry on with great pride. I would never be able to carry out any of these experiments without him. I thank him for being patient with me and my mistakes and for always being there to support me and to constantly encourage me in all my experiments.

The discussions with him are loaded with information and experience that there is no way the experiments can go haywire. I thank him once again for guiding me through my PhD I wish to express my sincere thanks to Dr. Pablo Radicella, Dr. Patrick Lomonte, Dr. Pierre Couble and Prof. Hans Geiselmann for their invaluable time spent on the evaluation of the thesis.

My heartfelt gratitude to the Model-In consortium and the European Communitys Seventh

Framework Programme FP7/2007-2013(222008)under which I was hired. This has indeed been a portal for me to exchange views and ideas with the many collaborators in the consortium.

My thanks to my collaborators Dr Ali Hamiche for providing me with the FACT protein complexes and Dr Tapas Kundu for his insights and suggestions. Special thanks to Dr. Zahari Peshev for helping us out with the Laser experiments. It is impossible to forget the many conversations we have had during the stop flow experiments.

My sincere thanks to Dr. Manu Shukla, for training me in the initial months of my stay in France. He has been an awesome teacher, friend and a selfless pedagogue with whom I had the privilege of learning the art of biochemistry. His cool nature, his experience and his words of advice helped me carry out experiments with ease. I am immensely glad to be trained under him and would love to convey my wishes and my prayers for becoming a great scientist.

My thanks, to Dr Sajad Hussain (Sajjus), the H1 from the mountains, for showering upon me, his protein gyan (knowledge). His smile and his helping tendency at any odd time makes him a wonderful teacher and a friend one can bank on. Being in his company I have learnt to keep things easy and simple. I would like to wish him luck in all his future endeavours.

My special thanks to my roomie, friend and philosopher Imtiaz Nisar Lone (Loney BABA) for being there always listening to me and sharing unforgettable moments here in France. He has always been like an elder brother to me and am grateful for his long discussions and critical reviews of my work. Being with him in the lab and his constant support and help is something I can never forget. All the best bro, wishing you loads of luck and success.

I would like to thank Lama Soudein (Lamoush), for being a wonderful lady she is, and for being the perfect personal secretary. Thank you, Lamoush for bearing with me and tolerating all my meaningful conversations and for keeping the atmosphere in the lab cheerful. I would also like to thank Mladen (Malaka) for being the ultimate snack provider every single day of his stay here in France. He has been a very good friend and brought smiles on everyone in the lab. Keep up with your hearty nature machan.

My thanks to Ognyan Tonchev(Gru) for capturing every single moment spent in the lab on his camera. No matter where we go he would be there like the paparazzi to capture the event.

My thanks, to Tala (Mozarts sister) for ending all her sentences with reallyyy and Watt and for forcing me to speak in French.

My special thanks to Elsa, our lab manager, for keeping the lab updated and not giving chance for the chemicals to run short of and always being there providing us with whatever we needed.

My thanks to Veronique Gerson, who has been instrumental in providing me with RSC, the wonder molecule that helped me complete my thesis and for her initial struggle in arranging all the paper works to get me here in France.

I would like to express my gratitude to Aude Marchand, for helping me out with all the official paper works and reimbursements.

A special word of thanks to mamma Olimpia and to Dane (Mr Chak-chak) for their kind help and support.

My thanks to Manju (Jhunjhu) and his wife Shwetha for making me feel like home here in France. My heartfelt gratitude to Sadhan Chandra Das for always being there in times of need and always providing an ear to all I had to say. I would like to thank my friends Farhan, Esber, Julia, Armel, Juliette, Monica, Loan, Meharnaz, Chandan and Xavier for making my stay here in France extremely wonderful and for being helpful. Thanks to Julie and Lydia for providing me a wonderful chance to showcase myself at the Todai Forum. Special thanks, to my buddies at the French class Georges, Malgorzata, Jancek, Olga and Adrian. Special thanks to my brother Charles Jacob Harrys Kishore for being instrumental in my career as a researcher. I have never hesitated in approaching him and have always banked on him. It is always he who puts me on the right track when I stray aside. Besides, being in his This would mean nothing if I could never thank my father Mr. Jacob Charles Philip Prasad who is not there with me but I do know that you are watching me daddy and I am now completing my Ph.D. and I know you are watching from heaven. You have taught me never to give up and be honest in all that I did. Dear dad, every single moment spent with you is something that is indelible and something that I will cherish for the rest of my life. Every single word of advice is reverberating through my mind, constantly reminding me of how great a dad you have been to my brother and me. I just feel sad that you are not around to see me now.

It goes without question that my mom, Mrs Esther Rani Charles deserves the best mention for going through all the pain and suffering without dad and for making my brother and me, what we are today. You have showered upon us your unconditional and selfless love and have never stepped back is providing us with what we need. Thank you mom for always being my best friend and listening to all I had to say every single day with patience, I knew that I was spoiling your sleep but my day would remain incomplete without talking to you. Mom thank you for everything that you have ever done and for your blessing that has always been on us.

Not a day would have gone by had it not been for your prayers. Thank you once again mom and love you loads.

I would like to express my thanks to the Lord for guiding me throughout my life. His grace and mercy has guided me through my PhD and has given me the energy, wisdom and courage to complete it in his name.

List of Figures

Chromatin Organisation and Chromatin architecture

General properties of Euchromatin and heterochromatin Incorporation of histone variants and the structural changes and perturbations incurred

Figure 7

The SWI/SNF Family of remodelers and their functions

The ISWI group of remodelers and their functions

The CHD family of Chromatin Remodelers

Figure 10

The INO80 family of remodelers

Figure 11

Chromatin Remodeler Domains

Electron microscopy reconstructions of ISWI class of remodelers

Figure 13

Biochemical properties of ATP dependant Chromatin Remodelers

Nucleotide Excision Repair Pathway

A schematic representation of both short-patch and long-patch base excision repair

B. Abstract

Résumé en Français

FACT est une protéine clé, qui joue de multiples rôles, y compris dans la transcription et la réparation!de!lADN!endommagé.!Néanmoins,!comment!!FACT!participe!à!la!réparation!et!à! la!transcription! de!la!chromatine!nest! pas! élucidé.!Dans! ce!travail!nous! avons! tout!dabord! étudié le rôle de FACT dans le processus de réparation par excision de base (BER). Nous avons! utilisé! des! nucléosomes! reconstitués! avec! de! lADN! les thymines sont partiellement substitués!avec!de!luracile.!Nous!avons!trouvé!que!lenzyme!UDG!est!capable!denlever!les! uraciles localisés du côté de la solution et pas les uraciles se trouvant en face de loctamer dhistones. La présence simultanée de FACT et de RSC (facteur de remodelage de la chromatine, impliqué dans la réparation) permet un enlèvement efficace des uraciles localisés du côté de loctamer dhistones par!lUDG.!De!plus,!laction!concertée!de!FACT!et! RSC contribue à!lenlèvement!de!la!lésion! oxydative 8-oxoG, autrement inaccessible, de la matrice!nucléosomale!par!lenzyme!OGG1.!Ce!résultat!est!obtenu!grâce!à!une!activité!« coremodelatrice » de la protéine FACT. Dans ce travail nous décrivons pour la première fois cette! nouvelle! propriété! de! FACT! et! ! nous! montrons! par! une! série! dexpériences! biochimiques! que! FACT! est! capable! de! stimuler! lactivité! de! remodelage! du! RSC.! Nos! expériences montrent que la présence de FACT!augmente!lefficacité!de!RSC!à!transformer! lénergie!libérée!par!lhydrolyse!de!lATP!en!travail! « mécanique ». Les données obtenues suggèrent une nature stochastique du BER in vivo, FACT étant un facteur clé dans le processus de réparation. Nous avons également! ! investigué! ! limplication! de! lactivité! coremodelatrice de FACT dans la fixation de NF-B! ! aux matrices nucléosomales. La production de nucléosomes remodelés, mais non -mobilisés!(remosomes)!nest!pas!suffisante!! pour promouvoir la fixation de NF-B. Pourtant, la mobilisation des nucléosomes par lintermédiaire!de!RSC!permet!une!interaction!efficace!entre!NF-B!! et!lADN!nucléosomal.!! Toutes ces données sont essentielles pour le décryptage du mécanisme moléculaire par lequel FACT agit dans le BER et dans la transcription médiée par NF-B.

Abstract in English

FACT is a vital protein which plays multiple roles in several processes including both transcription and repair of damaged DNA. However, how FACT assists repair and transcription remains elusive. In this work, we have first studied the role of FACT in Base Excision Repair (BER). We used nucleosomes containing DNA with randomly incorporated uracil. We found that the enzyme UDG is able to remove uracils facing the solution and not the uracils facing the histone octamer. The simultaneous presence of FACT and RSC (a chromatin remodeler involved in repair) allows, however, a very efficient removal of uracil facing the histone octamer by UDG. In addition, the concerted action of FACT and RSC permits the removal of the otherwise un-accessible oxidative lesion 8-oxoG from nucleosomal templates by OGG1. This was achieved by the co-remodeling activity of FACT, a novel property of this protein that we have discovered and analysed. The experiments reveal that the presence of FACT increases the efficiency of RSC to transform the energy released by!ATP!hydrolysis!into!mechanical!work.!The!presented!data!suggest!a!stochastic! nature of BER functioning in vivo, with FACT being a key factor in the repair process. The implication of the co-remodeling activity of FACT in NF-B factor binding to nucleosomal templates was also investigated. The generation of remodeled, but not mobilized nucleosomes (remosomes), was not sufficient to promote NF-B!binding. However, the RSCinduced nucleosome mobilization allows efficient NF-B!interaction with nucleosomal DNA. Nature is a grandmaster when it comes to building one atom and one molecule at a time.

The master works of nature include well ordered, well structured materials be it the synthesis of materials like bones, teeth and corals or the more complex molecular machines that patrol the human body making biological processes like replication, signaling, transcription, translation and DNA repair. These exquisitely designed molecular machines have left us pondering at the mystery of how they function and how they are made in the first place.

In this thesis, we make an attempt at studying the dynamics and function of one class of these machines termed chromatin remodelers and the role of histone chaperone FACT (Facilitates Chromatin Transcription) in assisting their function in both nucleosome mobilization and transcription factor NF-B!binding.

Chromatin Structure, Organization and Dynamics

Chromatin

Landmarks in the field of Chromatin

In the nucleus the DNA exists as a complex structure called chromatin, which is composed of DNA and proteins (The!word!chromatin!is!derived!from!the!Greek!word!Khroma!meaning! colored, based on its ability to stain with basic dyes).!The!term!Chromatin!was!suggested! for the first time by W Flemming. In the year of 1953, there was the discovery of the DNA double helix structure by Watson and Crick and two separate groups involving Wilkins and

Franklin [1; 2; 3]. In the year of 1959, Zubay and colleagues were able to prepare soluble chromatin [START_REF] Medawar | Preparation of nucleoprotein and deoxyribonucleic acid from small quantities of lymphoid tissue disintegrated by ultrasound[END_REF] and histones were fractionated by Johns [START_REF] Phillips | A study of the proteinase content and the chromatography of thymus histones[END_REF]. With the advent of the electron microscopy H. Davies went on to report the chromatin threads that were 30nm in dimensions and were isolated from chicken erythrocyte nuclei [START_REF] Everid | Electron-microscope observations on the structure of condensed chromatin: evidence for orderly arrays of unit threads on the surface of chicken erythrocyte nuclei[END_REF]. In the following years Klug was able to purify these chromatin preparation and also suggested the solenoid model [START_REF] Finch | Solenoidal model for superstructure in chromatin[END_REF]. Olins and Woodcock!reported!individually!the!beads!on!the!thread!which!the!former!called!as!v!bodies [8; 9]. In 1974, Roger Kornberg in collaboration with J.Thomas postulated a model for the chromatin structure and in the subsequent year Pierre Chambon went ahead to coin the term Nucleosome for the chromatin subunit [10; 11]. With the advance in technology and the boon of X-ray crystallographic studies we were able to profit from the crystal structure of the nucleosome [START_REF] Luger | Crystal structure of the nucleosome core particle at 2.8 A resolution[END_REF].

Chromatin and Chromosome Structure

The long strands of chromosomal DNA in the eukaryotic nucleus are approximately compacted 10000 to 50000 times in length. In spite of the tight packaging of the DNA the chromosome maintains its form and structure allowing regulatory proteins to access it, for transcription or replication. The molecular self-assembly takes place with DNA wrapping around Histones. These histones are positively charged molecules that have a strong affinity towards the negatively charged DNA. When visualised under an electron microscope they appear as beads on a string every 200 bp [START_REF] Aalfs | What does 'chromatin remodeling' mean?[END_REF]. Each bead is a nucleosome core particle that includes approximately 146 bp of DNA wrapped almost twice around a core histone octamer which comprises of a pair of each histone H2A, H2B, H3 and H4.

The Histone H1 (termed also as "linker" histone) binds to 40-70 bp of linker DNA, that separates adjacent core particles and helps compact the beads-on-a-string into fibers ~30 nm in diameter [7; 14]. When viewed under the electron microscope, these 30-nm fibers appear as helical structures with six nucleosomes per turn, an arrangement in which the DNA has been compacted ~40-fold in its linear dimension.

Laemmli and his colleagues [15; 16] took some stunning electron micrographs and provided enough insights into the organizations of the chromosomes. Their images mainly involved HeLa cell metaphase chromosomes stripped of histones showing DNA spooling out 30 to 90kb loops from a proteinaceous "scaffold" that is the X shape of the paired sister chromatids [START_REF] Paulson | The structure of histone-depleted metaphase chromosomes[END_REF]. If the histones were not stripped from the DNA then they appeared as loops of chromatin made up of approximately 180 to 300 nucleosomes coiled in the 30-nm fibre [START_REF] Marsden | Metaphase chromosome structure: evidence for a radial loop model[END_REF].

In cross section, the loops appear to radiate from the scaffold. Adjacent loop attachment sites are arranged in a helical spiral along the long axis of the metaphase scaffold [START_REF] Marsden | Metaphase chromosome structure: evidence for a radial loop model[END_REF]. Organizing 15 to 18 such loops per turn along the chromatid would account for ~1.2 million bp of DNA [START_REF] Nelson | The role of the nuclear matrix in the organization and function of DNA[END_REF]. This arrangement probably allows the stacking of loops into a cylinder of chromatin ~800 to 1000 nm in thickness, which is in good agreement with the diameter of the metaphase chromosome [15; 17]. This model also accounts for the dimensions of metaphase chromosomes, which are ~10,000-fold shorter and 400 to 500-fold thicker than the double stranded DNA helices contained within them.

Figure 1 shows the step wise breakdown of the chromosomes into the chromatin and subsequently nucleosomes. On the right panel we see the two models of the 30nm fiber, the solenoid and the zigzag model. Chromosomes are divided into two distinct domains based on their structural and functional properties. The chromosomal regions that do not undergo post mitotic decondensation were termed as heterochromatin and the chromosomes that decondense and spread out in the interphase were referred to as the euchromatin [START_REF] Passarge | Emil Heitz and the concept of heterochromatin: longitudinal chromosome differentiation was recognized fifty years ago[END_REF]. The general properties of euchromatin and heterochromatin are summarised in Figure 2.

The Nucleosome

The Nucleosome is the basic subunit of the chromatin and is interconnected by stretches of DNA called the linker DNA. Kornberg defined nucleosomes, as structures with about 200bp of DNA in close association with the core histones H2A,H2B, H3 and H4 [START_REF] Kornberg | Chromatin structure: a repeating unit of histones and DNA[END_REF]. In the absence of the linker histone the nucleosomes appear as beads on a string [START_REF] Olins | Spheroid chromatin units (v bodies)[END_REF], but in the presence of the linker histone they condense and the linker histone binds at the entry and exit site of the DNA. This in a way helps in the compaction of the lengthy DNA strands. The nucleosome is formed in vitro by reconstituting the DNA and histones and a gradual step wise dialysis from 2M salt to 10mM salt is carried out. Figure 3 describes how the nucleosome is assembled, first by the deposition of tetramer and then followed by the dimer on the DNA fragment.

The Histones

Histones were first described by A.Kossel in the year 1884 as an acid extractable material isolated from chicken erythrocyte. Histones are small basic proteins which are found in all eukaryotes and are highly conserved during evolution. There are 5 canonical forms of histones: H2A (14 kDa), H2B (14 kDa), H3 (15 kDa) and H4 (11 kDa) referred to as core histones and H1 (21 kDa) referred to as linker histone. The core histones have three domains namely the Histone fold domain, the N-terminal domain and accessory helices and less structured regions.

The histone fold domain consists of a helix-loop-helix motif. However, in the absence of DNA and in moderate salt conditions of 150mM, H3 and H4 form tetramers while H2A and H2B exist as dimers. In higher salt concentration of 2M an octamer of histones is formed.

Apart from the histone fold domains there are the C-terminal and the N-terminal domains.

The histone tails are prone to modifications and in turn are involved in the maintenance of the structure of chromatin. Linker histones help stabilize the higher order architecture. The linker histone does not resemble like the other histones and instead has a tripartite structure, made up of a globular domain flanked by a highly positive N and C terminal tails [START_REF] Wolffe | Histone H1[END_REF]. The

Complete histone Octamer with DNA

globular domain of histone H1 interacts with the entry and exit site of DNA in the nucleosome. Hydroxyl radical footprinting shows a particular signature when H1 is deposited on the nucleosomes [START_REF] Syed | Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome[END_REF].

Regulation of chromatin Dynamics

Replacement by Histone Variants

The nucleosome is a barrier to numerous vital cell processes that require access to free DNA [START_REF] Beato | Transcription factor access to chromatin[END_REF]. The cell uses incorporation of histone variants, histone modifications and ATP dependant chromatin remodeling complexes to overcome this nucleosome barrier (see Figure 4) [24; 25; 26]. on chromatin remodeling complexes [START_REF] Saha | Mechanisms for nucleosome movement by ATPdependent chromatin remodeling complexes[END_REF].

The histone variants are non-allelic isoforms of conventional histones which are expressed in very low amounts in the cell [START_REF] Henikoff | Assembly of variant histones into chromatin[END_REF]. In contrast to conventional histones, histone variants are incorporated into chromatin in a replication independant manner [START_REF] Henikoff | Assembly of variant histones into chromatin[END_REF].

Multiple variants of all the core histones except histone H4 have been reported. The H2A and the H3 variants are the well studied variants. These variants exhibit a similarity ranging from 45-90% [27; 28]. Previously it was reported that the carboxyl terminal of the H2A is essential for the stability of nucleosomal particles and that H2A-H2B and the tetramer interaction is hampered when the COOH terminus is truncated. Moreover, the remodeling and mobilization of the nucleosomes is reduced when the histone variants are incorporated [29; 30; 31; 32]. Some of the major histone variants of the histone H2A are H2A.X, H2A.Z, MacroH2A(mH2A) and H2A Barr Body deficient(H2A.Bbd) [START_REF] Ausio | Histone variants and histone modifications: a structural perspective[END_REF]. The major variants with the histone H3 which has been extensively studied are the H3.3 and the CENPA [34; 35]. The Figure 5 shows the conventional histones alongside the histone variants and their possible functions. The image is adapted from a review by Sarma and Danny Reinberg [START_REF] Sarma | Histone variants meet their match[END_REF].

Incorporation of histone variants confers novel properties to the nucleosome [START_REF] Campos | Histones: annotating chromatin[END_REF].

Incorporation of histone variants and how they affect the main properties of the nucleosome is depicted in Figure 6. This image is adapted from a review on histone variants [START_REF] Campos | Histones: annotating chromatin[END_REF]. An example of the interconnections among histone modifications is the finding that deacetylation of lysine K9, located nine amino acids from the amino terminus of histone H3, is a prerequisite for methylation of this same lysine. Methylation of K9, in turn, recruits the binding of repressor proteins, such as HP1 (Heterochromatin Protein 1) that helps in establishing a highly compacted and transcriptionally inactive region of chromatin known as heterochromatin. The inter-connectedness of histone modifications that collectively influence a web of regulatory events has led to the hypothesis for a "histone code" controlling chromatin dynamics [24; 37; 38]. Such a code would allow post-translational modifications of various amino acids within the core histones to carry informational content and instructions that help specify which genes are to be activated or repressed.

In addition to modifying the histones that wrap the DNA into nucleosomes and higher-order structures, the DNA itself can be modified, most notably by the addition of methyl groups to cytosines. A high level of methylation is typically correlated with gene silencing, and is particularly evident in the silencing of transposable elements and multi-copy transgenes. A variety of DNA methyltransferases exists to modify the DNA in a variety of patterns. Some DNA methyltransferases act primarily in conjunction with replication to perpetuate methylation patterns from "mother" strands to newly synthesized "daughter" strands of a chromosome. Other DNA methyltransferases can add methyl groups to DNA strands that have no pre-existing methylation. Methylation of DNA may silence genes by preventing the binding of transcription factors. However, it is likely that cytosine methylation exerts most negative effects on gene regulation via the involvement of other proteins that bind specifically to DNA when it is methylated. Indeed, a number of methyl-cytosine binding proteins have been identified and several are found in close association with one or more histone deacetylases. These findings suggest models, whereby cytosine methylation brings about local histone deacetylation, which could facilitate methylation of one or more deacetylated lysines on the histones and subsequent recruitment of repressor proteins that prevent transcription factors from gaining access to the given genes.

Some major protein domains involved with chromatin remodelling are as follows

L DNA methyltransferases (METs,CMTs,DRMs)

These are the enzymes that methylate DNA in various patterns.

L Methylcytosine Binding Domain Proteins (MBDs).

These proteins are thought to bind to methylated DNA to mediate other chromatin modifying events.

L Histone acetyltransferases (HACs).

These enzymes add acetyl groups to histones.

L Histone deacetylases (HDAs).

These enzymes remove acetyl groups from histones.

L Chromatin remodeling activities (CHR, CHB, CHC etc).

These large multi-protein complexes use energy derived from the hydrolysis of ATP to alter the positioning of nucleosomes on DNA.

L SET Domain containing proteins (SDGs).

SET domains are common within proteins that methylate histones.

L Chromodomain containing proteins.

Chromodomains are found in histone-binding repressor proteins such as Heterochromatin Protein 1.

L Bromodomain containing proteins

Bromodomains are found in proteins that bind acetylated lysines.

L High Mobility Group (HMG) Proteins HMG proteins are abundant non-histone chromosomal proteins that bind and bend DNA and provide for with "architectural" roles.

ATP dependant chromatin remodelling

The ATP dependant chromatin remodelers function either by repositioning nucleosomes, altering the nucleosomal conformations, changing histone compositions or ejecting histones from the DNA [START_REF] Hamiche | ATP-dependent histone octamer sliding mediated by the chromatin remodeling complex NURF[END_REF]. [START_REF] Mohrmann | Composition and functional specificity of SWI2/SNF2 class chromatin remodeling complexes[END_REF].

Likewise there are at least two SWI/SNF like remodelers in humans, BRG1/ hSWI/SNFA or hPBAF or hSWI/SNFB. A snapshot of the SWI/SNF family members and their functions is illustrated in Figure 7.

RSC (Remodels Chromatin Structure)

RSC is 15 subunit 1MDa protein isolated from yeast [START_REF] Cairns | RSC, an essential, abundant chromatin-remodeling complex[END_REF]. The RSC subunits, Sfh1, Rsc8 and Rsc6 have respective counterparts in SWI2/SNF2, SNF5, Swi3 and Swp73. The two complexes share actin related proteins namely Arp7 and Arp9 (also named Rsc11/Swp61 and Rsc12/Swp59). This particular remodeler is available in plenty compared to SWI/SNF [START_REF] Cairns | RSC, an essential, abundant chromatin-remodeling complex[END_REF].

There are some isoforms of RSC purified by RSC6 antibody short of 90 KDa and missing subunits RSC3 and RSC30 and this was referred to as RSCa. There are two other isoforms, one containing RSC1 and the other RSC2. While RSC2 containing isoform is more abundant, deletion of either of the genes leads to defects in growth and a double deletion is lethal [START_REF] Cairns | Two functionally distinct forms of the RSC nucleosome-remodeling complex, containing essential AT hook, BAH, and bromodomains[END_REF].

RSC unlike SWI/SNF is involved in mitosis and in repair of damaged DNA.

ISWI family of Remodelers

This group of ATP dependant chromatin remodelers get their name from their similarity to the SWI2 ATPase and hence Imitation SWItch. This ATPase is highly similar to Brahma and was first discovered in drosophila [START_REF] Elfring | Genetic analysis of brahma: the Drosophila homolog of the yeast chromatin remodeling factor SWI2/SNF2[END_REF]. ISWI family of remodelers have two SANT domains in the C-terminal region and do not have the bromodomain [53; 54]. They have a preference

to binding to nucleosomes with extra linker DNA than to nucleosome core particles. The ISWI group of remodelers play an important role in transcription activation and repression as well. A homozygous null mutation of ISWI leads to lethality in drosophila [START_REF] Deuring | The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo[END_REF]. This suggests that it plays an important role in gene expression. Apart from this it has also been shown to be involved in maintenance of higher order chromatin structure [START_REF] Deuring | The ISWI chromatin-remodeling protein is required for gene expression and the maintenance of higher order chromatin structure in vivo[END_REF]. The ISWI complex is composed of 2-4 subunits and are about 200-800 kDa in size. dNURF and dCHRAC were first identified in drosophila [56; 57]. Drosophila contains three ISWI complexes namely, NURF (NUcleosome Remodeling Factor), ACF (ATP-utilising Chromatin Factor) and CHRAC (CHRomatin Accessibility Complex). The ATPase activity of these remodelers is activated by nucleosomes and not by DNA. dNURF interacts with the histone tail of H4 and helps mobilizing the nucleosomes [START_REF] Georgel | Role of histone tails in nucleosome remodeling by Drosophila NURF[END_REF]. ACF has been reported as a regulator of nucleosome spacing [59; 60]. ACF is also capable of sliding the end positioned nucleosomes to centre position [START_REF] Eberharter | ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts[END_REF]. CHRAC is closely related to ACF and can perform like ACF in spacing the nucleosomes and sliding them [57; 62]. Based on the homology with drosophila ISWI, Tsukiyama and colleagues indentified two ISWI genes ISWI1 and ISWI2 in yeast [START_REF] Tsukiyama | Characterization of the imitation switch subfamily of ATP-dependent chromatin-remodeling factors in Saccharomyces cerevisiae[END_REF]. In higher eukaryotes such as Xenopus laevis, mouse and human there are ISWI complexes called RSF (Remodeling and Spacing Factor), hACF, WCRF(Williams Syndrome Associated Chromatin remodeling Factor), hCHRAC [64; 65; 66; 67; 68]. As in yeast two more genes in humans were identified and they were called hSNF2L and hSNF2H with almost 70% homology to the yeast genes [69; 70]. Figure 8 provides a summary of the ISWi family members and their general functions.

CHD family of Remodelers

This particular group of remodelers play an important role in development. It has been shown

that mutations in dMi-2 is lethal at the embryonic stage [START_REF] Khattak | Genetic characterization of Drosophila Mi-2 ATPase[END_REF]. The CHD (Chromodomain Helicase DNA-binding) or Mi-2 complexes contain ATPases with one or more chromodomains. The first CHD protein was identified in mouse and was isolated as a protein which showed the properties of both the SWI2/SNF2 group of remodelers and Polycomb/HP1 chromodomain family of proteins. The different CHD remodelers and their roles in the cell are summarised in Figure 9.

INO80 family of remodelers

This is again a large complex protein with 15 subunits and is known to be involved in DNA repair and transcription activation. One of the largest subunits INO80p has a conserved but discontinuous ATPase domain, with a huge spacer region in the middle. There are two conserved motifs TELY motif at the amino terminus and GTIE motif at the carboxy terminus [START_REF] Shen | A chromatin remodelling complex involved in transcription and DNA processing[END_REF]. There are also actin related proteins associated to this complex like Arp 4, 5 and 8.

Rvb1 and Rvb2 are found to be present in multiple copies per INO80 molecule and also have a helicase activity. Yeast mutants of INO80 show an increased hypersensitivity to DNA damaging agents and also defects in regulation of transcription. Also the Nhp10 subunit of INO80 was reported to be recruited in double strand break repair [73; 74]. A similarly large complex called SWR1 was found with 14 subunits and almost 4 subunits similar to INO80 such as Rvb1, Rvb2, Act1 and Arp4 [75; 76; 77]. SWR1 has been reported to play a role in DNA repair and also is involved in replacement of H2A/H2B dimers with H2A.Z /H2B dimers in an ATP dependant manner [START_REF] Mizuguchi | ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex[END_REF]. The involvement in cell events by INO80 family of remodelers is schematically described in Figure 10.

Domains involved in Remodeler Nucleosome Interaction

There are multiple remodeler domains that are involved in nucleosome recognition and interaction. However, it is not clear whether they act as individual domains or in concert with other domains. Some of the important and well characterised domains are as follows (See Figure 11).

Bromodomain

The bromodomain in the remodelers is distinct in its ability to recognise the acetylated lysines in the histones. In the case of SWI/SNF there exists such a bromodomain in the Cterminal region of the protein. Yeast, flies and humans all make two SWI/SNF related complexes and one of them has multiple bromodomains. These bromodomians can exist in a single protein called as polybromo or can exist in a number of proteins such as yRSC1/2/4/10 and open the possibility for cooperative recognition of modifications. Noteworthy, it has been reported that acetylated nucleosomes are easily mobilised by SWI/SNF [78; 79].

Chromo Domain (CHD)

The chromodomains is characteristic of the CHD family of remodelers. These remodelers have two chromodomains in their N terminal domains. These N-terminal domains behave as a structural unit to help the binding of the remodeler to a methylated lysine [80; 81; 82]. The presence of the chromodomains is highly necessary for the remodeler to both bind and remodel the nucleosomes [START_REF] Brehm | The many colours of chromodomains[END_REF].

Plant Homeodomains (PHD)

The Plant Homeodomain PHD finger has a methyl-lysine interaction motif. The PHD finger is reported to interact functionally with the bromodomains of the ISWI and the chromodomains of the CHD family remodelers [START_REF] Zhou | The PHD finger/bromodomain of NoRC interacts with acetylated histone H4K16 and is sufficient for rDNA silencing[END_REF]. However, in the case of dNURF the PHD finger of BPTF subunit interacts with the H3K4me3 stabilizing the BPTF/NURF interaction with active chromatin [START_REF] Wysocka | A PHD finger of NURF couples histone H3 lysine 4 trimethylation with chromatin remodelling[END_REF]. Apart from histone tails there are other epitopes for the recognition by the PHD domain. For example the PHD of dACF recognises the globular domain of the core histones [START_REF] Eberharter | ACF1 improves the effectiveness of nucleosome mobilization by ISWI through PHD-histone contacts[END_REF].

Hand SANT-SLIDE Domain

Several remodelers possess Hand SANT-SLIDE domain.This domain consists of three subdomains and is found in the C-terminal region of the ISWI remodelers. The SANT domain is also found in a lot of chromatin related proteins. The SANT is required for the function of ySWI/SNF and is present in ySWI3. Similarly this domain is necessary for the functioning of the yRSC and is present in the yRSC8. It is present in yAda2 and enhances the yGcn5 HAT activity [START_REF] Boyer | Essential role for the SANT domain in the functioning of multiple chromatin remodeling enzymes[END_REF]. While the SANT domain interacts with the histones, the SLIDE domain contacts the nucleosomal DNA. However, when the SLIDE domain is deleted in the dISWI there is loss of the ATPase and remodelling activities, in the contrary when the SANT domain is deleted, then there is reduced remodelling [START_REF] Grune | Crystal structure and functional analysis of a nucleosome recognition module of the remodeling factor ISWI[END_REF]. Thus we can say that this particular domain is characteristic of the ISWI family of remodelers and that they cooperate with each other in nucleosome recognition and stimulation of ATPase activities.

Figure 11: Chromatin Remodeler Domains

The image is adapted from [START_REF] Clapier | The biology of chromatin remodeling complexes[END_REF] and shows the various domains involved in the chromatin remodelers.

ATP dependant chromatin remodeling: Mechanism

The remodelers alter the chromatin structure making them more accessible to the DNA for processes like transcription and repair. In this part of the manuscript I will briefly summarize the data on the mechanism of nucleosome remodeling by the different remodelers.

Substrate Binding

The remodelers have the tendency to recognise the nucleosomes as their substrates. However, they interact both with the DNA and the histone part of the nucleosomes during their activity.

In the beginning DNA affinity columns were used for the isolation of SWI/SNF [START_REF] Kwon | Nucleosome disruption and enhancement of activator binding by a human SW1/SNF complex[END_REF]. The binding of the remodelers to the nucleosomal substrate was previously shown by band shift experiments [START_REF] Cote | Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex[END_REF]. The SWI/SNF remodelers show an affinity for DNA substrates in an ATP dependant fashion. In the case of ISWI remodelers there is lower affinity in binding with DNA in comparison with SWI/SNF remodelers [START_REF] Whitehouse | Evidence for DNA translocation by the ISWI chromatin-remodeling enzyme[END_REF]. There are also instances where the linker DNA helps in the remodelers to bind with the nucleosome [START_REF] Brehm | dMi-2 and ISWI chromatin remodelling factors have distinct nucleosome binding and mobilization properties[END_REF].

Evidence for the same is provided by cross-linking experiments [START_REF] Sengupta | The interactions of yeast SWI/SNF and RSC with the nucleosome before and after chromatin remodeling[END_REF] and electron micrographs [91; 92; 93; 94]. There are distinct places where the DNA and the histones in the nucleosomes come in contact with each other and the remodeler [40; 91; 95]. Leschziner used Orthogonal Tilt Reconstruction method (OTR) and showed that RSC posses a deep central cavity sufficient to fit in one complete nucleosome [START_REF] Leschziner | Conformational flexibility in the chromatin remodeler RSC observed by electron microscopy and the orthogonal tilt reconstruction method[END_REF] (see Figure 12).

ATP binding and Hydrolysis

The remodelers require ATP to carry out its functions of altering nucleosome structures. In case of SWI/SNF class of remodelers, their ATPase activity is stimulated by single stranded, double stranded or nucleosomal DNA all to the same extent [44; 50]. In strong contrast the ISWI group of remodelers exhibit ATPase stimulation to the nucleosomal substrates alone and not to DNA substrates [START_REF] Tsukiyama | Purification and properties of an ATP-dependent nucleosome remodeling factor[END_REF]. It was also shown that the SWI/SNF group of remodelers have a higher turnover of ATP than that of the ISWI group of remodelers. These remodelers belong to the SF2 family of helicases but lack the double strand displacement activity [START_REF] Cote | Stimulation of GAL4 derivative binding to nucleosomal DNA by the yeast SWI/SNF complex[END_REF].

Nucleosome Distortion/Disruption Processes

The ATP dependant remodelers function by giving rise to a number of events with an end product as a result of distortions and disruptions of the nucleosomal substrate. The DNA that is buried deep inside the nucleosome needs to be exposed so that transcription factors and repair enzymes can nudge their way to such patches of DNA. This is aided by the remodelers.

Some of the major nucleosomal disruption processes are summarised below [START_REF] Narlikar | Cooperation between complexes that regulate chromatin structure and transcription[END_REF] (Figure 13).

Superhelical Torsion

The presence of the helicases in remodelers substantiates the fact that the remodelers behave like helicases. A series of experiments done by Havas and colleagues [START_REF] Havas | Generation of superhelical torsion by ATP-dependent chromatin remodeling activities[END_REF] on different chromatin remodelers show that the remodelers have a tendency to generate a super helical torsion in DNA. They were able to show that the remodelers like SWI/SNF, BRG1 ISWI and Xenopus Mi-2 were all able to generate super helical torsion in an ATP dependant manner (Figure 13 h).

Nucleosome sliding

Non ATP dependant mobilization of nucleosomes along DNA or translational positioning can occur probably due to high temperature or ionic conditions [98; 99]. However, the strong interaction between the DNA and the histones makes such an event to occur unfavourable. This is where the ATP dependant remodelers come into play. It has been shown by mobility shift assays that the remodelers are able to mobilize the histone octamer along the DNA [START_REF] Langst | Nucleosome mobilization and positioning by ISWI-containing chromatin-remodeling factors[END_REF] (Figure 13 d). The sliding of histone octamer on DNA in an ATP dependant manner was first reported on drosophila extracts [101; 102]. Subsequently, the other remodelers such as NURF, ISWI and CHRAC also exhibited mono-nucleosomal sliding on longer DNA templates [39; 103]. Apart from nucleosome sliding the ISWI group of remodelers were able to generate regularly spaced chromatin templates suggesting their role in chromatin assembly [START_REF] Langst | Nucleosome mobilization and positioning by ISWI-containing chromatin-remodeling factors[END_REF] (Figure 13 e). The SWI/SNF group of remodelers on the other hand are able to mobilize the centrally positioned nucleosomes to end positioned nucleosomes (pushing them to the end of the DNA template) away from the thermodynamically favourable position [START_REF] Flaus | Dynamic properties of nucleosomes during thermal and ATP-driven mobilization[END_REF]. There is yet another event where the remodeler is capable of shifting the nucleosome 50 bp beyond the DNA template end and such species of nucleosomes are referred to as recessed [105; 106]. While all the above papers show in-vitro evidences of nucleosome sliding, there are also evidences of the same in in vivo. The IFN-"!promoter!when!infected! with viruses leads to the assembly of the enhancesome and pre-initiation complex. These factors always assemble on a nucleosome free promoter region. When the nucleosome position was analysed before and after the transcriptional activation it was seen that the TATA sequence for TBP binding which was blocked before, moved 35bp downstream allowing TBP to bind [107; 108]. Tsukiyama and group also showed nucleosome sliding in yeast by ISW2. Here, they used a galactose inducible allele of ISW2 to study the structural changes at the level of the promoter [START_REF] Fazzio | Chromatin remodeling in vivo: evidence for a nucleosome sliding mechanism[END_REF]. Figure 14 is a pictrographical representation of the different models of nucleosome sliding. 111 ; 112]. Experiments performed on the nucleosome core particle with no free linker DNA were also able to give similar results. In addition hSWI/SNF and ySWI/SNF introduced stable topological changes in closed circular arrays [87; 113]. Site specific cross linking experiments done on the octamer and DNA did not prevent the SWI/SNF remodelers from remodeling the nucleosomes [START_REF] Lee | hSWI/SNF disrupts interactions between the H2A N-terminal tail and nucleosomal DNA[END_REF].

Alteration in nucleosome composition

The disruption of the histone octamer with regard to the ATP dependant remodeling complex remains highly controversial. While in the beginning it was suggested that the remodelling by SWI/SNF leads to dissociation of H2A-H2B dimers or alters the histone folds [41; 44],

histone crosslinking experiments later showed that the octamer disruption is not necessary for restriction enzyme accessibility [START_REF] Boyer | Roles of the histone H2A-H2B dimers and the (H3-H4)(2) tetramer in nucleosome remodeling by the SWI-SNF complex[END_REF]. Some studies suggest that dimer expulsion is needed when the nucleosomes are pushed to the end of the template and they are off the DNA template they tend to have a loose dimer-tetramer interaction. Two independent works suggest the transfer of H2A-H2B dimers from a mononucleosomal substrate to H3 H4 tetramer [116; 117]. The sequences used for this study was the Mouse Mammary Tumour Virus Promoter(MMTV) and these sequences are prone to dimer loses [START_REF] Kelbauskas | Sequence-dependent variations associated with H2A/H2B depletion of nucleosomes[END_REF]. However, we cannot rule out the in vivo evidences that show the exchange or swap of H2A.Z-H2B dimers for H2A-H2B dimers [75; 76; 77].

Remosome

Recently our laboratory has made a detailed analysis of the mechanism of action of RSC [START_REF] Shukla | Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer[END_REF]. High resolution microscopy and novel biochemical techniques were used in this study.

Atomic Force Microscopy (AFM) and electron cryomicroscopy show that two types of products are generated during the RSC remodelling reaction of centrally positioned 601 nucleosomes: (i) stable non-mobilized particles, termed remosomes that contained about 180 bp of DNA associated with the histone octamer and, (ii) slid to the end of the nucleosomal DNA particles (Figures 15 and16). EC-M reveals that individual nucleosomes exhibit a highly irregular trajectory (Figure 15). Restriction accessibility analysis, DNase I footprinting and Exo III mapping clearly show that the histone-DNA interactions within the remosome are very highly altered, particularly at the nucleosomal dyad. The data suggest a two-step mechanism of RSC remodeling consisting of initial generation of a remosome, followed by its mobilization (Figure 16). It was further demonstrated in a series of biochemical experiments that the remosomes are intermediate products generated during the first step of the remodelling reaction that are further efficiently mobilized by RSC. The very recent data of our laboratory shows that, also SWI/SNF, but not ACF, was using the same two step mechanism for nucleosome remodelling (unpublished results).

Histone Chaperones

The! term! molecular! chaperone! was! first! coined! by! Ron! Laskey! [119; 120]. He used the word to describe the nuclear proteins in the extracts of frog oocytes that prevented the incorrect interactions between histones and DNA instead it would end up as aggregates.

Lately, the term chaperones has been used to describe the molecules which prevents incorrect interactions. Similarly the histone chaperones protect the interaction between the negatively charged DNA and the positively charged histones.

Histone chaperones are key molecules that play an important role in the assembling and disassembling of the nucleosomes (Figure 17). It has been established that the H3-H4 dimers are assembled into the H3-H4 tetramers on the DNA. This conformation of the H3-H4

tetramer is formed with the help of the histone chaperone and it is this histone chaperone that deposits the tetramer on the DNA. This particular structure! is! called! the! tetrasome! [121; 122].!To!this!Tetrasome!two!dimers!of!H2A!and!H2B!are!incorporated!thus!giving!rise!to! the nucleosome core particle. What is important in this step of dimer deposition is that they need to be in the opposite direction and hence are deposited in a step wise fashion. While doing so, there are instances, where the presence of a single H2A-H2B dimer is termed as a Hexasome.! Histones! and! DNA! do! not! assemble! into! nucleosomes! under! physiological! conditions as there is a higher tendency of the histones to no specifically bind to the DNA and leading to aggregates [121; 122]. This is exactly where the histone chaperones come to play their role of aiding in the molecular assembly and disassembly of the nucleosomes.

These histone chaperones help in regulating the assembly and disassembly in a less energy consuming fashion.

Histone chaperones are acidic in nature and it is a common trait between most of the chaperones. However they have very little sequence similarity. The histone chaperone Asf1 has a hydrophobic beta sheet on the edge and this mediates with the histones [START_REF] English | Structural basis for the histone chaperone activity of Asf1[END_REF]. However, on the other hand the histone chaperone Chz1 has an irregular chain and alpha helices which interacts with the H2A.Z and H2B [START_REF] Zhou | NMR structure of chaperone Chz1 complexed with histones H2A.Z-H2B[END_REF]. Histone chaperones and their functions in particular are very diverse and in order to understand them the chaperones are classified into three categories.

1.

Chaperones that bind and transfer histones alone without involving additional partners, for example, Asf1.

2.

Chaperones that combine several other histone chaperones subunits, for example, CAF-1.

3.

Chaperones that have histone binding capacity and are harboured within large enzymatic complexes, for example Arp-4 in INO80 Chromatin remodelling complex) [125; 126].

.

FACT (Facilitate Chromatin Transcription) complex

Human FACT complex was first identified in the year 1998 as an essential factor for transcription elongation in the chromatin landscape [START_REF] Orphanides | FACT, a factor that facilitates transcript elongation through nucleosomes[END_REF]. The heterodimeric complex is composed of two proteins Spt16 and SSRP1. The SSRP1 protein has a homologous counterpart in yeast called Pob3 and a HMG protein called Nhp6. Together they form the yeast FACT. Figure 18 shows the alignment and the domain breakup of the FACT complex in human and in yeast. This Image is adapted from a recent review from Winkler and Luger [START_REF] Winkler | The histone chaperone FACT: structural insights and mechanisms for nucleosome reorganization[END_REF].

FACT has been linked to activation of the tumour suppressor protein p53 and to histone variant H2AX-H2B in instances of DNA damage [129; 130]. However, in all three instances be it transcription, replication or repair, FACT functions by reorganising nucleosomes through the disruption of histone-histone and histone-DNA interactions. The FACT heterodimer also has the ability to deposit the H2AH2B dimer and H3-H4 tetramer on the DNA [START_REF] Belotserkovskaya | FACT facilitates transcription-dependent nucleosome alteration[END_REF]. It is also worthwhile to mention here that FACT aids in chromatin dynamics during certain critical processes in the cell and also has a histone chaperone activity.

The histone modifications are an important step and are involved wherever the chromatin dynamicity or histone exchange is needed. Modifications of the core histones in the nucleosomes lead to alterations in the structural architecture of the chromatin. This may in turn lead to recruitment of the transcription machinery or a blockage of the machinery. These epigenetic modifications are carried out with the help of factors such as FACT, CHD1, SWI/SNF and ACF [START_REF] Campos | Histones: annotating chromatin[END_REF]. CHD1 a chromatin remodeler is always localised throughout the coding region of actively transcribing genes with the help of a signal from the trimethylation mark on the Lys-4 of histone H3 (H3K4me3) [81; 82]. CHD1 and FACT physically interact with each other on the nucleosomes in the ORFs of active genes. Similarly, H2BK120ub1 is another epigenetic mark and is associated with the PAF complex and FACT [START_REF] Pavri | Histone H2B monoubiquitination functions cooperatively with FACT to regulate elongation by RNA polymerase II[END_REF]. FACT has also been implicated in repair and is involved in exchange of histone variants such as H2AX.

This however is triggered by the phosphorylation of H2AX and this aid in easy exchange of variant histones through FACT [133; 134].

There are also instances where there can be direct modification on the FACT itself. For example poly(ADP-ribosylation) of the Spt16 subunit of FACT by PARP1 (poly(ADPribose) polymerase 1 and this type of modified FACT cannot bind on nucleosomes in vitro [START_REF] Huang | Modulation of nucleosome-binding activity of FACT by poly(ADP-ribosyl)ation[END_REF]. Similarly, modification to the SSRP1 subunit of FACT alters the FACT activity levels.

There is evidence of direct phosphorylation of the SSRP1 subunit by interaction with CK2 (casein kinase 2) [START_REF] Keller | A DNA damage-induced p53 serine 392 kinase complex contains CK2, hSpt16, and SSRP1[END_REF]. This phosphorylated subunit of FACT does not bind to the nucleosomes. Instead, when such phosphorylated SSRP1 is accumulated in a storage pool and can lead to sudden activation in vivo. Thus, we can say that the modification on FACT and its subunits moderates the nucleosome organisation and chromatin dynamics.

FACT functional models

There are two widely supported mechanisms of how FACT may function. While, on one hand! the! Dimer! eviction! model! suggests! that! the! histone! H2A-H2B dimers are displaced thereby providing access to the nucleosomal DNA [131; 137; 138]. This model comprises of three steps namely FACT binding to nucleosomes followed by FACT mediated H2A-H2B displacement and finally the reinsertion of the H2A-H2B dimers. This window between the displacement and the replacement of the dimers gives an ample amount of time for the RNA polymerase II to perform its function. In-vitro experiments suggest that the FACT complex has maximal activity when the FACT to the nucleosome ratio is near 1:1.

On the other hand the!Global!accessibility!or!non-eviction!model!suggests!that!it!is!just!the! reorganisation of the nucleosomes, where the histone DNA contacts are lost, giving a sufficient window for accessibility to factors without histone eviction [139; 140]. This fairly recent model again includes three main steps. The first step involves the binding of Nhp6 which leads to small perturbations and this in turn is an essential step for the recruitment of endogenous damage happens to be that of the deamination of cytosine to Uracil. The other deamination of adenine to hypoxanthine occurs at a lower frequency than that of the former [START_REF] Seeberg | The base excision repair pathway[END_REF].Metabolism also gives rise to certain reactive oxygen species also referred to as ROS that result in oxidation leading to fragmented or open ring structures of the bases or oxidised aromatic derivatives and could give rise to a possible mismatch. S-adenosylmethionine, a metabolic byproduct, is a genotoxic agent that attacks nucleophylic sites on the DNA and generates N-methyl purines and several alkylation lesions [START_REF] Seeberg | The base excision repair pathway[END_REF]. UV and ionising radiations result in the production of radicals, lesions and bulky adducts in the DNA. The above described mutations and damages might lead to genomic instability, cancer and subsequent organism death. All organisms have created very efficient strategies, the DNA repair pathways, to correct these aberrations and to maintain genome integrity and cell viability.

Excision and repair of the DNA damage

The cell has created two major mechanisms for excision of the DNA damage, namely Nucleotide Excision Repair (NER) and Base Excision Repair (BER). NER is a very complicated repair pathway which allows the removal of bulky adducts and photoproducts, attached to the nucleotides. NER functions via a complex, termed nucleotide excision repairosome, which consists of at least 30 proteins. To repair the lesion, the repairosome excises about 30 nucleotides during the repair process [START_REF] Friedberg | DNA damage and repair[END_REF].

BER is involved in the repair of small alterations of the bases and uses fewer enzymes.

Importantly, in human cells 10-20000 DNA lesions are generated per day, which are repaired by BER [START_REF] Seeberg | The base excision repair pathway[END_REF]. 8-oxoG is the most common base lesion corrected by BER [START_REF] Peterson | Cellular machineries for chromosomal DNA repair[END_REF]. The glycosylase required for this is OGG1. BER is also implicated in the removal of uracil formed in the DNA by either deamination of cytosine to uracil, thus resulting in U:G mismatch or by misincorporation of dUMP opposite to adenine residues. The glycosylase implicated in the removal of uracil is UDG. We will further describe in more details the mechanism of BER action in both naked DNA and chromatinized templates.

Nucleotide Excision Repair

Nucleotide excision repair has an elaborate mode of lesion detection. There are two major pathways in NER: Global Genome NER and Transcription coupled NER (Figure 20). The GG-NER screens first on the basis of disrupted base pairing instead of lesions per se like a patrolling inspector. In Transcription coupled NER, the lesions which prove as a hindrance for the polymerase to carry out transcription are repaired. While the modes of repair in GG-NER and TCR NER are strikingly different in their modes of detection and initial repair, there are overlapping or identical stages in the later part of the repair. There are 25 or more proteins playing a role in NER which are assembled step by step at the site of the lesion and then after the repair is done the complex is disassembled again.

There are three main characterised syndromes which arise due to inborn defects in NER.

They are namely Xeroderma pigmentosum (XP), Cockayne Syndrome (CS) and Trichothiodystrophy (TTD). They are all outcomes of extreme sun sensitivity [START_REF] Lehmann | The xeroderma pigmentosum group D (XPD) gene: one gene, two functions, three diseases[END_REF].

Xeroderma pigmentosum arises from mutations in any one of the seven genes XPA through XPG and increases the frequency of tumours and induces neurodegeneration. In Cockayne Syndrome, a mutation in the CSA or CSB results in dysmyelination and physical impairment (dwarfism). There is also premature ageing due to increased apoptosis events. With regard to TTD (has some symptoms similar to Cockayne Syndrome) there is brittle hair, brittle nails and scaly skin [START_REF] Hoeijmakers | Genome maintenance mechanisms for preventing cancer[END_REF].

Figure 20: Nucleotide Excision Repair Pathway

This image is adapted from [START_REF] Hoeijmakers | Genome maintenance mechanisms for preventing cancer[END_REF].

Base Excision Repair

BER in naked DNA

The different steps of BER on naked DNA templates were well characterized in vivo by using purified recombinant proteins (Figure 21) [START_REF] Jagannathan | Base excision repair in nucleosome substrates[END_REF]. BER consists of two main steps.

During the first step a specific glycosylase recognizes the modified base and removes it. This results in the generation of apyrimidic/apurinic (AP) site intermediates [START_REF] Parikh | Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA[END_REF]. This step of the BER pathway is realized in both short patch BER, which involves the removal of only one nucleotide or long patch BER, which is implicated in the replacement of 2-13 nucleotides (Figure 21) [START_REF] Jagannathan | Base excision repair in nucleosome substrates[END_REF]. An AP endonuclease activity then cleaved the AP site resulting in the formation! of! a! 3-hydroxyl! and! 5-deoxyribose phosphate or alternatively by an AP lyase producing!a!5-phosphate!and!a!3-fragmented deoxyribose. In the short patch BER, the 5-

dRP

.2.2 BER in nucleosomes

The nucleosomes, as expected, impede BER. However, the BER enzymes involved in the repair of distinct lesions are differently affected. For example, 8-oxoG was very unefficiently repaired when it was inserted close to the dyad axis of centrally positioned 601 nucleosomes [START_REF] Menoni | ATP-dependent chromatin remodeling is required for base excision repair in conventional but not in variant H2A.Bbd nucleosomes[END_REF]. Indeed, OGG1, APE1 and Polymerase " activities were strongly reduced in such nucleosomes. However, SWI/SNF stimulated the processing of 8-oxoG by each one of the three BER repair factors to efficiencies similar to these for naked DNA. Interestingly, SWI/SNF induced remodelling, but not mobilization of conventional nucleosomes, was required to achieve this effect [START_REF] Menoni | ATP-dependent chromatin remodeling is required for base excision repair in conventional but not in variant H2A.Bbd nucleosomes[END_REF]. Recently, BER was also studied in dinucleosomal templates with or without linker histone H1 [START_REF] Menoni | Base excision repair of 8-oxoG in dinucleosomes[END_REF]. A single 8-oxoG was inserted either in the linker or the core particle DNA within the dinucleosomal template. It was found that in the absence of histone H1 the glycosylase OGG1 removed 8-oxoG from the linker DNA and cleaved DNA with identical efficiency as in the naked DNA. In contrast, the presence of histone H1 resulted in close to 10-fold decrease in the efficiency of 8-oxoG initiation of repair in linker DNA independently of linker DNA length. The repair of 8-oxoG in DNA within the core particle was very highly impeded in both absence and presence of histone H1.

Chaperone-induced uptake of H1 restored the efficiency of the glycosylase induced removal of 8-oxoG from linker DNA, but not from the nucleosomal DNA. However, the removal of histone H1 and nucleosome remodeling are both necessary and sufficient for an efficient removal of 8-oxoG in nucleosomal DNA.

The situation appeared, however, to be quite different for the activity of uracil DNA glycosylase (UDG) which recognises and excises uracil bases from DNA [START_REF] Cole | Uracil DNA glycosylase activity on nucleosomal DNA depends on rotational orientation of targets[END_REF]. A recent study used a set of model nucleosome substrates in which single thymidine residues were replaced with uracil at specific locations and a second set of nucleosomes in which uracils were randomly substituted for all thymidines. It was found that UDG efficiently removes uracil from internal locations in the nucleosome where the DNA backbone is oriented away from the surface of the histone octamer, without significant disruption of histone-DNA interactions. However, uracils at sites oriented toward the histone octamer surface were excised at much slower rates, consistent with a mechanism requiring spontaneous DNA unwrapping from the nucleosome. In contrast to the nucleosome core, UDG activity on DNA outside the core DNA region was similar to that of naked DNA. Association of linker histone reduced activity of UDG at selected sites near where the globular domain of H1 is proposed to bind to the nucleosome as well as within the extra-core DNA. These results indicate that some sites within the nucleosome core and the extra-core (linker) DNA regions represent hot spots for repair that could influence critical biological processes.

Transcription Factor NF-B!(Nuclear Factor Kappa B) 1.6.1 NF-B

The transcription factor NF-B! plays an important role in many cellular processes and remains a hot area of interest. This transcription factor by the name of NF-B!(Nuclear Factor Kappa B) was first described and reported by Baltimore and Sen as a transcription factor that binds to the enhancer element controlling the formation of the immunoglobin kappa light chain and its expression [START_REF] Sen | Inducibility of kappa immunoglobulin enhancer-binding protein Nf-kappa B by a posttranslational mechanism[END_REF]. It derives its name since it was first reported to be found in B cells. Since then NF-B!has been vividly reported in many journals as a molecule playing an important role in inflammatory responses, immune reactions and in tumorigenesis and cancer.

25 years since its discovery it is one molecule that has been extensively studied and a lot needs to be understood as to how it plays such a crucial role.

The Family Tree of NF-B!proteins

NF-B! is the most widely encountered member! of! a! family! of! transcription! factors,! the! dimers!of!Rel!proteins. In mammalian cells, the NF-B/Rel family contains five members:

RelA (p65), c-Rel, Rel B, NF-B1 (p50 ; p105) and NF-B2 (p52 ; p100) [START_REF] Li | NF-kappaB regulation in the immune system[END_REF] (Figure 22). Three of the family members, Rel A(p65), c-Rel(Dorsal and Dif in Drosophila), and Rel B, have a transactivation domain(TD) at the C-terminus (Figure 22). The transactivation domains consists of regions rich in serine, acidic and hydrophobic residues which are essential for trans-activation activity. NF-B1/p105 and NF-B2/p100 are the inactive precursors of the p50 and p52 proteins, respectively and in an un-stimulated state; these proteins are localized to the cytoplasm. These proteins are processed proteolytically and their C-terminal domains are removed, thereby providing these proteins admission inside the nucleus [START_REF] Silverman | NF-kappaB signaling pathways in mammalian and insect innate immunity[END_REF].

p50 and p52 usually form homodimers or heterodimers with one of the three proteins that has a transactivation domain. RelA and p50 exist in a wide variety of cell types, while c-Rel expression is confined to hematopoietic cells and lymphocytes (Figure 22). The expression of Rel B!is!limited!to!highly!specific!sites,!such!as!the!thymus,!lymph!nodes!and!Peyers!patches [START_REF] Wilkins | Molecular structure of deoxypentose nucleic acids[END_REF]. In addition to the heterodimers p50/p65 homodimers of these Rel proteins also exists. It has been reported that p50 and p52 homodimers do not induce transcription. They are thought to be used as post-induction repressors, following invasion by pathogens [154; 155].

However, it has been demonstrated in vitro that p50/p50 has kB site dependant transcriptional activation [START_REF] Lin | Differential transcriptional activation in vitro by NFkappa B/Rel proteins[END_REF].

NF-B!! is induced by stimuli such as pro-inflammatory cytokines and bacterial toxins such as LPS and exotoxin B and a number of viruses/ viral products (HIV-1,HTLV-1,HBV, EBV, Herpes Simplex) as well as pro apoptic and necrotic stimuli(oxygen free radicals, UV light and gamma irradiation)

Although each NF-B! dimer has a different DNA-binding affinity for kB sites bearing the consensus sequence GGGRNNYYCC (R, purine : Y, pyrimidine : N, any base) [START_REF] Hayden | Signaling to NF-kappaB[END_REF], their functions often overlap. NF-B complexes composed solely of family members lacking transactivation domains, such as p50 homodimers, are thought to impose transcriptional repression [START_REF] May | Rel/NF-kappa B and I kappa B proteins: an overview[END_REF]. Irradiation of DNA with conventional UV light sources produces mainly two types of monophotonic lesions: cyclobutane pyrimidine dimers (CPDs) and pyrimidine pyrimidone monoadducts ( (6-4) PPs). The efficiency of induction of these photolesions might be affected by the presence of specifically bound proteins. This has been exploited to map in vivo the interactions of different transcription factors with their cognate DNA sequence [159; 160; 161; 162]. UV irradiated DNA was digested with T4 endonuclease V (for detection of CPD's) or treated with hot piperidine (for (6-4) PP detection). The sites of the cleavage were found by ligation mediated PCR, followed by separation on a sequencing gel. The above two types of monophotonic lesions can also be induced by using UV laser irradiation, and this was used by Geiselmann and coworkers [START_REF] Engelhorn | In vivo interaction of the Escherichia coli integration host factor with its specific binding sites[END_REF] to study the interactions of integration host factor of E. coli with DNA.

Laser irradiation induces both monophotonic and laser specific oxidative lesions. However, by changing irradiation conditions one can create mostly one type of lesions only. For example, upon high intensity laser irradiation essentially oxidative lesions are formed, the quantum yield of monophotonic lesions being strongly reduced. Two well studied laser-induced oxidative lesions are: 8-oxodG and oxazolone [164; 165] (Figure 24). These lesions are very sensitive to local helical DNA conformation and they can Biphotonic products: The UV laser irradiation could be used for both photofootprinting and protein-DNA crosslinking be analyzed and quantified at the nucleotide level by treatment with specific reagents. 8-oxodG is quantitatively cleaved by Fpg protein, while oxazolone is removed upon hot piperidine treatment [START_REF] Cadet | Oxidative damage to DNA: formation, measurement, and biological significance[END_REF] (Figure 24). Separation of the cleaved irradiated DNA allows one to find the positions and the relative yield of the lesions [START_REF] Spassky | Influence of the local helical conformation on the guanine modifications generated from one-electron DNA oxidation[END_REF]. The chemical mechanism of formation of these two types of lesions is well documented. These lesions originate from the competitive transformation process of the transient guanine radical cations precursor generated by a biphotonic absorption and ionization mechanism. The majority stable product 8-oxodG is formed through hydration at position 8 while the less favourable oxazalone is formed upon deprotonation of the radical cation precursor. [START_REF] Spassky | Influence of the local helical conformation on the guanine modifications generated from one-electron DNA oxidation[END_REF].

Mapping of DNA lesions

I.8 Objectives

FACT is an important protein with diverse roles in different vital for the cell processes.

Despite the impressive efforts invested how FACT assists, however, DNA repair and transcription remains elusive. The current view is that FACT is facilitating these two processes by modulating the structure of the nucleosomes, but the available data are We have addressed these questions in vitro by using a number of molecular biology and biochemistry methods and reconstituted nucleosomal templates. We discovered that FACT exhibits! a! co-remodeling! activity! and! it! is! able to boost the remodelling activity of the chromatin remodelers RSC and ACF. Our in vitro experiments demonstrate that FACT facilitates both BER and NF-B binding to nucleosomes via its co-remodeling activity. We anticipate that this property of FACT is essential for its in vivo function.

Summary

FACT protein, in addition to its role in transcription, is involved in the repair of damaged DNA. Although, how FACT assists repair remains elusive. In this work, we have studied the role of FACT in Base Excision Repair (BER). As a model system we have used positioned nucleosomes containing uracil randomly incorporated in nucleosomal DNA. UDG, in agreement with the available data, was able to remove uracils facing the solution, but not uracils facing the histone octamer. FACT alone has no effect on the UDG-mediated removal of uracil. The simultaneous presence of FACT and RSC (a chromatin remodeler involved in repair) allows, however, a very efficient removal of uracil facing the histone octamer by UDG as well as the removal by OGG1 of the otherwise un-accessible oxidative lesion 8-oxoG from nucleosomal templates. This was achieved thanks to the un-expected novel property of FACT to greatly enhance both the remodeling and mobilization activity of RSC.

The experimental results reveal that the presence of FACT increases the efficiency of RSC to transform the energy freed by ATP hydrolysis into! mechanical! work. The presented data suggest a stochastic nature of BER functioning in vivo with FACT being a key factor in the repair process.

Introduction

DNA is packaged under the form of chromatin in the eukaryotic nucleus. The nucleosome, the repeating unit of chromatin, consists of an octamer of core histones (two each of H2A, H2B, H3 and H4), around which DNA is wrapped in close to two superhelical turns [START_REF] Van Holde | chromatin[END_REF].

The DNA, connecting the consecutive nuclesomes, is called linker DNA and a fifth histone, termed linker histone, interacts with it [START_REF] Van Holde | chromatin[END_REF]. Each core histone contains a structured domain, the histone fold, and an unstructured NH2-terminus [167; 168; 169]. Both the linker histone and the core NH 2 -termini are involved in the assembly and maintenance of both the 30 nm chromatin fiber [14; 170] and the mitotic chromosomes [171; 172].

The nucleosome is a barrier for numerous processes requiring access to the naked DNA underlying sequences [START_REF] Beato | Transcription factor access to chromatin[END_REF]. The three main strategies that the cell uses to overcome the nucleosome barrier are the posttranslational modifications of core histones [START_REF] Strahl | The language of covalent histone modifications[END_REF], the incorporation of histone variants in chromatin [31; 173] and the chromatin remodelers [25; 86]. The chromatin remodelers are very sophisticated nanomachines able to perturb the histone-DNA interactions and to mobilize the histone octamer along DNA by using the energy freed by the hydrolysis of ATP [25; 100; 174; 175]. Depending on the type of ATPase that the chromatin remodelers contain, they are classified in at least four main groups, namely the SWI/SNF, ISWI, CHD and INO80 families [100; 176; 177]. The chromatin remodeler RSC belongs to the SWI/SNF family and it is involved in the repair of damaged DNA [50; 177]. RSC contains a central cavity sufficient for binding of a single nucleosome [START_REF] Chaban | Structure of a RSC-nucleosome complex and insights into chromatin remodeling[END_REF]. We have recently analyzed the mechanism RSC-induced nucleosome mobilization and have shown that RSC generates initially an ensemble of particles with highly altered histone-DNA interactions, which are further mobilized by RSC [START_REF] Shukla | Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer[END_REF].

Base Excision Repair (BER) is the major pathway that the cell uses to repair lesions induced upon oxidative stress. The different steps of BER functioning on naked DNA are well understood, but how BER functions on chromatinized templates remains elusive. The reported data show that the presence of nucleosomes interferes strongly with BER, although the different BER enzymes are affected in distinct manner [146; 150; 178; 179; 180; 181]. For example, the accessibility in nucleosome DNA of uracil DNA glycosylase (UDG) is reduced down to 30-fold and the removal of histone octamer facing uracil, in contrast to the removal of solution facing uracil, is greatly (up to 3 orders of magnitude) inhibited [START_REF] Cole | Uracil DNA glycosylase activity on nucleosomal DNA depends on rotational orientation of targets[END_REF]. No such rotational position dependence of the BER enzyme OGG1 removal of 8-oxoG, the major oxidative lesion, was, however, observed [START_REF] Menoni | ATP-dependent chromatin remodeling is required for base excision repair in conventional but not in variant H2A.Bbd nucleosomes[END_REF]. Indeed, in this later case the nucleosome remodeling by SWI/SNF was a prerequisite for efficient repair [148; 149].

Human FACT (Facilitates Chromatin Transcription) protein consists of two subunits, hSpt16 and SSRP1, which are both required for functionality [START_REF] Orphanides | The chromatinspecific transcription elongation factor FACT comprises human SPT16 and SSRP1 proteins[END_REF]. FACT exhibits a histone chaperone activity, it makes a complex with the H2A-H2B dimer and the (H3-H4) 2 tetramer and is able to deposit them on DNA [START_REF] Belotserkovskaya | FACT facilitates transcription-dependent nucleosome alteration[END_REF]. In addition to its role in transcription, FACT appeared also to be implicated in both replication and repair of damaged DNA [START_REF] Wittmeyer | The Saccharomyces cerevisiae DNA polymerase alpha catalytic subunit interacts with Cdc68/Spt16 and with Pob3, a protein similar to an HMG1-like protein[END_REF]. FACT was found in a complex with casein kinase 2 (CK2), which phosphorylates p53 in a DNA damage dependent manner resulting in an increase of p53 activity [129; 136]. FACT was also involved in both phosphorylation and exchange of histone variant H2A.X, two critical events related to the repair of DNA damage [START_REF] Heo | FACT-mediated exchange of histone variant H2AX regulated by phosphorylation of H2AX and ADP-ribosylation of Spt16[END_REF]. No other data are, however, available on the direct role of FACT in the processes of repair of damaged DNA.

In this work in a series of in vitro experiments we have analyzed the role of FACT in BER of uracil containing nucleosomes and we discovered a novel function of FACT in this process.

Our data show that FACT greatly facilitates the removal of uracil by UDG from nucleosomal DNA by boosting the activity of the involved in DNA repair chromatin remodeler RSC. This suggests that in vivo FACT might act in a concert with RSC to repair damaged DNA.

Results

Effect of FACT on the efficiency of UDG removal of uracil from nucleosomal DNA

To address the potential role of FACT in BER we have used reconstituted centrally positioned nucleosomes. FACT was purified to homogeneity from HeLa cells by double immuno-affinity procedure (Supplementary Figure 1C and Materials and methods). The histone octamer was assembled with highly purified recombinant core histones (Supplementary Figure 1A) and the nucleosome reconstitution was carried out by using the 601 sequence, containing randomly incorporated uracil [START_REF] Nilsen | DNA base excision repair of uracil residues in reconstituted nucleosome core particles[END_REF]. The reconstitution conditions were optimized and essentially all DNA was assembled into nucleosomes (Supplementary figure 1B). The OH and DNAse I footprintings (Figures 2A,4) showed clear 10 bp cleavage pattern further confirming the proper wrapping of the nucleosomal DNA around the histone octamer in the reconstituted samples. The reconstituted nucleosome samples were then incubated with increasing amount of UDG (Figure 1, lanes 3-6), DNA was isolated and after alkali treatment (to cleave the DNA phosphor-backbone at the abasic site generated upon removal of uracil by UDG) the cleaved DNA was run on a PAGE under denaturing conditions. The cleavage pattern of the free DNA arms of the nucleosome DNA is identical to the cleavage pattern of the naked DNA (Figure 1, bottom of the gel, compare lanes 3-6 with lanes 21-24). Although, the cleavage pattern of the nucleosomal DNA strongly differed from this of naked DNA (upper part of the gel, compare lanes 3-6 with lanes 21-24). Comparison with the OH footprinting of the nucleosome particles (lanes 1 and 25) shows that even at the highest concentration of UDG used, cleavage in nucleosomal DNA is only observed at the sites facing the solution which are also accessible to OH° radicals. This result is in perfect agreement with the reported data Figure 1. The simultaneous presence of both FACT and RSC, but not FACT alone, is required for efficient UDG excision of uracil at nucleosomal DNA sites oriented towards the histone octamer. Centrally positioned nucleosomes were reconstituted by using 32 P!5-labeled 255 bp 601 DNA fragment containing randomly incorporated uracil residues. (A) Lanes 3-6: analysis of the UDG enzymatic activity within the nucleosomal and linker DNA. The nucleosome solution was incubated with the indicated increasing (nine fold step) amount of UDG for 60 minutes at 30°C and the cleavage pattern of the isolated DNA was analyzed using PAGE under denaturing conditions; lane 2, no UDG added lane 1; OH radical footprinting of the nucleosomes. (B) Lanes 8-12: RSC induces a highly efficient UDGmediated excision of uracil at inward facing sites within the nucleosome. Nucleosomes were incubated with increasing (two-fold step) amount of RSC for 50 minutes at 30°C, and after arresting the reaction they were treated with 9 -2 units of UDG and the isolated cleaved DNA analyzed on denaturing PAGE; lane 8, control with no RSC added in the reaction. (C) Lanes 14-19: FACT facilitates the RSC-dependent UDG excision of uracil at inward facing sites within the nucleosome. Nucleosomes were incubated with increasing (2 fold step) amount of FACT in the presence of 0.2units of RSC and, after arresting the reaction they were treated with 9 -2 units of UDG. The cleaved purified DNA was analyzed on denaturing PAGE; lane 13, control containing 1.6pmol of FACT with no RSC added. Note that this highest used in the experiment concentration of FACT does not affect the excision of uracil by UDG. (E) UDG cleavage pattern of the naked 255 bp 601 fragment. The experiment was carried out as described in (A), but with nine-fold smaller concentration of UDG an each respective point; on the left is shown schematics of the nucleosome.

(REF) and illustrates the inability of UDG to remove uracil from sites facing the histone octamer. Noteworthy, the presence of 1.6 pmol FACT in the reaction mixture does affect neither the efficiency nor the pattern of removal of uracil by UDG (compare lane 13 with lane 4; note that in both cases the same concentration of UDG (9-2units) was used Pretreatment of the nucleosome samples with increasing amount of RSC changed, however, completely the UDG cleavage pattern of nucleosomal DNA , i.e. it became qualitatively indistinguishable from this of naked DNA (compare lanes 8-12 with lanes 21-24). Thus, the RSC induced remodeling of the nucleosomes renders all uracil residues (including the ones facing the histone octamer) accessible to UDG. Intriguingly, the same effect was observed when a very small amount (0.15 Units) of RSC (this amount of RSC was unable to change the UDG accessibility to uracil, see lane 8) and increasing amount of FACT was used for the pretreatment of nucleosomes (compare lanes 14-19 with lanes [START_REF] Wolffe | Histone H1[END_REF][START_REF] Syed | Single-base resolution mapping of H1-nucleosome interactions and 3D organization of the nucleosome[END_REF][START_REF] Beato | Transcription factor access to chromatin[END_REF][START_REF] Strahl | The language of covalent histone modifications[END_REF]. This shows that in vitro FACT and RSC act in concert to facilitate the UDG removal of uracil from nucleosomal DNA and suggests that in vivo they could act similarly to assist BER.

FACT boosts both the remodeling activity and the capacity of RSC to mobilize the nucleosomes

To analyze how FACT affects the remodeling activity of RSC we have used DNase I footprinting. Briefly, we have incubated 601 end-positioned nucleosomes with RSC either in the absence or presence of FACT and after arresting the remodeling reaction, the samples were treated in controlled manner with DNase I. The cleaved DNA was then purified and analyzed on a PAGE under denaturing conditions (Figure 2A). The presence of FACT alone, as expected, did not change the clear 10 bp cleavage pattern of nucleosomal DNA (Figure 2A, compare lane 1 with lane 2). This reveals that under the conditions of the experiment, FACT does not alter the structure of the nucleosome. When incubated with 0.2 units of RSC nucleosomes exhibited some alterations in the DNase I cleavage pattern, testifying for some relatively small perturbations in the histone-DNA interactions induced by RSC (compare lane 3 with lanes 1 and 2). Remarkably, the treatment of the nucleosomes with the same amount of RSC, but in the presence of 1.6pmol of FACT, resulted in very pronounced alterations in the DNase I digestion pattern (lane 4). The same altered DNase I digestion pattern was observed with 5-fold more RSC in the absence of FACT (1unit, lane 5). We conclude that FACT exhibits! strong! co-remodeling! activity and is able to boost no less that 5-fold the remodeling activity of RSC.

The effect of FACT on the efficiency on nucleosome mobilization by RSC was studied by EMSA (Figure 2B,C). Treatment with 0.2 units of RSC led to mobilization of a very small part of the nucleosomes (not exceeding 20%, see Figure 2B and quantification on Figure 2C).

The presence of increasing amount of FACT in the reaction mixture led to a strong increase of the amount of slid nucleosomes and already at the highest concentration (1.6 pmol) of (A) DNase I footprinting. End-positioned nucleosomes, reconstituted on 32 P! 5-labeled 241 bp 601 DNA fragment, were incubated with 0.2 units of RSC in the absence (lane 3) or in the presence of 1.6 pmol of FACT (lane 4) for 50 minutes at 30°C; lane 5, same as lane 3, but with 1 unit of RSC; After arresting the remodeling reaction, the samples were digested with 0.1 units of DNase I for 2 minutes, the cleaved DNA was isolated and run on 8% PAGE under denaturing conditions; lanes 1 and 2, controls showing the DNase I cleavage pattern of nucleosomes (lane 1) alone or incubated with 1.6pmol FACT under the conditions described above. (B) The presence of FACT increases the efficiency of RSC-induced nucleosome mobilization. Centrally positioned nucleosomes were incubated with 0.2 units of RSC in the presence of increasing amount of FACT, the reaction was arrested and the samples were run on native PAGE. The position of the unslid and slid nucleosomes were indicated; lane 1 control nucleosomes; lane 2, nucleosomes incubated with RSC alone (in the absence of hFACT) (C) quantification of the data presented in (B). FACT, the slid nucleosomes represent 57% from the overall nucleosome population (Figure 2C). Similarly, the presence of FACT in the remodeling mixture led to strong increase in the time-course of the mobilization reaction (Supplementary Figure 2). Therefore, FACT boosts the RSC ability to mobilize the nucleosomes.

FACT efficiently assists RSC to generate nucleosome-like structures exhibiting high accessibility to restriction enzyme

Our recent high-resolution microscopy and biochemical data suggested an intriguing two step-mechanism for RSC nucleosome remodeling (REF). The first step consists in a formation of a stable, non-mobilized particle, termed remosome, which contains ~ 180-190 bp of DNA loosely attached to the histone octamer. The remosome is formed by RSCpumping of ~ 15-20 bp from each end of the free DNA linkers of the nucleosome without repositioning of the histone octamer. During the second step, the remosome is mobilized by RSC [START_REF] Shukla | Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer[END_REF].

The main characteristic of the remosome is the higher accessibility of its DNA to restriction enzymes. To test if FACT was able to facilitate the generation of remosomes by RSC we have! used! the! recently! developed! in! gel! one! pot! assay [START_REF] Shukla | Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer[END_REF]. This approach detects quantitatively the alterations in histone-DNA interactions with a 10 bp resolution all along the nucleosomal DNA (Figure 3 + hFACT ( 7)

#
were excised and in gel digested with HaeIII. The digested DNA was purified from the gel and run on an 8% PAGE under denaturing conditions. A similar experiment using either control nucleosomes or nucleosomes treated with FACT (in the absence of RSC) was also performed. The gel was then dried and the product bands were visualized by exposure on a PhosphorImager and quantified. As seen (Figure 3C, D), and in agreement with the reported data [START_REF] Shukla | Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer[END_REF], the accessibility of the control particles (in the absence of FACT) to the restriction enzyme strongly decreases from d 7 to d 0 . FACT does not affect this behavior of the HaeIII digestion pattern. In fact, d 7 and d 6 behaved differently compared to the other positions and showed a high percent of cleavage (up to 50% in the case of d 7 ). In contrast, the internally located positions (from d 4 to d 0 ) were poorly cleaved. This is in agreement with the reported data and reflects the mode of association of the histone octamer with the nucleosomal DNA [START_REF] Wu | A 'one-pot' assay for the accessibility of DNA in a nucleosome core particle[END_REF]. Noteworthy, these results reveal that FACT alone in the concentrations used does not affect the histone-DNA interactions within the nucleosome.

However, upon incubation with both RSC and FACT, all sites along nucleosomal DNA exhibited highly altered accessibility (Figure 3C,D). The accessibility of d 7 decreased relative! to! the! control! particles! (this! effect! is! due! to! the! pumping! of! linker! DNA! in! the! nucleosome [START_REF] Shukla | Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer[END_REF], while that of the other positions strongly increased. This increase in the HaeIII accessibility profile paralleled the increase of FACT concentration used in the remodeling reaction. Since this altered HaeIII accessibility profile is a remosome specific structural! signature [START_REF] Shukla | Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer[END_REF], we conclude that FACT assists RSC in perturbing the histone-DNA interactions in the nucleosome and in the subsequent generation of remosomes.

UDG removes histone octamer faced uracil from both remosomes and slid nucleosomes with the same efficiency

Since FACT increases impressively the capacity of RSC to generate remosomes, its involvement in repair might be mainly associated with this specific to it property. If this was the case, one should expect damaged DNA to be easily repaired within the remosome. We have addressed this question by studying the ability of UDG to remove uracil from remosomes (Figure 4). In agreement with the data in Figure 1, UDG was unable to excise histone octamer facing uracil from the control nucleosome (Figure 4,! nucleosomes).!

Although, even at the lowest concentration of UDG (0.003 units) used in the experiments, in addition to solution facing uracil, the histone octamer facing uracil was rather efficiently

The presence of FACT increases the efficiency of RSC to transform the energy freed by ATP!hydrolysis!into!mechanical work

The above presented data reveals that FACT assists very efficiently RSC to both alter the histone-DNA interaction and to mobilize the nucleosome in an ATP-dependent manner. To achieve this, FACT could either act on the nucleosomal substrate or on RSC or on both of them. To differentiate between these possibilities we have carried out the RSC nucleosome mobilization assay at increasing concentration of FACT, but at low ATP concentration (120 µM). At this low concentration of ATP it is possible to precisely measure the amount of ATP hydrolyzed by ATP and thus, to precisely determine the percentage of nucleosomes mobilized!by!the!hydrolysis!of!a!unit!of!ATP.!Under!the!conditions!of!the!experiment,!the! increase of the FACT concentration results in a 3 fold increase (from 20% to 60 %) of the slid by RSC nucleosomes (Figure 5A). Remarkably, under the same experimental conditions no changes in the amount of hydrolyzed ATP was detected (Figure 5B). This demonstrates that the presence of FACT does not affect the ATPase activity of RSC, while it boosts very strongly the generation of slid nucleosomes, i.e. ~4-5-fold more slid nucleosomes are generated per unit of hydrolyzed ATP in the presence of the highest amount of FACT

Discussion

FACT is involved in several processes including transcription, replication and DNA repair (recently reviewed in [START_REF] Winkler | The histone chaperone FACT: structural insights and mechanisms for nucleosome reorganization[END_REF]. In this work we have studied how FACT assists BER. The presented data reveal that FACT alone has no effect on the removal of uracil by UDG from nucleosomal!DNA.!Although,!FACT!exhibits!a!strong!co-remodeling!activity and it is able to increase many-fold the efficiency of the involved in DNA repair chromatin remodeler RSC to both remodel and mobilize the nucleosomes. FACT does not affect the ATPase activity of RSC, but instead makes the nucleosomes easier to be remodeled and mobilized, i.e. it increases the efficiency of transformation of the energy freed by the RSC-induced hydrolysis of ATP into mechanical work. This allows, in turn, very low amount of RSC to be sufficient to strongly alter the histone-DNA interactions as well as to slide the nucleosomes and thus, the lesions in chromatin DNA to be efficiently repaired. Our data suggest that in vivo FACT acts indirect in BER, via RSC, in increasing the efficiency of repair of DNA lesions. FACT (at the concentrations used in the experiments) does not affect nucleosome structure as judged by both DNase I footprinting and the very sensitive restriction enzyme accessibility assay (one pot assay). In addition, no stable binding of FACT to the nucleosome was detected by EMSA. Then how does FACT act on the nucleosome substrate to make it much easily remodelable?! It! is! difficult! to! answer! to! this! question.! Obviously,! some! transient! FACTnucleosome interactions during the RSC remodeling process should be generated allowing the remodeler to function with much higher efficiency. These transient FACT-nucleosome interactions might require at least some flexibility of the histone octamer, since crosslinking of!the!histone!octamer!interferes!with!the!co-remodeling!activity!of FACT (Supplementary Figure S3). Interestingly, FACT assists also very efficiently the nucleosome mobilization by ACF (Supplementary Figure S4), a remodeler belonging to the ISWI family. FACT was also found stably recruited to the CENP-A nucleosomal complexes [START_REF] Song | A high-resolution map of nucleosome positioning on a fission yeast centromere[END_REF] and RSF, a chromatin remodeler, is also a member of this complex [START_REF] Perpelescu | Active establishment of centromeric CENP-A chromatin by RSF complex[END_REF]. In addition, FACT was shown to physically interact with CHD1, another chromatin remodeler [START_REF] Krogan | RNA polymerase II elongation factors of Saccharomyces cerevisiae: a targeted proteomics approach[END_REF]. Thus, the cell appears to use the FACT nucleosome reorganization ability to control the activity of distinct remodelers belonging to different families for different purposes in different processes.

Noteworthy, genetic analysis in yeast has revealed evidences for functional relationship between the N-terminal domain of Spt16, one of the FACT subunits, and the docking domain of H2A [START_REF] Vandemark | Structural and functional analysis of the Spt16p Nterminal domain reveals overlapping roles of yFACT subunits[END_REF]. The proper folding and integrity of the docking domain of H2A is, however, required for chromatin remodeler dependent nucleosome mobilization [START_REF] Shukla | The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling[END_REF]. For example, nucleosome reconstituted with deleted docking domain H2A or with the histone variant H2A.Bbd (which possesses defective docking domain) cannot be mobilized by remodelers from both SWI/SNF and ISWI family [START_REF] Shukla | The docking domain of histone H2A is required for H1 binding and RSC-mediated nucleosome remodeling[END_REF]. This suggests that FACT could, via a transient Spt16-mediated interaction with the H2A docking domain, reorganize the nucleosomes in a way to make them easier remodelable.

As mentioned above, we have not observed structural changes within the nucleosomes due to the presence of FACT alone. Although, data are reported that FACT alone is able to alter the overall nucleosome structure and to destabilize both histone-DNA and histone-histone interactions without any histone eviction [START_REF] Xin | yFACT induces global accessibility of nucleosomal DNA without H2A-H2B displacement[END_REF]. In these last experiments the molar ratio FACT: nucleosome exceeded, however, at least 50 fold the ratio of FACT: nucleosome used in our experiments. (It is worthwhile to mention that the FACT used by the Formosa group consists of at least 50 fold excess of the Nhp6 with respect to the other components of the yeast FACT (Spt16-Pob3) and we are close to equal ratios of the nucleosome and the FACT).

In addition to facilitating UDG removal of uracil, FACT is also able to efficiently assist the OGG1-mediated excision of 8-oxoG (the major oxidative lesion found in vivo) from nucleosomal templates in a RSC-dependent manner (Supplementary figure 5). This reveals that FACT can facilitate the function of distinct BER enzymes via specific reorganization of the nucleosome structure, probably during the required for BER nucleosome remodeling. Importantly, we found that BER acts very efficiently on remosomes and not on nonremodeled nucleosomes. All this suggests that BER could function in a stochastic manner with FACT playing an important role. We propose the following mechanism for BER functioning in vivo (see schematics, Figure 5). The chromatin remodelers from the SWI/SNF family with the help of FACT generate remosomes throughout the genome in a random way.

The! BER! enzymes! scan! genome-wide chromatin and when they detect a lesion within a remosome, they repair it. The remosomes, relatively metastable structures, after being repaired are either spontaneously or with the help of remodelers converted into conventional nucleosomes.!This!process!of!remosome!birth!and!death!is!taking!place!permanently!in!the! cell nucleus and it requires only very small changes in the overall chromatin structure (the remosomes are generated by just pumping 15-20 bp of DNA from both flanking the nucleosome linkers [START_REF] Shukla | Remosomes: RSC generated non-mobilized particles with approximately 180 bp DNA loosely associated with the histone octamer[END_REF]. Such a simple scenario may explain how the 10-20 thousands oxidative lesions generated in a single cell per day are efficiently repaired without the necessity of the BER enzymes to be targeted to the lesions.

Experimental procedures

Preparation of DNA fragments

1. The 255 bp 601 DNA probe used for reconstitution of centrally positioned nucleosomes was PCR amplified from pGEM-3Z-601.1! plasmid! (kindly! provided! by! J.! Widom).! 5! end! labelling was performed by using 32P-labeled primer in PCR.

2.

For! One! Pot! Restriction! enzyme! Assay! a! set! of! eight! pGEM-3Z-601.2 mutants were utilized, each containing HaeIII site at a different superhelical location, as described before [START_REF] Wu | A 'one-pot' assay for the accessibility of DNA in a nucleosome core particle[END_REF];! note! that! the! dyad! 7! fragment! contains! an! additional! HaeIII site located at 4 bp away from the d7 site). Briefly, a 281 bp fragment was amplified using primers targeting the vector specific sequence flanking the 601.2 sequence. Labelling of the fragment was done as described above.

The fragments were subsequently digested with SphI to get a fragment of 255 bp with 57 and 51 bp linker DNA on left and right side respectively. All the fragments were purified on 6% native acrylamide gel prior to use for nucleosome reconstitutions.

3.

For repair assays the 601 sequence was incorporated with uracil by substituting 0.25% with dUTPs in the normal dNTPs mix comprising dATP, dGTP, dCTP and dTTP with a concentration 25mM.1µl of this new dNTP mix was used in a PCR reaction and random incorporation of uracil is achieved.

4.

For end positioned nucleosomes we used the p199.1 plasmid to generate a 250bp fragment with a single linker length of 100bp and the nucleosome at the end.

SDS PAGE electrophoresis of FACT purified from HeLa cell extract

In!order!to!study!FACT!and!its!combined!effect!with!remodelers!we!characterised!the!protein! FACT on an 18% SDS PAGE. The FACT is purified from HeLa cell extracts and hence this form of hFACT consists of two subunits Spt16 (Suppressor of Ty protein 16) and SSRP1 (Structure Specific Recognition Protein 1). The yeast FACT comprises of two homologous subunits called Spt16 and Pob3. The molecular weight of the 140kDa and 80kDa correspond to the higher molecular weight Spt16 and the lower molecular weight SSRP1. The SDS gel shows the presence of the two subunits of FACT.

SDS PAGE electrophoresis of recombinant histones and octamer

The recombinant core histones were purified to homogeneity and the octamer was reconstituted in equimolar ratios at 2M NaCl. When reconstituted with the DNA fragment of choice the nucleosomes are assembled on the DNA. Here we see the individual hisotnes separated on an 18% gel and a lane showing the reconstituted octamer and its individual histones in equimolar ratios. (Alongside is a native chicken histone and its histone composition).

Band shift /EMSA of reconstituted centrally positioned nucleosomes

Nucleosome reconstitution was performed by the salt dialysis procedure.

In order to carry out the salt dialysis nucleosome reconstitution 2.5 -3.0 µg of core histones was mixed with 2.5 µg of carrier DNA and the labelled DNA probe in 2 M NaCl10 mM TrisHCl (pH7.4), 1 mM EDTA (pH8.0), 10 mM!"-mercaptoethanol, 1mg of Bovine serum albumin per ml in a total volume of 90µl.

The reaction mixtures was thoroughly mixed and transferred to a dialysis tubing and dialysed at 4°C against 10 mM Tris-HCl (pH 8.0)1 mM EDTA (pH 8.0)10 mM "-mercaptoethanol containing 1.2, 1.0, 0.8, and 0.6 M NaCl. Each dialysis step was carried out for 2 h. Finally, the reconstituted material was dialyzed overnight against 10 mM Tris-HCl (pH 8.0)1 mM EDTA (TE) and 10 mM NaCl.

The reconstituted nucleosomes were analyzed on a 5% native polyacrylamide (acrylamide/bisacrylamide, 29:1)0.25× TBE gel. Under optimal conditions, more than 90 95% of the 32 P-labeled fragment was usually nucleosome reconstituted.

In the case of centrally positioned nucleosomes the nucleosomes are assembled on 601 255bp fragment. Here they are able to form a nucleosome with two linker lengths of 52 and 56bp respectively. The gel shift assay/EMSA shows that the nucleosomes are formed and migrate slower compared to free DNA because of its structural retardation in the gel.

DNAse I footprinting of centrally positioned nucleosomes and DNA

Once the reconstituted nucleosome particles are ready we next characterised the nucleosomes by performing a DNase I footprinting. The nucleosomal samples and the DNA fragments were subjected to partial DNase I digestion and then run on a 8% denaturing PAGE -Urea gel. We are able to observe the repeats every 10 base pairs showing the intact DNA and Histone contact points in the case of the nucleosomes and a ladder of bands in the case of the DNA as it is exposed everywhere and is unlike the nucleosomes with the histone octamer protecting the DNA sequence.

OH radical foorprinting of centrally positioned nucleosomes and DNA

The same reconstituted nucleosomes were also subjected to the OHº radical footprinting. In order to perform the hydroxyl radical footprinting on the mononucleosomes, it was carried out in a 15 µl reaction volume. The hydroxyl radical reaction was carried out by mixing 2.5 µl each of 2 mM FeAMSOゆ and 4 mM EDTA, 1M Ascorbate and 0.12 % H 2 O 2 in a drop on the side of the tube cap before mixing it with the reaction solution. The reaction was stopped by adding 0.1% SDS, 25 mM EDTA, 1% glycerol and 100 mM Tris pH 7.4. The DNA was purified by phenol chloroform and then run on an 8% denaturing gel. The hydroxyl radical footprinting performed on the 601 255 bp fragment gave rise to a pattern characteristic of the nucleosome. The OH radical performed on the nucleosomes gives rise to a structured repeat of the nucleosomes which are 14 in number and represent the superhelical turns in the nucleosomes, 7 on each side from the dyad. In the case of the DNA we can see a ladder of the sequences showing its overall exposure to the radicals.

Nucleosome remodeling reaction

Typical remodeling reactions were performed with 30 ng of nucleosomes and the appropriate amount of RSC in remodeling buffer (RB) 10 mM Tris pH 7.4, 5% glycerol, 1 mM rATP, 2.5 mM MgCl2, 1 mM DTT, 100 mg/ml BSA, 50 mM NaCl, 0.01% NP40) in a volume of 7.5 µl at 29°C for 50 minutes. While RSC is used to slide centrally positioned nucleosomes to end position and ACF is used to slide the end positioned nucleosomes to central positions. The reactions were stopped with 0.1 units of apyrase and 1µg of plasmid DNA pUC18/pUC19.

BER Initiation Assays

The uracil incorporated DNA was formed by PCR and the nucleosomes were reconstituted on this fragment. For carrying out the repair assay the nucleosomes were either subjected to either RSC or FACT or a combination of the two. A fixed concentration of the UDG was used and the samples were incubated in UDG at 29 ºC for an hour. They were then purified by phenol chloroform and precipitated with ethanol and then the reaction was completed using an AP-lyase. We used APE-1 incubated with the DNA to complete the reactions. The fragment was once again phenol chloroformed and ethanol precipitated before running on an 8% denaturing PAGE.

Another method of cleaving the apurinic sites was by treatment with piperidine once the UDG treatment was carried out. In a reaction volume of 9 µl for all the samples 1µl of piperidine was added and heated at 90ºC for 15 minutes. After 15 minutes the tubes were spun at high speed for a short time to bring down the vaporised samples and the heating repeated for another 15 minutes. Once this is done the samples are subjected to phenol chloroform and then ethanol precipitated. The samples were thoroughly suspended in formamide dye heated at 90 degrees for 5 minutes before loading on an 8% denaturing gel.

In the case of BER initiation by hOGG1 the DNA fragment used is 601 DNA but is subjected to UV irradiation before reconstitution. This gives rise to 8-OxoG lesions at places in the DNA fragment. We use human OGG1 to cleave the lesions and then followed by APE-1 digestion to cleave the DNA at the apurinic sites. The DNA fragment is then phenol chloroformed and ethanol precipitated. Then it is thoroughly resuspended in formamide buffer and loaded on an 8% denaturing gel.

In Gel One Pot assay

A sliding reaction was carried out in the presence of increasing concentrations of FACT.

Here, the amount of RSC used gave rise to 10% slid and with increase in FACT the nucleosomes are mobilized. Prior to loading on 5% native polyacrylamide gel, 6.25 pmol of not radioactively labelled 255 bp 601 middle positioned nucleosomes were added to each reaction as a carrier in order to maintain stability during subsequent procedures. They are resolved on 5 % native polyacrylamide gel. Bands corresponding to control unremodeled and unmobilized remodeled nucleosomes were excised, collected in siliconized eppendorf tubes, crushed very gently and immersed with 50 µl restriction buffer (10 mM Tris pH7.6, 10 mM MgCl 2 , 50 mM NaCl, 1 mM DTT and 100 µg/ml BSA) containing increasing amount of HaeIII (0.03, 0.12, 0.50, 2.0, 8 units/µl) for 5 minutes at 29°C. The reaction was stopped by addition of an equal volume (50 µl) of stop buffer containing 0.2% SDS and 40 mM EDTA.

DNA was eluted from the gel slices, purified as described above, and run on 8% denaturing gel. The quantification of extent of accessibility at different superhelical locations in the nucleosome was performed using Multi-Gauge Software (Fuji).

Gel excision of nucleosomes, remosomes and slid fractions

Centrally positioned 601 nucleosomes were incubated with RSC in the remodeling reaction as described above. Reaction was stopped at 50 minutes by addition of 0.1 units of Apyrase and 1 µg of plasmid DNA, as under these conditions the non-mobilized fraction contains essentially remodelled nucleosome particles. Reaction products were resolved on 5% native polyacrylamide gel. Bands, corresponding to plain nucleosome fraction from 0, un-mobilized fractions from 50 minute, and mobilized fraction from 50 minute reaction time points were excised. Excised bands were then cut in small pieces and soaked in 80 µl Elution Buffer (EB) containing Tris 10 mM pH7.4, 0.25 mM EDTA and 10 mM NaCl, at 4°C for 3 hours with gentle shaking. 0.75 nmol of cold 601 255 bp nucleosomes were added in the elution buffer to maintain the stability of eluted nucleosomes. Eluted nucleosomes were filtered through glass fibre filter under low speed centrifugation (200g) to remove acrylamide particles, washed and concentrated using 100 kDa cut-off spin filters. Eluted nucleosomes, remosomes and slid particles were divided into equal aliquots, were further subjected to repair by UDG and hOGG1 in the following experiments. Refer to the DNA repair assays for more details.

ATPase assays

The ATPase assays were carried out in the remodeling reaction buffer whose composition is mentioned above. However, to slow down the reactions we used a lower concentration of ATP such as 100µM and the ATPase assay was performed on the naked DNA, nucleosomes (centre and end positioned with their respective remodelers). To the reaction mixture 1µl of the source 32P ATP was added. After the remodelling reaction is carried out at 29 degree Celsius for 50 minutes, the reaction was stopped by adding formamide dye and 0.1% SDS. A fraction of the sample was loaded on a 13% denaturing PAGE and migrated at 150 volts for 35 minutes. The gel was dried on a gel drier and exposed. Supplementary Figure 5: Remosomes are capable of performing the same extent of Base excision repair initiation as that of slid. Centrally positioned nucleosomes were reconstituted on 32 P!5!labeled!255bp!601!DNA!fragment!which!is!UV!irradiated.(A)!Analysis!of!hOGG1! enzymatic activity within the nucleosomal and the linker DNA. The nucleosomal species including nucleosomes, remosomes and slid were gel eluted and then subjected to enzymatic treatment by increasing amounts of hOGG1. The nucleosome species were incubated with hOGG1 at 29ºC for 90 minutes. The DNA was purified before treating with APE1. This enzymatic cleavage of the apurinic sites was carried out at 37ºC for 2 hours. The cleavage pattern of the isolated DNA was analysed using an 8% denaturing PAGE.(B) RSC promotes hOGG1 cleavage of the lesions that are at the edge and are exposed to the solution. Lane numbers 17 and 23 represent a hydroxyl radical footprint of the nucleosomes and helps us determine the parts of the fragment that are facing inside at the octamer and those that are facing the solution.(C) Increasing amounts of hOGG1gives rise to a signature cleavage pattern in the DNA. Lanes 18-22 represent the DNA that is subjected to hOGG1treatment as mentioned in (A). As noted in the introduction (Chapter1), FACT is actively involved in transcription [188; 189; 190; 191]. For example, nucleosomes assembled on a high affinity DNA sequence present a polar barrier to transcription of Polymerase II and FACT assists Polymerase II to overcome the nucleosome barrier [START_REF] Bondarenko | Nucleosomes can form a polar barrier to transcript elongation by RNA polymerase II[END_REF]. In addition, FACT facilitates the polymerase driven transcription by destabilizing nucleosomal structure so that one histone H2A-H2B dimer is removed during enzyme passage [START_REF] Belotserkovskaya | FACT facilitates transcription-dependent nucleosome alteration[END_REF]. We observed from our previous data that FACT has a co-remodeling activity with chromatin remodelers such as ACF and RSC. FACT acts as efficient fuel conservers and assists the remodeling (generation of remosomes) and mobilization of the nucleosome species with the least energy spent.

With the above in mind, we also wanted to see how the presence of FACT affects the transcription factor interaction with nucleosomes. We hypothesized that FACT, by boosting

Results

Substitution of the NF-B! binding site in the 601 sequence does not alter the nucleosome characteristics.

We have inserted the binding sequence (GGGGATTCCCC) of NF-B! at the nucleosomal dyad (at the center of the nucleosomal DNA, refered here as NF-B D 0 , Figure 1). The nucleosomes were reconstituted by serial salt dialysis. The efficiency of reconstitution was checked by using native PAGE (Figure 2A). As seen, no free DNA was observed in the different samples thus evidencing that under the conditions of reconstitution, all the DNA is associated with the histone octamer. Importantly, both reconstituted nucleosomes exhibited the same DNAase 1 and the hydroxyl radical footprinting patterns (Figure 2B). We concluded that the incorporation of the NF-B!binding sites does affect neither the translational nor the rotational positioning of the 601 nucleosomes. This has allowed us to analyze further with high accuracy the interaction of NF-B!with the reconstituted nucleosomal templates. We have analyzed the binding of NF-B to naked nucleosomal DNA, control (non RSCtreated) nucleosomes, remosomes and slid nucleosomes. The schematic on Figure 3 shows the protocol for isolation of the different species from native PAGE. The species eluted from the gel were allowed to interact with NF-B!and then irradiated with a single 266 laser pulse.

GGGGATTCCCC

DNA was isolated, treated with Fpg protein, and the cleaved samples were resolved on a denaturing gel. The binding of NF-B!to the different probes was also analyzed with EMSA.

As seen, the EMSA shows that NF-B! is able to bind to all of the studied samples (naked DNA, control nucleosomes, remosomes and slid nucleosomes). Indeed, in all four cases higher molecular complexes are formed with much slower electrophoretic mobility (Figure 3). Noteworthy, EMSA does not differentiate between specific and non-specific association of NF-B!with the respective samples. The use of the UV laser footprinting allows, however, to differentiate between specific and non-specific binding (see Chapter 1.7 for more details).

Analysis of the UV footprinting data reveals that only naked DNA and slid nucleosomes exhibited the NF-B UV laser footprinting signature (see Figure 3). No footprints were observed for both control nucleosomes and remosomes (Figure 3). This evidences that NF-B! is able to specifically interact only with naked DNA and slid nucleosomes, i.e. even the perturbed histone-DNA interactions within the remosomes represent a sufficiently strong barrier for NF-B to interact with its cognate sequences.

FACT boosts RSC mediated mobilization of nucleosomes and aids in the binding of transcription factor NF-B on the nucleosomes

We hypothesized that FACT would be able to assist the binding of NF-B to nucleosomal templates through boosting the remodeling activity of RSC. To test this we have incubated 601 NF-B!D0 nucleosomes with either 0.2 u or 1.4 u of RSC (at both these concentrations of RSC a very small part of the nucleosomes was slid) in the presence or absence of FACT.

After arresting the mobilization reaction, NF-B was added to the reaction mixture and allowed to interact with the different templates. Next, the different sample solutions were irradiated with a single UV laser pulse and DNA was isolated and treated with Fpg. The cleaved DNA was then run on a denaturing PAGE (Figure 4). The control naked DNA shows a very clear footprinting reflecting the specific binding of NF-B to its cognate sequence (Figure 4, compare lane 3 with lane 4). In agreement with the previous data (Figure 3), no binding of NF-B was observed to the control nucleosomes (Figure 4, lane 6). Importantly, FACT alone was unable, as expected, to help NF-B to bind to the nucleosome (Figure 4, lane 8). Incubation with 0.2 units of RSC alone or in combination with FACT has again no effect on the capacity of NF-B to interact specifically with the nucleosomes (Figure 4, lanes 9-12). Although, the concerted action of 1.4 units of RSC and FACT leads to the appearance of a footprinting and thus, to some binding of NF-B (Figure 4, lanes 13-16).

Acknowledgements

The FACT is an important molecule that is involved in transcription, replication and DNA repair.

We have in this work tried to understand how FACT assists in Base excision repair and transcription. The data presented demonstrates that the FACT by itself is not able to bring about any changes in the structure of the nucleosome but through its co-remodelling activity it is able to increase the efficiency of the remodelers to both remodel and mobilize nucleosomes and thus to facilitates BER. FACT turns out to be a fuel conserver and acts in the efficient conversion of the energy freed by the hydrolysis of ATP by the remodeler in order to have maximum work done. This scenario was shown to be valid for both remodelers RSC and ACF. However, since FACT function is realized by making the remosomes easier remodelable!we!predict!that!FACT!would!be!also!able!to!boost!the!activities!of!chromatin! remodelers belonging to other families. This remains to be tested in future.

The presented data show that in vitro FACT acts in concert with RSC to assist BER. If this was also happening in vivo, one should expect FACT to be associated within the cell with different repair proteins as well as with remodelling factors involved in repair of damaged DNA. We would like to test this hypothesis. Briefly, we plan to establish stable cell lines expressing double tagged (FLAG and HA) SSRP1 (one of the subunits of FACT) and then to purify the FACT complex by using double immuno-purification procedure. The members of the immuno-purified FACT complex will be identified by mass spectrometry. Their identity will be further confirmed by Western blotting using specific antibodies. If we find some remodelling factors involved in DNA repair and/or repair proteins associated with FACT, this will be a strong evidence for the FACT operating mechanism in DNA repair.

The proposed mechanism for FACT functioning in DNA repair predict that FACT should be physically present at the site of DNA damage at least at the beginning of the repair process to allow efficient chromatin remodelling. We will study this by using cell lines expressing GFP-FACT. We expect GFP-FACT to be recruited to the sites of laser-induced damage of DNA. We will follow in real time both the recruitment and the time-course of GFP-FACT

SI Text

  shadow and seeing him work has made him my inspiration and role model. Your constant encouragement has driven me to become what I am today. I love you bro and I thank you for being my friend and critic scientist. Thanks a zillion for always being there. Here is me wishing you a totally fantastic and fabulous research life ahead.

Figure 3 Figure 4 Nucleosome Dynamics Figure 5 Histone

 345 Figure 3Assembly of Histones on the DNA to form the nucleosome Figure 4 Nucleosome Dynamics Figure 5 Histone Variants and their functions Figure 6

Figure 14 Models for Nucleosome Sliding Figure 15 Picture showing the experimental evidences of remosomes Figure 16

 141516 Figure 14Models for Nucleosome SlidingFigure 15Picture showing the experimental evidences of remosomesFigure 16Schematic representation of the two-step, RSC induced nucleosome mobilization

Figure 17 HistoneFigure 18 Figure 19 Two

 171819 Figure 17Histone chaperones aid in step wise assembly and disassembly of the nucleosomesFigure 18Domain distribution and structural alligment of hFACT against yFACTFigure 19Two Models for FACT meadiated Nucleosome Reorganization

Figure 20

 20 Figure 20

Figure 22 NF-B!family Figure 23 Figure 24

 222324 Figure 22NF-B!familyFigure 23Illustration depicting the biphotonic mechanism of UV laser induced photo lesionsFigure 24Chemical and enzymatic mapping of DNA lesions generated by UV laser biphotonic ionization chemistry

  Our data are instrumental in deciphering the molecular mechanism of FACT implication in BER and NF-B mediated transcription. C. Keywords a. Mots clés en Français Remodelage de la chromatine, nucléosome, FACT, RSC, UDG, chromatine, réparation, ADN, NF-B b. Keywords in English Chromatin remodelling, nucleosome, FACT, RSC, chromatin, repair, DNA, NF-B!! D. Details of the laboratories of thesis preparation a. Laboratory of affiliation

Figure 1 :

 1 Figure 1: Chromatin organisation and chromatin architecture [18].

Figure 2 :

 2 Figure 2: General properties of Euchromatin and heterochromatin[19].

Figure 3 :

 3 Figure 3: Assembly of Histones on the DNA to form the nucleosome.

Figure 4 :

 4 Figure 4: Nucleosome Dynamics: where (a) Incorporation of Histone variants (b) Modication of Histones and (c) chromatin remodeling. This image is adapted from a review on chromatin remodeling complexes [40].

Figure 5 :Figure 6 :

 56 Figure 5: Histone Variants and their functions

Figure 7 :

 7 Figure 7: The SWI/SNF Family of remodelers and their functions.

Figure 8 :

 8 Figure 8: The ISWI group of remodelers and their functions.

Figure 9 :

 9 Figure 9: The CHD family of Chromatin Remodelers.

Figure 10 :

 10 Figure 10: The INO80 family of remodelers.

Figure 12 :

 12 Figure 12: Electron microscopy reconstructions of ISWI class of remodelers

Figure 13 :

 13 Figure 13: Biochemical properties of ATP dependant Chromatin Remodelers

Figure 14 :

 14 Figure 14: Models for Nucleosome Sliding (a) Twist Diffusion model (b) The Loop/Bulge propagation model. This image is adapted from [110].

Figure 15 :

 15 Figure 15: Picture showing the experimental evidences of remosomes.

Figure 16 :

 16 Figure 16: Schematic representation of the two-steps, RSC induced nucleosome mobilization.

Figure 17 :

 17 Figure 17: Histone chaperones aid in step wise assembly and disassembly of the nucleosomes [121].

Figure 18 :

 18 Figure 18: Domain distribution and structural alligment of hFACT againstyFACT.This Image is adapted from a recent review from Winkler and Luger[START_REF] Winkler | The histone chaperone FACT: structural insights and mechanisms for nucleosome reorganization[END_REF].

  the ySpt16-Pob3. The second step involves the tethering of the yeast FACT to the histone components in the nucleosome. The final step involves the restoration of the whole nucleosome. A model endorsing the two pathways on FACT is shown in the Figure 19.

Figure 19 : 1 . 5 DNA damage and Repair Systems 1 . 5 . 1

 1915151 Figure 19: Two Models for FACT mediated Nucleosome Reorganization.

  can be removed by the dRPase activity of polymerase b (pol b) and the 3-abasic terminus left by AP lyase is cleaved by the 3-diesterase activity of polymerase b (pol b) and the 3 -abasic terminus left by AP lyase is cleaved by the 3 -diesterase activity of AP endonuclease. The resulting gap is #lled by pol b and the remaining nick is sealed by DNA ligase I or III. In the case of long patch BER, however, additional factors such as FEN1, PCNA and RFC are involved. After the formation of the AP site by AP endonuclease, pol g or pol e extend the DNA strand from 3-OH which is accompanied by displacement of the strand containing 5-dRP for several nucleotides. The resulting $ap is removed by the activity of FEN1 producing a nick that is ligated by DNA ligase I. The accessory factors such as PCNA and RFC enhance the activity of DNA pol g and pol e in this pathway (Figure 21)..

Figure 21 :

 21 Figure 21: A schematic representation of both short-patch and long-patch base excision repair

  These proteins have homology and possess a structurally conserved 300 amino acid sequence called the REL region, a structurally similar motif, referred to as the Rel Homology Region (RHR, sometimes also referred as RH). This Rel Homology Region is responsible for locating the binding sequence on the DNA, dimerization with other Rel Proteins and most importantly nuclear localization. Rel proteins are utilized by many eukaryotic organisms and the RH domain remains highly conserved amongst them. While all Rel proteins share a similar motif, they can still be divided into two classes based upon the sequence at the C-terminal side of the RH. The first class of proteins consists of a long chain of repeats that inhibit their function. The second class of Rel proteins contains a transcription activator region on the C-terminal end of the RH.

Figure 22 : NF-B!family 1 . 7

 2217 Figure 22: NF-B!family

Figure 23 :

 23 Figure 23: Illustration depicting the biphotonic mechanism of UV laser induced photo lesions.

Figure 24 :

 24 Figure 24: Chemical and enzymatic mapping of DNA lesions generated by UV laser biphotonic ionization chemistry.

  contradictory and do not allow to have a clear picture of FACT activities. The two main objectives of the present study are: (a) To decipher the molecular mechanism of FACT functioning in Base Excision Repair. (b) To understand how FACT modulates the structure of the nucleosome and gives easier access to the underlying nucleosome DNA sequence by studying the interaction of the transcription factor NF-B!with nucleosomal templates.

Figure 2 .

 2 Figure 2. FACT facilitates both RSC-induced remodeling and mobilization of nucleosomes. (A) DNase I footprinting. End-positioned nucleosomes, reconstituted on 32 P! 5-labeled 241 bp 601 DNA fragment, were incubated with 0.2 units of RSC in the absence (lane 3) or in the presence of 1.6 pmol of FACT (lane 4) for 50 minutes at 30°C; lane 5, same as lane 3, but with 1 unit of RSC; After arresting the remodeling reaction, the samples were digested with 0.1 units of DNase I for 2 minutes, the cleaved DNA was isolated and run on 8% PAGE under denaturing conditions; lanes 1 and 2, controls showing the DNase I cleavage pattern of nucleosomes (lane 1) alone or incubated with 1.6pmol FACT under the conditions described above. (B) The presence of FACT increases the efficiency of RSC-induced nucleosome mobilization. Centrally positioned nucleosomes were incubated with 0.2 units of RSC in the presence of increasing amount of FACT, the reaction was arrested and the samples were run on native PAGE. The position of the unslid and slid nucleosomes were indicated; lane 1 control nucleosomes; lane 2, nucleosomes incubated with RSC alone (in the absence of hFACT) (C) quantification of the data presented in (B).

  and[111; 183]. Briefly, eight mutated32 P-end labeled 255 bp 601.2 sequences were used to reconstitute centrally positioned nucleosomes. A single HaeIII restriction site (designated as d 0 to d 7 , where the subscript refers to the number of helical turns from the nucleosome dyad) was inserted within each of these sequences. The equimolar mixture of the eight reconstituted nucleosomes was incubated with appropriate amount of RSC either alone (to produce ~15% of slid nucleosomes) or in the presence of

Figure 3 .

 3 Figure 3.! In! gel! one! pot! assay! analysis! of! the! effect! of! FACT! on! the! DNA! accessibility! towards HaeIII along the length of nucleosomal DNA in control and RSC treated nucleosomes. (A, B) Effect of hFACT on RSC-induced remosome generation. (B) Preparative PAGE. Centrally positioned nucleosomes were treated with increasing amount of FACT in the presence of 0.2 units of RSC and after arresting the reaction they were separated on native PAGE; last lane, nucleosomes treated with 5-fold higher amount (1 unit) of RSC, in the absence of hFACT; the first three lanes, untreated, and treated with hFACT and with 0.2 units of RSC nucleosomes, respectively. The indicated bands (from 1 to 9) were excised from the gel and in-gel digested with 8 units of HaeIII for 10 minutes at 30°C. The cleaved DNA was then isolated and separated in 8% PAGE under denaturing conditions (B). The positions at the cleavage of the different dyads are indicated on the left; the numbers of each lane refers to the respective excised bands from the preparative PAGE (see A); ctrl, control, non digested DNA; DNA, naked DNA used for reconstitutions of the nucleosomes digested with HaeIII. (C) Quantification of the data presented in (B). The # indicates a fragment that corresponds to a HaeIII! site!present! only! in! dyad!7!601.2! fragment!and!located! at! 4!bp!from! the!dyad!7! (d7) site

  removed!(Figure!4,!remosomes).!!Upon!increasing!the amount of UDG, the efficiency of removal increases and achieves saturation at ~5x6 -1 units! of!UDG!(Figure!4,!remosomes).! Excision of uracil from slid nucleosomes exhibits essentially the same behavior (Figure 4, slid).! These! results! reveal! that! the! alterations of the histone-DNA interactions in the remosome are sufficient for achieving efficient repair.

Figure 4 .

 4 Figure 4. Efficient UDG excision of uracil from RSC-generated remosomes and slid nucleosomes. 32 P! 5-labeled 255 bp 601 DNA fragment containing randomly incorporated uracil residues was used for reconstitution of centrally positioned nucleosomes. The nucleosomes were treated with RSC either in the absence of ATP (control particles) or in the presence of ATP to produce ~ 50% mobilized particles. The remodeling reaction was arrested and the samples were separated on native PAGE. The slid nucleosomes and the nonmobilized nucleosomes (containing the remosome fraction) as well as the control nucleosomes were eluted from the gel slice. The particles were then treated with the indicated increasing concentrations of UDG, the cleaved DNA was isolated and run on 8% PAGE under denaturing conditions; DNA, naked 255 bp 601 DNA fragment digested with UDG; first and last lane, OH radical footprinting of native nucleosomes; on the right part of the figure is shown a schematics of the reconstituted nucleosome.

( 1 .

 1 6pmol) used in the experiments. In other words, the presence of FACT allows RSC to transform much more efficiently the energy freed by the ATP hydrolysis into mechanical work. Since FACT does not affect the ATPase activity of RSC, it should act on the nucleosomes making them more prone to remodeling.

Figure 5 .

 5 Figure 5. FACT increases the efficiency of the freed by the RSC-mediated hydrolysis of ATP energy used for nucleosome remodeling (A) Nucleosome mobilization assay. Centrally positioned nucleosomes were incubated with 0.3 units of RSC at 120 mM of ATP in either the absence or presence of increasing amount of FACT for 50 minutes at 30°C. After arresting the reaction, the samples were run on native PAGE. The bands corresponding to the centrally and end-positioned nucleosomes are indicated. The lower panel represents the respective quantified data (B) ATPase hydrolysis assay. Centrally positioned nucleosomes were incubated with RSC (0.3 units) and increasing amount of FACT in the presence of 120 mM of ATP and 3.3µM of32 P[gATP]. The products of the ATP hydrolysis were analyzed on 15% PAGE under denaturing conditions. Lower panel shows the respective quantified data.

  chromatin remodeling and sliding, might also allow an easier access of transcription factors to nucleosomal templates. If this was really the case, transcription factors should be able to bind to either remodeled nucleosomes (remosomes) and/or slid nucleosomes generated by the concerted action of FACT and RSC. We carried out some biochemical experiments to validate our hypothesis. We decided to use the transcription factor NF-B!in our studies. The transcription factors NF-B! has 150 gene targets but how NF-B! invades! the! nucleosomal! templates remains still elusive.We have analyzed how the transcription factor NF-B interacts with the nucleosomes by inserting the NF-B!recognition sequence within nucleosomal DNA. The interaction of NF-B! (p50/p50 dimer) with control nucleosomes, and RSC-generated remosomes and slid nucleosomes was studied by using EMSA, DNase I and UV laser footprinting. The combination of these three techniques allowed us to demonstrate that NF-B! binds specifically only to slid, but not to both control nucleosomes and remosomes.

Figure 1 :

 1 Figure 1: Incorporation of NF-B! binding site in the nucleosome. Schematic showing the wild type 601 sequence and the mutated 601 sequence, containing the NF-B!binding sites at the dyad of the nucleosome (center of the nucleosomal DNA).

Figure 2 :

 2 Figure 2: Characterization of the centrally positioned nucleosomes formed on the new templates 601 NF-B! D 0 . (A) EMSA on reconstituted nucleosomes using WT-or mutated 601-NF-B!D 0 sequence, which contains the inserted GGGGATTCCCC NF-B!! recognition sequence at the nucleosomal dyad. Note that there is no change in pattern or migration of the abovementioned nucleosomes with no free DNA formed. (B) OHº radial footprinting of 601 WT and 601 NF-B!D 0 nucleosomes. (C) DNase 1 footprinting of 601 WT-and 601 NF-B! D 0 nucleosomes. The position of the nucleosome is indicated on the schematics between panel (B) and panel (C).

  Laser Footprinting demonstrates that the RSC-induced mobilization of nucleosomes, but not their remodeling, is sufficient for NF-B! binding to its cognate DNA sequence

Figure 3 :

 3 Figure 3: NF-B!binding on the Nucleosome species. (A) Schematics of the isolation of the different nucleosome species. DNA, control nucleosomes and RSC treated mobilized and non mobilized (remosomes) fractions were run on native gel, excised from the gel and after gel elution they were allowed to bind with NF-B. The NF-B! complexes! were!then irradiated with the UV laser and treated with Fpg. The cleaved DNA was run on denaturing gel. (B) UV laser footprinting (lower panel) and EMSA (upper panel) of the studied samples. The inset (right) shows an enlarged part of the gel containing the binding sequence of NF-B.! Above the inset, the position of the control and the slid nucleosomes are presented.

  !research!leading!to!these!results!has!received!funding!from!the!European!Communitys! Seventh Framework Programme FP7/2007-2013 under grant agreement number 222008 hFACT

Figure 4 :

 4 Figure 4: Transcription factor binding in the presence and absence of FACT. Lanes 1 and 17 10 bp DNA ladder. The bottom panel shows the enlarged NF-B binding sequence. On the right is shown the schematic presentation of the nucleosome..

Figure S1 .

 S1 Figure S1. Expression and purification of H2A COOH-terminal deletion and chimeric

Figure S2 .

 S2 Figure S2. One pot restriction accessibility assay of conventional and H2A.Bbd

Figure S3 .極

 S3 Figure S3. Scans of hydroxyl radical cleavage profile (See Figure 4B) for H2A, H2A.Bbd

Introduction The nucleic-acid system that operates in terrestrial life is optimized (through evolution) chemistry incarnate. Why not use it to allow human beings to sculpt something new, perhaps beautiful, perhaps useful, certainly unnatural. Roald Hoffmann, writing in American Scientist, 1994.

	List of Abbreviations MTA 1.1	Metastasis associated Antigens
	NaCl		Sodium Chloride
	ACF NCP		ATP-dependent Chromatin assembly and remodeling Factor Nucleosome Core Particle
	AFM NER		Atomic Force Microscopy Nucleotide Excision repair
	ALS NF-B!!		Amyotrophic Lateral Sclerosis Nuclear Factor Kappa-light-chain-enhancer of activated B cells
	AP-site NHEJ		Apurinic Site Non Homologous End Joining
	ATP nm		Adenosine-5'-triphosphate Nano meter
	BAP NURD		Bramha Associated Protein NUcleosome Remodeling and Deacetylation
	Bbd NURF		Barr Body Deficient Nucleosome Remodeling Factor
	BER OH		Base excision Repair Hydroxyl radical
	bp PAGE		Base Pair Polyacrylamide Gel Electrophoresis
	BRG PBAP		Brahma Related Protein-1 Poly Bramha Associated Protein
	BSA PCR		Bovine Serum Albumin Polymerase Chain Reaction
	CENPA PHD		Centromere Protein A Plant Homeo Domain
	CHD ROS		Chromodomain Helicase DNA-binding Reactive Oxygen Species
	ChIP RSC		Chromatin Immuno Precipitation Remodels Structure of Chromatin
	CHRAC SDS		CHRomatin Accessibility Complex Sodium Dodecyl Sulfate
	CK2 UDG		Casein Kinase 2 Uracil DNA Glycosylase
	COPD UV		Chronic obstructive Pulmonary Disorder Ultra Violet
	COX2		Cyclooxygenase 2
	CS		Cockayne Syndrome
	Da		Dalton (1g/mol)
	DMS		Dimethylsuberimidate
	DNA		Deoxyribo Nucleic Acid
	DTT		Dithiothreitol
	ECM		Electron Cryo Microscopy
	EDTA		Ethylene Diamine Tetra Acetic acid
	EM		Electron Microscopy
	EMSA		Electrophoretic Mobility Shift Assay
	ERK		Extracellular signal related Kinase
	FACT HAC	Fascilitates chromatin transcription Laboratoire de Biologie Moléculaire de la Cellule Histone Acetyltransferases
	HAD Hae III	Histone deacetylases UMR5239-ENS de Lyon-CNRS Haemophilus aegypticus III
	HeLa		École Normale Supérieure Henrietta Lacks
	HEPES		4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
	HMG		46!allée!dItalie High Mobility Group
	hOGG1 iNOS		human Oxoguanine Glycosylase 69364 Lyon cedex 07 Inducible nitric oxide Syntase
	ISWI KDa		Imititation SWItch FRANCE Kilo Dalton
	LPS		Lipopolysaccharide
	MAP		Mitogen Activated Protein
	MBD		Methylcytosine Binding Domain

1.3.2 Post translational modification of histones

	Chromatin modifications and gene regulations are an important branch of chromatin
	biochemistry. Core histones can be reversibly modified by acetylation, methylation,
	phosphorylation, ubiquitination or ADP-ribosylation and these modifications have
	consequences for gene activation, gene repression and chromosome replication. Lysines at
	the amino-terminal ends of the core histones are the predominant sites of known regulatory
	modifications. Active genes are preferentially associated with highly acetylated histones
	whereas inactive genes are associated with hypoacetylated histones. Histone acetylation and
	deacetylation are thought to exert their regulatory effects on gene expression by altering the
	accessibility of nucleosomal DNA to DNA-binding transcription activators, with the help of
	other chromatin modifying enzymes or multi-subunit chromatin remodeling complexes
	capable of displacing nucleosomes.

1.3.3.1 Chromatin remodeler Families 1.3.3.1.1 SWI/SNF Family of Chromatin remodelers

	This family of chromatin remodelers have been named the SWI/SNF family mainly because
	of the discovery of the first member ySWI/SNF [41; 42; 43]. The yeast SWI/SNF complex
	consists of 11 subunits namely, SWI2/SNF2, SWI3, SNF5, SNF6, SNF11, SWP82, SWP73,
	SWP29, ARP7 and ARP9 [43; 44; 45; 46]. Most of the SWI/SNF components have similar
	homologous counterparts in other SWI/SNF like remodelers, thus suggesting some kind of
	conservation of function. For example, SWI1p shows homology to OSA and Baf250p of
	dBrahma and hSWI/SNFa. SWI/SNF like remodelers have a tendency to bind to naked DNA
	and to nucleosomal DNA in an ATP dependant manner [47; 48]. A striking similarity of these
	SWI/SNF remodelers is with High Mobility Group Box HMGB containing proteins in their
	ability to bind to DNA structures and not to sequence specific DNA [47; 48]. Moving
	towards higher organisms there is the presence of SWI/SNF like complexes in drosophila
	called BAP(Brahma Associated Protein) or PBAP (Polybromo associated BAP)

Cell Cycle Progression; Chromosome Segregation and cohesion RNAPII Elongation and Termination Metamorphosis and Immune System Function Transcription Elongation; Activator Targeting Signaling; Differentiation and Development; Tumor Supressor; Viral Integration and Expression

	Mitotic Gene Expression;
	DNA Repair;
	Activators, Corepressor
	Targeting; Replication;
	Transcrition Activation,
	Elongation and Termination
	ySWI/SNF		DNA Repair; Activator Targeting;
			RNAII and RNAPII Regulation;
	m/hpBAF	yRSC	Cell Signaling; Spindle Checkpoint Assembly;
	SWI/SNF
	Family
	m/hBAF	dBAP
	dPBAP

INO80 SWR1 INO80 Family H2A.Z Incorporation; DNA Repair by p-H2A Recruitment DNA Repair by p-H2A Recruitment; Core Histone Removal; Homologous Recobination; Replication; Telomere Length Regulation; Transcription Regulation; RNAPII Regulation

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

21 22 [START_REF] Beato | Transcription factor access to chromatin[END_REF] 24 25 RSC -------.15 .3 .6 1.2 2.4 -. 2 .2 .2 .2 .2 .2 ------hFACT ------------1.6 -.1 .2 .4 .8 1.6 ------•OH•OH UDG -9 -3

Acknowledgements

I would like to thank my guide Dr. Stefan Dimitrov for giving me an opportunity to work with him as a PhD student. It gives me immense pleasure to mention that it is a privilege to work with him and I am indebted to him for his role in shaping me into a scientist. I must say

Acknowledgements

The!research!leading!to!these!results!has!received!funding!from!the!European!Communitys! Seventh Framework Programme FP7/2007-2013 under grant agreement number 222008

Supplementary information for sequences used in chapters 2 and 3

Appendix II

Publications

Crosslinking with DMS

DMS is water soluble, imidoester crosslinker. The imidoester functional group is one of the most specific acylating groups available for modification of primary amines. Moreover, the imidoamide reaction product does not alter the overall charge of the protein. The crosslinking of the histones in the nucleosomes were carried out as follows. The nucleosome in itself helps in bringing the histones in the octamer closer to each other for the crosslinking reaction to progess. The free DNA suggests there are no free floating histones in the mixture. DMS is freshly prepared with a concentration of 50mg/ml. The nucleosomes are buffered against 0.3M HEPES pH 10 to remove any remaining traces of Tris. The nucleosome concentration is kept at 50ng/µl to prevent intra nucleosome cross-linking. For a 5 µg nucleosome mix in a reaction volume of 100 µl at pH 10 we add 20 µl of the DMS stock solution. The reaction mixture is incubated at room temperature for 1 hour. The cross linking reaction is stopped by the addition of 2 µl of 1M Tris pH8.0 followed by a buffering the solution against 25 mM Tris for 90 minutes and then against 10 mM Tris overnight. The amount of cross linked histones is checked by running the TCA precipitated histones on a 5-20 % gradient gel. recruitment to damaged DNA by using fluorescence microscopy. If we find that FACT is recruited as predicted to the sites of DNA damage, we will next ask whether the time-course of recruitment of FACT is similar to these of the other identified proteins of the FACT complex (see above). Of particular interest will be to analyse the kinetics of recruitment to damaged DNA of the expected chromatin remodeler(s) associated with FACT in vivo.

Supplementary Figures

FACT and NF-B!binding to chromatin

The presented in chapter 3 data suggest that FACT could be involved in the activation of NF-B! dependent genes via boosting the remodelling of the chromatin structure at their promoters. In addition, the available literature data suggest that FACT should assist transcription by allowing an easier Polymerase II passage through the nucleosome. We will study these questions by ChIP using either specific antibodies against FACT or cells which express tagged (HA-and FLAG) versions of FACT (see 4.1.1). In the last case the commercially available high affinity anti-HA and anti-FLAG antibodies would be very helpful. We will analyze both the presence and the time of recruitment of FACT to both the promoters and the coding sequences of early and late NF-B!responsive genes.

Appendix