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Contents lists available at ScienceDirect

DNA Repair

jo ur nal home p age: www.elsev ier .com/ locate /dnarepai r

ini review

hromatin structure in double strand break repair

nastas Gospodinova,∗∗, Zdenko Hercegb,∗

Institute of Molecular Biology, Bulgarian Academy of Sciences, Acad. G. Bonchev Str. 21, 1113 Sofia, BulgariaEpigenetics Group, International Agency for Research on Cancer (IARC), 150 Cours Albert Thomas, F-69008 Lyon, France

r t i c l e i n f o

rticle history:eceived 10 July 2013ccepted 11 July 2013vailable online xxx

eywords:NA repairhromatin

a b s t r a c t

Cells are under constant assault by endogenous and environmental DNA damaging agents. DNA doublestrand breaks (DSBs) sever entire chromosomes and pose a major threat to genome integrity as a resultof chromosomal fragment loss or chromosomal rearrangements. Exogenous factors such as ionizingradiation, crosslinking agents, and topoisomerase poisons, contribute to break formation. DSBs areassociated with oxidative metabolism, form during the normal S phase, when replication forks collapseand are generated during physiological processes such as V(D)J recombination, yeast mating typeswitching and meiosis. It is estimated that in mammalian cells ∼10 DSBs per cell are formed daily. If

istone modificationsSBsancer

left unrepaired DSBs can lead to cell death or deregulated growth, and cancer development. Cellularresponse to DSB damage includes mechanisms to halt the progression of the cell cycle and to restore thestructure of the broken chromosome. Changes in chromatin adjacent to DNA break sites are instrumentalto the DNA damage response (DDR) with two apparent ends: to control compaction and to bind repairand signaling molecules to the lesion. Here, we review the key findings related to each of these functionsand examine their cross-talk.

© 2013 Elsevier B.V. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. The DNA damage response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. Accessibility and positioning of damaged chromatin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

3.1. ATP-chromatin remodeling in DSB repair. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.2. Post-translational modifications in control of chromatin accessibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003.3. Higher-order chromatin organization and DSB repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

4. Provision of binding interfaces . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.1. H2AX as a key spatial organizer of the DDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.2. Ubiquitination and methylation of histones in DDR . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004.3. Recruitment of chromatin remodelers by poly(ADP-ribosyl)ation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

5. Crosstalk and interdependence of DSB-induced chromatin changes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006. Chromatin modifiers, DSB repair and cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 007. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Please cite this article in press as: A. Gospodinov, Z. Herceg, Chromahttp://dx.doi.org/10.1016/j.dnarep.2013.07.006

Funding source . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

∗ Corresponding author. Tel.: +33 4 72 73 83 98; fax: +33 4 72 73 83 29.∗∗ Corresponding author. Tel.: +359 2 979 26 47.

E-mail addresses: agg@bio21.bas.bg (A. Gospodinov), herceg@iarc.fr (Z. Herceg).

568-7864/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.dnarep.2013.07.006

tin structure in double strand break repair, DNA Repair (2013),

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. Introduction

Double strand breaks (DSBs) sever entire chromosomes and theesulting loss of chromosomal fragments or chromosomal rear-angements pose a grave threat to genome integrity. Exogenousactors such as ionizing radiation, crosslinking agents, as well asopoisomerase poisons, contribute to break formation. DSBs aressociated with oxidative metabolism, form during normal S phasehen replication forks collapse, or are generated deliberately dur-

ng processes such as V(D)J recombination, yeast mating typewitching and meiosis. It is estimated that in mammalian cells ∼10SBs per cell are formed daily. If left unrepaired, DSBs can lead toell death or deregulated growth and cancer development.

The cellular response to DSBs consists of mechanisms to halt the

Please cite this article in press as: A. Gospodinov, Z. Herceg, Chromahttp://dx.doi.org/10.1016/j.dnarep.2013.07.006

rogression of the cell cycle and to restore the structure of the bro-en chromosome (Fig. 1). Changes in chromatin adjacent to DNAreak sites are integral to the DNA damage response (DDR) with

ig. 1. A simplified scheme of the DNA damage response. (A) Following break induction tho H2AX phosphorylation, binding of MDC1 and amplification of the damage signal. (B) Fond BRCA1 and depending on the cell cycle stage and other factors, DSB ends are either eime for repair the cell cycle is blocked, as Chk2 is phosphorylated by ATM (C.1.) and phosphinases and cell cycle transitions. Additionally, Chk2 phosphorylation of p53 ellicits transcs the major DSB repair mode in mammals. It is initiated by the binding of Ku proteins

NA-PK regulates limited processing of DNA ends by the Artemis nuclease (D.3.) and br DNA ligase IV complexed with XRCC4 and XLF (D.4.). (E) Homologous recombination reingle-strand DNA overhangs are first covered by RPA, later displaced by Rad51 in an exucleoprotein filament searches for and invades the homologous duplex and following eouble helix is restored (E.4.).

PRESSepair xxx (2013) xxx– xxx

two apparent ends: to control compaction, and to bind repair andsignaling molecules to the lesion (Fig. 2). Here, we review the keyfindings related to each of these functions and examine the rela-tionship of chromatin changes during DSB repair with the nuclearstructure and oncogenesis.

2. The DNA damage response

As repair takes time, it is essential to first block cell cycle pro-gression to prevent cell cycle events that might aggravate theadverse effects of DNA damage. Mammalian cells have three DNAdamage checkpoints/brakes: G1/S, intra-S and G2/M [1], whererepairs occur. Checkpoint pathways consist of DNA damage sen-

tin structure in double strand break repair, DNA Repair (2013),

complex is one of the first factors to bind DSBs [2]. MRN recruits theataxia-telangiectasia-mutated (ATM) checkpoint kinase through itsbinding to the Nbs1 subunit [3]. ATM is the major checkpoint kinase

e MRN complex binds the DSB. Recruitment and activation of ATM by the DSBs leadllowing additional histone modifications facilitated by MDC1, recruitment of 53BP1xtensively resected or not, which determine repair pathway choice. (C) To provideorylates and inactivates Cdc25 phosphatases (C.2.), thus blocking cyclin-dependentriptional events and may induce apoptosis (C.3.). (D) Non-homologous end joining

(D.1.), which in turn, bind DNA-PKcs (D.2.) and activate DNA-PKcs kinase activity.ings about the recruitment of the factors that carry out rejoining of the DNA endspair requires extensive processing of the DSB ends into 3′-ssDNA overhangs (E.1.).change reaction dependent on Rad51 paralogues and Rad52 (E.2.). Rad51-coveredxtension (E.3.) of the invading strands and Holliday junction resolution the broken

Please cite this article in press as: A. Gospodinov, Z. Herceg, Chromatin structure in double strand break repair, DNA Repair (2013),http://dx.doi.org/10.1016/j.dnarep.2013.07.006

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Fig. 2. Schematic representation of chromatin changes associated with specific steps of DSB repair. (1) Following induction of a DSB, chromatin in the regions surrounding thelesion rapidly decondenses to a more open configuration in an energy dependent manner. This ATP dependent chromatin-remodeling event likely facilitates the subsequentrecognition and processing of damaged DNA. (2) Chromatin perturbations or DSBs trigger phosphorylation and activation of TIP60 acetyltransferase, which binds methylatedH3K9, acetylates and activates ATM kinase. (3) ATM is recruited by the MRN complex and phosphorylates its targets, key among them histone H2AX. H2AX phosphorylationcauses MDC1 to bind stably, and the latter recruits more of the ATM forming an autoamplification loop to spread the modification. (4) MDC1 serves as an interaction platformfor the binding of further histone modifying activities, TRRAP-TIP60 acetyltransferase complex among them. Histone acetylation by this complex provides access to thedownstream repair factors. Recruitment of RNF8 and RNF168 is promoted by (5) p400 (likely as part of TRRAP–TIP60 complex) and (6) CHD4 of the NuRD complex. RNF8ubiquitin ligase (7) (likely aided by other factors) initiates a series of histone ubiquitination events. PARylation by PARP1 (8) recruits SMARCA5 (9), the catalytic subunitof ISWI chromatin remodeling complexes and SMARCA5 promotes RNF168 accumulation at DSBs (10). RNF168 amplifies the ubiquitin signal to levels needed for 53BP1and BRCA1 recruitment by targeting lysine residues in H2A/X and H2B. Recruitment of 53BP1 requires simultaneous histone ubiquitination and histone methylation. (11)MMSET methyltransferase increases H4K20 methylation at DSBs and is required for efficient repair. 53BP1 binding may also rely on other methyl marks, exposed after DSBformation. (12) Chromatin remodeling events by INO80 and SMARCAD1 are required for efficient 5’–3’ resection prior to HR. (13) Yeast SWI/SNF chromatin remodeler disruptschromatin structure at the donor locus and facilitates strand invasion. (14) Histone deacetylases 1 and 2 are rapidly recruited at sites of DSBs and promote NHEJ. Deacetylationby HDACs is likely followed by hyperacetylation (15) and p300 and CBP acetyltransferases have been shown to promote recruitment of Ku proteins. Recruitment of Ku70 isalso facilitated by SWI/SNF chromatin remodeler.

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ctivated in response to double strand breaks. In S-phase, a relatedIKK family member, ATR, is recruited when its interaction partnerTRIP (ATR-interacting protein) is bound to RPA-coated single-tranded DNA [4]. The PIKK kinases phosphorylate and activate theownstream effector kinases: checkpoint kinases 1 and 2 (Chk1 andhk2). These target cell cycle progression via inactivation of Cdc25hosphatases, which are critical regulators of CDK activity [5,6].

Cells utilize two major pathways to repair DSBs: non-omologous end-joining (NHEJ) and homologous recombinationHR). In mammalian cells NHEJ is the principal DSB repair mech-nism. The process is initiated by the Ku70 and Ku80 proteins,ollowing limited DSB end processing by the MRN complex. Kueterodimer recruits the DNA-dependent protein kinase catalyticubunit (DNA-PKcs), as well as the Artemis nuclease and the ligaseomplex (DNA ligase IV, together with XRCC4 and XLF) [7]. NHEJ isctive throughout the entire cell cycle [8], but as it is not guided by

template, it is considered error-prone.During late S and G2 phases an intact homologous sequence

ecomes available on the sister chromatid and it is used as a tem-late for the restoration of DSBs by the homologous recombinationepair [9]. HR repair is initiated when broken ends are processedo long single-stranded DNA (ssDNA) in a two-step reaction inhich Mre11 and Sae2 (CtIP in mammals) first remove a small

ligonucleotide from DNA ends. Further bulk resection is carriedut by Exo1 and/or BLM and Dna2 to generate extensive tractsf ssDNA [10] in a mechanism conserved from yeast to mammals11]. Replication protein A (RPA) complex binds the resulting ssDNAverhangs, and is later replaced by Rad51. Rad51-covered nucleo-rotein filament carries out the homology search and catalysestrand exchange to prime the DNA repair synthesis [12]. Formationf the Rad51 nucleoprotein filament is enhanced by interactionsith Rad51 paralogues [13–15] and Rad52 [16–18]. Rad51 invasion

nto the donor duplex is ATP-dependent [19–21] and is stimulatedy Rad54 [22,23]. The invading strand is extended by replicativeNA polymerases and branch migration leads to restoration of theenetic information spanning the break [12,13].

The choice between HR repair and NHEJ is established at thetep of DNA end resection–its initiation prevents NHEJ and pro-otes HR repair, and is dependant on the cell cycle phase and the

ype of DNA ends, among others [24]. Thus, the proteins involvedn resection are direct phosphorylation targets of cyclin dependentinases [25,26]. The abundance of CtIP is cell cycle regulated, withhe highest levels in S and G2 phases and lower in GI [27,28]. Impor-antly, the BRCA1 and 53BP1 proteins are involved in an interplayhat controls DNA end resection–BRCA1 stimulates end resection29,30], while 53BP1 inhibits resection by protecting DNA breaknds [31].

. Accessibility and positioning of damaged chromatin

It is believed that DNA compaction in chromatin hinders theNA metabolism processes, and that DNA repair requires mech-nisms to open compacted chromatin structures. In line withhis thinking, early observations of nuclease sensitivity kineticsf newly synthesized DNA in UV irradiated cells suggested thateversible changes of chromatin accessibility are a part of the repairrocess, which led to the “access, repair, restore” model for DNAepair [32]. Ample evidence indicates that multiple mechanismsooperate to provide damage accessibility during DSB repair asell.

Please cite this article in press as: A. Gospodinov, Z. Herceg, Chromahttp://dx.doi.org/10.1016/j.dnarep.2013.07.006

.1. ATP-chromatin remodeling in DSB repair

In an elegant study in living cells, Kruhlak et al., foundhat seconds after break induction, chromatin undergoes an

PRESSepair xxx (2013) xxx– xxx

energy-dependent local expansion, suggesting the involvementof ATP-dependent chromatin remodelers. These complexes func-tion by weakening the histone–DNA interactions at the expense ofATP hydrolysis and can slide or evict individual nucleosomes. Thechange corresponded to a 30–40% reduction in the density of chro-matin fibers in the vicinity of the DSB [33]. A more recent studyimplicated poly(ADP-ribose) polymerase (PARP) activity in theprocess, as chromatin expansion at the site of damage could be sup-pressed not only by ATP depletion but also by PARP inhibitors [34].These data suggest a model in which immediately after DSB for-mation, chromatin is decondensed by ATP-dependent chromatinremodeling (likely assisted by other activities), which facilitatesthe subsequent recruitment of DNA damage checkpoint and repairproteins [33]. Similarly, a number of ATP-dependent chromatinremodelers have been shown to participate in DSB repair, mostlyat its early stages. Thus, SWR1 participates in yeast NHEJ by facil-itating recruitment of the Ku proteins [35]. Early events in NHEJ,in mammals, depend on SWI/SNF chromatin remodeling – theBRM1 subunit of the SWI/SNF complex is required for recruitmentof Ku70 and efficient NHEJ [36]. Knock-down of this protein orthe BRG1 subunit of SWI/SNF impairs H2AX phosphorylation andsensitizes cells to DSB-inducing agents [37]. Yeast SWI/SNF chro-matin remodeler has been shown to participate in HR repair bydisrupting heterochromatin at the donor locus facilitating strandinvasion [38].We have found that mammalian INO80 protein, thecore subunit of the chromatin remodeler of the same name, isrequired for the initial steps of DSB ends processing. It associateswith chromatin within 10 kb of a defined break site and its knock-down results in deficient HR repair. INO80 depletion impairs focalrecruitment of 53BP1 but does not impede Rad51 foci formation,suggesting that it is required for the initial steps of repair. Fur-ther analysis indicated that INO80-deficient cells are compromisedin 5′-3′ resection of DSB ends [39]. Similarly, yeast INO80 com-plex is recruited to DSBs and strains deficient in its componentsare less effective in the initial 5′-3′ resection at DSB ends priorto strand invasion [40]. These data suggest an evolutionary con-served role for INO80 in DSB end processing. Recently, the yeastSaccharomyces cerevisiae Fun30 protein and its human counterpartSMARCAD1, two SNF2-family ATP-dependent chromatin remod-ellers, were found to be directly involved in the DSB response.Fun30 physically associates with DSB ends promoting Exo1- andSgs1-dependent end resection through a mechanism involvingits ATPase activity. SMARCAD1 is also recruited to DSBs, and thekinetics of its recruitment are similar to that of Exo1. The loss ofSMARCAD1 impairs end resection and recombinational repair, ren-dering cells hypersensitive to DNA damage [41]. Taken together,these data suggest that ATP-dependent chromatin remodelers playkey roles in initiating the DSB repair process and establishing thechromatin environments required for downstream signaling andrepair events. This notion however, needs to account for the find-ings of a very recent report that a number of yeast remodelersrequire DSB end processing and Rad51 for recruitment to DSBs [42].Together with studies [43–45] on factors affecting mobility of dam-aged chromatin (see below), this may implicate these remodelersin promote chromatin mobility during HR repair.

3.2. Post-translational modifications in control of chromatinaccessibility

Chromatin compaction is also controlled by histone acetylation.Acetylation of the positively charged lysine and arginine residuesof histone N-terminal tails weakens their interaction with the neg-

tin structure in double strand break repair, DNA Repair (2013),

atively charged DNA backbone. Early yeast studies showed thatN-terminal deletion of histone H3 impaired DSB repair. GCN5,the enzyme which catalyzes acetylation of N-tail lysines of his-tone H3, was found to be recruited to DSBs, and GCN5 deletion

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utants lost viability following induction of a single DSB [46].utation of any of the four N-terminal lysines of histone H4 abol-

shed repair of both DSB and UV lesions and the defect was linkedo the NuA4 acetylase complex [47]. The mammalian homologuef yeast NuA4, TRRAP/TIP60 complex is required for DSB repair asell. Ectopic expression of mutated TIP60 lacking acetyltransferase

ctivity led to defects in DSB repair and apoptosis [48]. Depletionf TRRAP/TIP60 subunits TRRAP and RUVBL1 and 2 resulted in HRepair defects that could be overcome by forced chromatin relax-tion, indicating that TRRAP/TIP60 is needed to mediate chromatinccessibility [49,50]. The evidence obtained by Dr. Herceg’s groupas revealed that TIP60 is recruited to sites of DSB in a TRRAP-ependent manner [49]. The resulting hyperacetylation of histone4 is important for loading a subset of repair proteins, namely3BP1, Rad51 and BRCA1 but not MDC1, indicating that some pro-eins are recruited in an acetylation-independent way, while thethers require chromatin relaxation mediated by TRRAP/TIP60 [49].he finding that MDC1 is recruited independently is in agreementith the role of MDC1 as a binding platform for many repair factors,

ncluding TIP60 [51]. This may indicate that efficient binding of allR repair factors downstream of the TIP60 complex is acetylation-ependent.

TIP60 acetyltransferase, likely alone or as part of a differentomplex, is required to activate ATM kinase. TIP60 binds his-one H3 trimethylated on lysine 9 and this interaction activatests acetyltransferase activity. Depletion of H3K9me3 blocks TIP60ecruitment and ATM activation [52]. A very recent paper reportshat to acetylate and activate ATM, TIP60 needs to be phosphory-ated on Tyr44 by c-Abl and, importantly, that chromatin relaxationnduces this modification [53]. Thus, this mechanism may link theery fast ATP-dependent chromatin relaxation upon induction of aSB [33] and the activation of downstream ATM signaling.

Histone acetylation stimulates NHEJ through p300 and CBP.hese acetyltransferases localize to DSBs, mediate acetylation ofistones H3 and H4 and promote recruitment of the Ku proteins36]. HR and NHEJ are stimulated by yet another histone acetyl-ransferase – MOF [54]. Its activity increases H4K16 acetylation inesponse to DSBs [55,56]. Deletion of MOF delays �-H2AX foci for-ation [54] and impairs recruitment of BRCA1 and 53BP1 [54,55].

he latter suggests that MOF participates at the step which estab-ishes the choice between either NHEJ or HR repair [57]. In additiono local changes, a recent study showed global acetylation of H3K14ollowing induction of DSBs in an HMGN1-dependent manner.cetylated H3K14 is needed for ATM to bind to damaged chromatin

58].However, the acetylation level of chromatin around DSBs in

ammalian cells changes both up and down during the coursef DSB repair and factors that promote compaction, in addition tohose that relax chromatin, are involved in the process. Initially,amburini and Tyler showed that the acetylation status of histone3 and H4 lysines varies during HR repair and that histone deacety-

ases (HDACs) Rpd3, Sir2, and Hst1 are recruited to a site-specificSB [46]. In mammalian cells, HDAC1 and HDAC2 are quickly

ecruited to DSBs, deacetylate H3K56 and H4K16 and their deple-ion impairs NHEJ [56]. In contrast to the yeast study, deacetylationy HDAC1 and 2 in mammalian cells preceded hyperacetylationt the lesions. These data suggest that the simple “access, repair,estore” model may not be so straightforward, and raise the ques-ion why compaction-promoting factors are needed for repair. Oneossible explanation may be that deacetylation represses localranscription and prevents its interference with repair. Compactedhromatin may be needed to stop Ku proteins from sliding away

Please cite this article in press as: A. Gospodinov, Z. Herceg, Chromahttp://dx.doi.org/10.1016/j.dnarep.2013.07.006

which they do on naked DNA) and keep them concentrated at theSB ends [56].

The issue of compaction during DSB repair is further com-licated by results indicating the need of heterochromatin

PRESSepair xxx (2013) xxx– xxx 5

components in repair. Heterochromatin forms a compacted chro-matin structure harboring repressive histone marks such astrimethylation of histone H3 at lysine 9 and H3K9me3-associatedheterochromatin proteins [59]. DSBs are repaired with slowerkinetics in heterochromatin than in euchromatin. The majority of�-H2AX foci are located outside of, or close to, heterochromatindomains, which suggests that heterochromatin limits the accessof DDR proteins to DNA [60,61]. HP1 protein variants are centralin heterochromatin organization and, as could be expected, havebeen shown to be inhibitory to repair [62,63]. Surprisingly how-ever, after initial dispersion, all three HP1 variants (HP1 ˛, ̌ and �in mammals) accumulate at DNA damage sites [64–66] and in thecase of HP1� this is strictly dependent on p150CAF1, the largestsubunit of chromatin assembly factor 1 [67]. Depletion of HP1�in human cells led to decreased survival after ionizing radiationand defects in the recruitment of DDR factors, including 53BP1and BRCA1, and impaired DNA end resection [67]. While the rolesof the HP1 variants during DSB repair seems not to overlap, theyemerge as important regulators of HR repair [68]. ATM kinase andthe downstream components of ATM signaling have a crucial rolein counteracting the inhibitory effect of condensed chromatin, andindeed may only be needed to modify and support repair in tightlycompacted heterochromatic parts of the genome [63,69]. This issupported by the fact that depletion of factors that promote com-paction such as KAP-1, HP1 and histone deacetylases bypassed therequirement for ATM signaling. Following phosphorylation by ATM,KAP-1 disperses throughout chromatin after phosphorylation, andis thought to promote global relaxation [70]. In parallel, phos-phorylated KAP-1 forms foci, dependent on 53BP1 and other DDRmediator proteins, although these are dispensable for pan-nuclearKAP1 phosphorylation [69]. Taken together, heterochromatin com-ponents contribute to repair in multiple, seemingly opposite ways,indicating that repair does not require a DNA region depopulated ofproteins to provide access for repair proteins, but rather a “repair-permissive state” involving specific structures with varying degreesof compaction [71]. In support of such a notion are the findingsthat while some NuRD complex components are rapidly recruitedto DSBs (e.g. HDAC1 and 2 [56]), KAP-1 phosphorylation resultsin the dispersal of the CHD3 subunit of the same complex fromheterochromatin [72]. This indicates that NuRD complex functionis not just either stimulatory or inhibitory to repair but instead itsfunction depends on the chromatin context [72].

3.3. Higher-order chromatin organization and DSB repair

On top of the changes in chromatin accessibility that occur inthe vicinity of the lesion, there seems to exist an extra level ofcontrol entailed by the higher-order chromatin organization. Yeastgenetic screens found functional links between DNA repair andcomponents of the nuclear pore complex (NPC) [73] – large macro-molecular structures that mediate intracellular trafficking betweenthe nucleus and cytoplasm. A later study found that DSBs associatewith NPCs through a mechanism dependent on the nucleoporinNup84, the action of the Slx5–Slx8 SUMO-targeted ubiquitin ligasecomplex and checkpoint kinases [74]. Remarkably, peripheral relo-calization and anchoring of DSBs is directed by Htz1–the yeasthomologue of histone variant H2AZ [75]. Telomerase machineryhas also been implicated in the recruitment of DSBs to the nuclearperiphery [76]. A recent study has demonstrated that local chro-matin remodeling and nucleosome eviction by the yeast INO80complex increased large-scale chromatin movements by enhancingthe flexibility of the chromatin fiber during both transcription and

tin structure in double strand break repair, DNA Repair (2013),

DSB repair [43], further suggesting that chromatin structure may bean important regulator of the mobility of a damaged chromosomallocus. Other authors have found that increased mobility follow-ing DSB induction, requires Rad51, the ATPase activity of Rad54,

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he ATR homologue Mec1 and the DNA-damage-response medi-tor Rad9 (homologue of 53BP1) [44,45]. In yeast, silent matingype loci HMR and HML cluster together in the nucleus. The pro-ess requires phosphorylated H2A(X) and HR repair proteins, but isndependent of telomeres and the cell cycle. In unperturbed cells,he silenced domains were enriched in phosphorylated H2A(X) asell as in SMC proteins, which likely mediate the long range inter-

ctions [53]. Taken together, these data strongly suggest that HRepair machinery and the associated chromatin regulators, in addi-ion to their primary function, may be important determinants ofhe three-dimensional genomic organization.

Still, it is not clear to what extent enhanced mobility of dam-ged chromatin is conserved in higher eukaryotes. A recent workn Drosophila described striking dynamic behaviors of hetero-hromatic DSBs repaired by HR repair. Proteins involved in DSBesection get rapidly recruited to DSBs within heterochromatin,ut late repair foci of Rad51 are assembled only after the damagedequences themselves have moved outside of the heterochromaticP1 domains. This movement is crucially dependent on checkpointinases [77] and may serve two important purposes: (1) to facilitateccess of the repair machinery to the damage site; (2) to minimizehe risk of illegitimate joining during homologous recombinationepair among the abundant repeat sequences found in heterochro-atin [78]. An alternative explanation to an active relocalization

rocess could be offered by a model in which the rapid movementf DSBs to the periphery of the heterochromatic regions represents

passive effect of heterochromatin relaxation in the vicinity of aSB [79]. Indeed, several studies suggest very limited mobility ofSB-containing chromatin in mammalian cells. For instance, liveell imaging of an endonuclease-induced break (recognition sitembedded within bacterial operators, labeled with GFP derivative-agged repressors) indicated that that broken ends are positionallytable and their lack of mobility depended on Ku80 protein [80].nother study using histone H2B tagged with photoactivatable GFPnd laser induced damage to follow mobility of damaged chro-atin did not detect significant movement of chromatin regions

ontaining DSBs over longer time periods, including up to 4 hfter irradiation [33]. On the other hand, it has been reported thateprotected telomeres (resembling DSB ends) are more mobile andample larger territories within the nucleus, which facilitates NHEJ.his dynamic behavior depended on 53BP1 and ATM [81]. A recenttudy using fluorescently tagged 53BP1, in living mammalian cellsound that chromatin domains containing DSBs are 2–3 times more

obile than intact chromatin. The increased DSB mobility was ATPependent and could be reduced by ATM inhibition [82]. Finally,

n support of a model that increased mobility of damaged DNA iseeded to place DNA ends in a temporary repair compartment, ishe recent finding implicating the bromodomain protein Brd4 asn insulator that limits propagation of DNA damage signaling inhromatin [83].

. Provision of binding interfaces

.1. H2AX as a key spatial organizer of the DDR

In addition to providing control of chromatin compaction andccessibility, chromatin changes provide proper binding sitesor repair factors. The phosphorylation of �-H2AX is at the heartf a sequence of histone post-translational modifications thatlay vital roles in recruiting DNA repair factors to chromatin.hosphorylation of histone variant H2AX on serine 139 (in yeast,

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129 of H2A is phosphorylated) is one of the earliest eventsnduced by DNA breaks [84]. H2AX is a substrate of the ATM [85] orNA-PK kinases [86,87] in response to DSBs and ATR in signaling of

eplication stress, induced by DNA lesions [88,89]. In mammalian

PRESSepair xxx (2013) xxx– xxx

cells phosphorylated H2AX encompasses large chromatin domainsand forms prominent nuclear foci that could easily be visualized byimmunostaining [84]. The spreading of �-H2AX in megabase-sizedregions surrounding the break is attributed to an amplificationmechanism involving the MDC1 protein. �-H2AX-tethered MDC1binds Nbs1 and stabilizes MRN complex at the DSBs [90], whichrecruits more ATM [91,92]. ATM induces further H2AX phospho-rylation and establishes a positive feed-back loop to spread themodification [93]. A similar interaction of MDC1 with TopBP1-bound ATR anchors the ATR kinase to chromatin under conditionsthat favor generation of ssDNA [94]. MDC1 plays a determiningrole in the interaction of phosphorylated H2AX with its down-stream partners as it acts as an interaction platform for otherDDR components including ATM, Chk2, Rad51, Nbs1, RNF8 andTopo II.

4.2. Ubiquitination and methylation of histones in DDR

MDC1 recruits the RNF8 E3 ubiquitin ligase to repair foci [95],initiating a series of H2A/X ubiquitylation events. Histone ubiquity-lation is an essential regulator of the DNA damage response, neededto bring about BRCA1 and 53BP1 recruitment. RNF8 cooperateswith its E2 ubiquitin-conjugating enzyme UBC13 to catalyze theformation of K63-linked ubiquitin chains [95–97] in a process facil-itated by HERC2, which stabilizes the interaction between the E2and E3 enzymes [98,99]. An additional E3 ligase – RNF168 amplifiesthe ubiquitin signal to a level needed for 53BP1 and BRCA1 recruit-ment by targeting lysine residues in H2A/X and H2B [100,101]. Therecruitment of RNF168 is fully dependent on functional RNF8 asit associates with histones ubiquitylated by the latter [100]. Still,RNF8 itself, needs an initiating ubiquitylation activity. It is believedthat RNF2 mediates monoubiquitylation (of �-H2AX [102]), whichis required for further addition of ubiquitin residues by RNF8 andRNF168. Ubiquitylated histones are bound by the RAP80 protein,which contains two ubiquitin interacting motifs. RAP80 and severalother proteins form a complex that bridges the interaction betweenBRCA1 and modified histones [103–107].

Polyubiquitylation of H2A/X is required for the recruitment of53BP1 [95,108], but until recently, 53BP1 was thought to bindmethylated histones and not polyubiquitin chains (which arenonetheless essential for the process). Initially, it was found that53BP1 binds methylated H3K79, however as the level of this modifi-cation does not change in response to damage it has been proposedthat DSB-induced changes in the chromatin structure expose themark [109]. Accumulation of 53BP1 in repair foci requires dimethy-lation of H4K20. Deletion of Set9 methylase in S. pombe resulted inan impaired ability of Crb2 (RAD9 and 53BP1 homologue) to relo-cate to the sites of damage [110]. Transition to monomethylation ofH4K20 in mice, following knockout of Suv4-20 h methyltransferaseresulted in increased sensitivity to DNA damage [111]. Similarto H3K79 methylation, it has been suggested that H4K20me2does not change in response to DNA damage, but instead getsexposed and promotes 53BP1 binding. However, recent resultsindicate that H4K20 methylation increases locally upon inductionof DSBs and that it is mediated by the MMSET methyltransferasein mammals. Downregulation of MMSET significantly decreasesH4K20 methylation and subsequent accumulation of 53BP1 atDSBs [112]. A recent study has elucidated how vertebrate 53BP1is recruited to the chromatin that flanks DSB sites. 53BP1 recog-nizes mononucleosomes containing dimethylated H4K20 and H2Aubiquitinated on Lys 15, the latter being a product of RNF168 actionon chromatin. 53BP1 binds to nucleosomes using its previously

tin structure in double strand break repair, DNA Repair (2013),

characterized methyl-lysine-binding Tudor domain [109] and acarboxy-terminal extension, termed the ubiquitination-dependentrecruitment (UDR) motif, which interacts with the epitope formedby H2AK15ub. 53BP1 is therefore a bivalent histone modification

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eader that recognizes a histone ‘code’ produced by DSB signaling113].

.3. Recruitment of chromatin remodelers byoly(ADP-ribosyl)ation

Poly(ADP-ribosyl)ation – PARylation- is yet another post-ranslational modification involved in DSB response. PARP-1 carriesut the bulk of the modification with ∼85% of PARP activity [114],ut PARP-2 and PARP-3 also participate. PARP1 binds with highffinity to SSBs and DSBs [53] and gets auto-PARylated. PARyla-ion was initially linked to repair of single-stranded breaks, as it isequired for the recruitment of XRCC1, however evidence suggesthat PAR synthesis promotes recruitment of MRE11 to HU-inducedoci [115] and sites of laser-induced DNA damage [116]. Avail-ble data implicate PARP1 and 2 in promoting alternative NHEJ,hile PARP3 promotes APLF binding during canonical NHEJ by sta-

ilizing the XRCC4–LigIV complex on broken chromosomes [114].nterestingly, PARP activity is required to efficiently recruit vari-us chromatin remodelers to the sites of damage. ALC1 is such ahromatin remodeler, which gets rapidly recruited to DNA damageites by a PAR-dependent mechanism [117,118]. CHD4, a subunitf the NuRD complex, promotes the rapid PAR-dependent recruit-ent of the complex to DNA damage sites. CHD4 promotes repair

f DNA double-strand breaks and cell survival after DNA dam-ge and is a phosphorylation target for ATM [119]. Finally, PARPctivity, promotes spreading of SMARCA5 – the ATPase of severalistinct ISWI chromatin remodeling complexes – which in turnromotes RNF168 accumulation at DSBs [34]. PARP activity is also

nvolved in the initial ATP-dependent expansion of chromatin atNA damage sites [33] as its inhibition reduces chromatin expan-

ion at sites of laser-induced DNA damage [34]. These data indicatehat PARylation (of histone and non-histone) proteins is anotherost-translational modification participating in the DNA damageesponse by providing binding sites for the chromatin remodelingctivities needed for efficient DSB repair.

. Crosstalk and interdependence of DSB-inducedhromatin changes

Various damage-related post-translational modifications oremodeling events influence downstream chromatin changes andany examples of crosstalk between them have been described.

hus, it has been shown that activation of ATM kinase dependsn ATM being acetylated by the TIP60 histone acetyltransferase120]. The acetyltransferase activity of TIP60 in turn, is activatedy the interaction of the chromodomain of TIP60 and histone H3ri-methylated on lysine 9 at DSBs, which becomes accessibleollowing displacement of HP1� [52]. In addition to roles inctivating ATM, TIP60-mediated histone acetylation is involvedn �-H2AX dephosphorylation. In Drosophila, TIP60 acetylateshosphorylated H2Av (the fly orthologue of H2AX) and drives

ts exchange for unmodified histones [121]. In HeLa cells, TIP60cetylates �-H2AX at K5 and promotes its ubiquitylation andelease [122]. A separate study found that H4 acetylation byhe TIP60 complex is required for dephosphorylation of H2AX123]. Chromatin remodeling also participates in the maintenancef H2A(X) phosphorylation levels in the vicinity of DSB. YeastNO80 and SWR1 complexes function antagonistically at chro-

atin surrounding a DSB, as they promote the incorporation ofifferent histone H2A variants–H2A(X) and H2AZ, respectively

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124]. Another crosstalk concerning ATM activity at the sites ofamage is the recently described role of the SWI/SNF chromatinemodeler in H2AX phosphorylation. BRG1 is a subunit of theammalian SWI/SNF complex and it binds to acetylated H3 but

PRESSepair xxx (2013) xxx– xxx 7

only that in �-H2AX nucleosomes, not in bulk chromatin. BRG1stimulates the recruitment of the GCN5 acetyltransferase andcauses further acetylation of �-H2AX domains. Increased chro-matin accessibility augments recruitment of ATM kinase, whichin turn promotes additional H2AX phosphorylation, and the threeactivities form an auto-amplification loop. Cells deficient forBRG1 and therefore lacking this mechanism have impaired H2AXphosphorylation [37].

In yeast, phosphorylated H2AX serves as an entry point of theINO80 subfamily of chromatin remodelers NuA4, SWR1 and INO80[125–127]. In mammalian cells H2AX phosphorylation recruitsMDC1 [128]. MDC1 recruitment to sites of damage and down-stream chromatin changes depend on the dephosphorylation ofY142, since MDC1 could only bind H2AX unphosphorylated at Y142[129]. NuRD remodeling complex subunits MTA1 and CHD4 bindto DSBs in a PARP and RNF8 dependent manner and in turn, aid therecruitment of RNF168 and BRCA1 to DSBs [130]. SMARCA5 (a sub-unit of ISWI chromatin remodelers) directly interacts with RNF168and also requires PARP1 activity. SMARCA5 promotes RNF168 accu-mulation at DSBs, and SMARCA5 depletion results in DSB repairdefects [34]. Taken together, these data indicate that chromatinremodeling and post-translational modifications of histones, aswell as non-histone players in DDR, are closely interdependent andeach event promotes the next. There is no doubt that the knowncrosstalk between chromatin modifiers will increase as we get adeeper understanding of the DNA damage response.

6. Chromatin modifiers, DSB repair and cancer

Although our present knowledge of how the activities of chro-matin modifiers/remodelers are altered in cancer is still limited[131], the importance of these epigenetic regulators and their abil-ity to “drive” changes linked with tumor progression is alreadyrecognized. Genome wide sequencing studies of human cancershave reported ∼300,000 mutations. Yet only about 120 drivergenes (that is, genes, that when mutated, directly or indirectly con-fer selective growth advantage to the cell) have been identified.Nearly half of these genes encode proteins that directly regulatechromatin through modification of histones or DNA [132]. Thesediscoveries highlight the key importance of these epi-drivers intumorigenesis and hold the key to mechanistic understanding ofthe epigenetic changes that are rampant in tumors [133]. While,most functions of chromatin regulators in cancer are linked withtranscriptional control and epigentic reprograming of cells, theirparticipation in DNA repair certainly plays a role since genomeinstability is a fundamental feature of cancer [134]. In relationto DSB repair, chromosomal instability is seen in most solidtumors, where it is fueled by increased proliferation rates, DNAreplication stress and DSB formation [135]. There are a numberof examples of chromatin modifiers/remodelers implicated bothin aberrant reprogramming during tumorigenesis and chromatinchanges needed for efficient DSB repair. As already noted p300and CBP stimulate NHEJ [36]. Mutations of p300 and CBP arepresent in hepatocellular, breast, colorectal, and gastric cancers[136]. Oncogenic fusions of MLL-p300 and MLL-CBP, have beenobserved in a variety of hematologic malignancies [137]. Reversalof histone acetylation occurs by HDAC1 and HDAC2, which promoteNHEJ. Within the NuRD complex, HDAC1 and 2 function togetherwith the ATP-dependent remodeling subunits CHD3 and CHD4,metastasis-associated (MTA1/2) proteins and methyl CpG-bindingdomain proteins [138]. MTA1 is upregulated in a variety of can-

tin structure in double strand break repair, DNA Repair (2013),

cers [139]. SWI/SNF complexes have been implicated in NHEJ [36],HR [140,141] and V(D)J recombination [142–144] and several oftheir subunits are mutated in a number of tumors [145]. SMARCB1and SMARCA4 genes are mutated in malignant rhabdoid tumors

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146,147], a rare yet lethal tumor diagnosed in children. SMARCA4s also mutated in Burkitt’s lymphoma [148] in lung cancer [149]nd medulloblastoma [150]. Experiments in mouse models alsoupport a role for SWI/SNF components as tumor suppressors145]. In these tumors, accompanying aberrant repair events, andncreased mutability, will likely further exacerbate tumorigenesisnitiated by transcriptional reprogramming. On the other hand, inhe limited number of cases, in which these chromatin regulatorsr their subunits are overexpressed (e.g. MMSET [151] or MTA1152–154]), their therapeutic targeting would probably be highlyffective, since it would simultaneously restore transcriptional con-rol and render cancer cells more sensitive to cytotoxic treatments.

. Concluding remarks

The efficient repair of DSBs requires significant changes of chro-atin structure at lesion sites, together these form the chromatin

esponse to DNA damage. Acetylation and ATP-dependent chro-atin remodeling control accessibility of the players involved in

he process. Phosphorylation of H2AX, ubiquitylation of H2A as wells H2B [155,156] and methylation of H3/H4 provide binding inter-aces for the sequential recruitment of repair factors and likely anyf the chromatin modification/remodeling events is dependent onthers, either pre-existing before damage induction or induced byreak formation. Despite the enormous progress in understand-

ng the DDR in the last 15 years, the number of interdependenciesetween chromatin factors in it continues to grow. DSB repair needso function in a very diverse environment, with certain remod-lers likely needed in particular chromatin configurations, whicheems to be the reason behind this complexity. The formation of

temporary repair compartment around the break, including itse-localization within the nucleus possibly reflects the need tooordinate repair with other processes such as transcription anduclear transport. Alternatively, this may be necessary to avoidbnormal chromosomal rearrangements or for optimal efficiency.s the list of chromatin changes and the players that carry themut is already long, elucidating their repair specificities at differ-nt genomic locations and the co-ordination with on-going nuclearrocesses remains for the future.

As DSBs are among the primary means to induce apoptosis inancer cells, it is within the set of factors and interactions that makep DSB repair, that potential targets for therapy are sought. Due tohe pleiotropic functions of chromatin regulators, they are promis-ng in this respect. Granted, we have sufficient understanding ofhe DDR as well as the pathways deregulated in the specific tumor,herapies could perhaps be tailored to attack cancer cells exploi-ing the interactions of the target (or the process it promotes) withther deregulated participants. As proof of principle we alreadyave the example of PARP inhibitors which highly sensitize cellsith BRCA1 and 2 mutations [147] and have shown encouraging

esults in clinical trials [146].In addition to cancer therapy, understanding the chromatin

spects of DNA repair may have implications for gene therapy asell. With the advent of zinc-finger nucleases (ZFN) it is now pos-

ible to “edit” the mammalian genomes to correct disease-causingutations. ZFNs or TALE nucleases could be designed to target a

pecific genomic sequence and when co-delivered with an appro-riately designed gene-targeting vector, they could stimulate geneeplacement through HR repair [157]. Therefore, besides the devel-pment of delivery methodologies, one way to increase efficiencyould be to find ways to increase the probability of cutting the tar-

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et sequence and channel the repair of the cut through homologousecombination with the correcting sequence.

Both the complete understanding of the chromatin response toamage and its practical applications remain for the future, one

PRESSepair xxx (2013) xxx– xxx

that hopefully, is rapidly approaching due to the efforts of thoseworking in the field.

Conflict of interest

A conflicting interest exists when professional judgment con-cerning a primary interest (such as patient’s welfare or the validityof research) may be influenced by a secondary interest (such asfinancial gain or personal rivalry). It may arise for the authors whenthey have financial interest that may influence their interpretationof their results or those of others. Examples of potential conflicts ofinterest include employment, consultancies, stock ownership, hon-oraria, paid expert testimony, patent applications/registrations,and grants or other funding.

Funding source

All sources of funding should also be acknowledged and youshould declare any involvement of study sponsors in the studydesign; collection, analysis and interpretation of data; the writingof the manuscript; the decision to submit the manuscript for publi-cation. If the study sponsors had no such involvement, this shouldbe stated.

Acknowledgements

We thank Dr Lyubomira Chakalova for critically reading themanuscript. The work of the IARC Epigenetics Group is supportedby grants from the National Cancer Institute (NIH), United States;l’Association pour la Recherche sur le Cancer (ARC), France; la LigueNationale Contre le Cancer, France; the Bill and Melinda Gates Foun-dation, European Union grants No. 308610 entitled “Exposomics:Enhanced exposure assessment and omic profiling for high priorityenvironmental exposures in Europe”; and a French National Can-cer Institute (INCa) grant “Biomarkers of B vitamins, Epigenome,genetic polymorphisms, and breast cancer risk in the EuropeanProspective Investigation into Cancer and Nutrition (EPIC) Study”(to Z.H.). A.G. acknowledges the support of grant DMU 03/9 ofthe Bulgarian National Science Fund and ICGEB research grantCRP/12/005.

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