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gH2AX and its role in DNA double-strand break repair1 Jeffrey Fillingham, Michael-Christopher Keogh, and Nevan J. Krogan

Abstract: One of the earliest responses to a DNA double-strand break (DSB) is the carboxy-terminal phosphorylation of budding yeast H2A (metazoan histone H2AX) to create gH2A (or gH2AX). This chromatin modification stretches more than tens of kilobases around the DSB and has been proposed to play numerous roles in break recognition and repair, although it may not be the primary signal for many of these events. Studies suggest that gH2A(X) has 2 more direct roles: (i) to recruit cohesin around the DSB, and (ii) to maintain a checkpoint arrest. Recent work has identified other factors, including chromatin remodelers and protein phosphatases, which target gH2A(X) and regulate DSB repair/recovery. Key words: Checkpoint recovery, chromatin, double-strand break repair, gH2AX, H2A, homologous recombination. Re´sume´ : Une des re´ponses les plus pre´coces a` un bris d’ADN double-brin (BDB) est la phosphorylation carboxy-terminale de l’histone H2A chez la levure a` bourgeonnement (histone H2AX chez les me´tazoaires) afin de cre´er la forme gH2A (ou gH2AX). Cette modification de la chromatine s’e´tend sur plusieurs dizaines de kilobases autour du BDB et pourrait jouer plusieurs roˆles dans la reconnaissance et la re´paration du bris, quoiqu’elle puisse ne pas eˆtre le signal initial de ces e´ve´nements. Des e´tudes sugge`rent que gH2A(X) exerce 2 roˆles directs : (i) en recrutant la cohe´sine autour du BDB et (ii) en maintenant un point de controˆle. Des travaux re´cents ont identifie´ d’autres facteurs, incluant des agents remodelant la chromatine et des prote´ine-phosphatases, qui ciblent gH2A(X) et re´gulent la re´paration des BDB et le re´tablissement des fonctions. Mots cle´s : Point de controˆle, chromatine, re´paration du bris double-brin, gH2A, H2A, recombinaison homologue. [Traduit par la Re´daction]

Introduction Cells must deal with thousands of DNA lesions per day. The majority are mediated not by exogenous agents, such as chemicals or ionizing radiation, but rather by endogenous damage, created from the byproducts of metabolism, including reactive oxygen species, spontaneous depurination of the DNA strands, and DNA single- and double-strand breaks (SSBs and DSBs, respectively) from deoxyribose oxidation or replication fork collapse (Lindahl 1993, 2000; Lindahl and Wood 1999). Failure to repair these lesions leads to del-

eterious mutations, genomic instability, cell death, and in higher eukaryotes, contributes to oncogenesis. Of all damage types, DSBs are particularly toxic. If unrepaired, the centromere-distal segment of the chromosome will be lost, which is generally a lethal event. Not all DSBs are accidental: those programmed by specific endonucleases play a role in immunoglobulin V(D)J recombination (recombination activating gene (RAG)-induced), crossing-over during mitosis and meiosis (Spo11-induced), and yeast matingtype switching (homothallic (HO)-endonuclease induced) (reviewed in: Paˆques and Haber 1999). DSBs activate highly

Received 24 March 2006. Revision received 9 May 2006. Accepted 10 May 2006. Published on the NRC Research Press Web site at http://bcb.nrc.ca on 11 August 2006. Abbreviations: ATM, ataxia telangiectasia mutated; ChIP, chromatin immunoprecipitation; CPT, camptothecin; DSB, double-strand break; DSBR, double strand break repair; gH2A, S129p Saccharomyces cerevisiae H2A; gH2AX, S139p metazoan H2AX; HAT, histone acetyltransferase; HTP-C, histone H2A phosphatase complex; HR, homologous recombination; IR, ionizing radiation; MMS, methylmethanesulfonate; NHEJ, nonhomologous end joining; SSB, single-strand break. J. Fillingham. Banting and Best Dept of Medical Research, University of Toronto, Toronto, ON M5S 1A8, Canada. M.C. Keogh.2 Dept of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02138, USA. N.J. Krogan.3 Dept of Cellular and Molecular Pharmacology, UCSF, San Francisco, CA 94143, USA. 1This

paper is one of a selection of papers published in this Special Issue, entitled 27th International West Coast Chromatin and Chromosome Conference, and has undergone the Journal’s usual peer review process. 2Corresponding author (e-mail: [email protected]). 3Corresponding author (e-mail: [email protected]). Biochem. Cell Biol. 84: 559–568 (2006)

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conserved signal transduction pathways to induce DNA damage checkpoints that delay cell-cycle progression and allow time for repair (reviewed in Nyberg et al. 2002). In metazoan cells, checkpoint signaling can also direct damaged cells to undergo apoptosis and eliminate potentially catastrophic mutations (Rouse and Jackson 2002; Zhou and Elledge 2000). Budding yeasts have distinct G1, intra-S, and G2/M checkpoints, depending on the source and timing of the damage (Kolodner et al. 2002; Rouse and Jackson 2002). While many of the conserved checkpoint regulators (e.g., MRX complex, Rad24 complex, Mec1, Rad9, and Rad53) participate in all 3 responses, others (e.g., Mrc1, Chk1, and Rad14) have been ascribed roles in specific checkpoints (Wysocki et al. 2005 and references therein). There are 2 genetically separable pathways to repair DSBs: homologous recombination (HR) and nonhomologous end joining (NHEJ) (reviewed in: Jackson 2002; Moore and Krebs 2004; Paˆques and Haber 1999). During HR, the 5’-terminated strand is resected, and the resulting single-stranded 3’ sequence invades an intact homologous duplex, priming off it to reconstitute the broken strand by DNA replication (reviewed in: Krogh and Symington 2004; Symington 2002) (Fig. 1). During NHEJ, the 2 broken ends of the DSB are directly rejoined, without any requirement for homology, and typically, with some loss of sequence (reviewed in Daley et al. 2005). In yeast, if homologous sequences or sister chromatids are available, the principle repair pathway is HR (Moore and Haber 1996; Rudin and Haber 1988; Wilson 2002), particularly in diploid cells (Kegel et al. 2001). The three Rs of eukaryotic DNA metabolism, replication, reading, and repair, must function in the context of chromatin. The basic repeating unit is the nucleosome core particle: 146 bp of DNA wrapped around a core histone octamer composed of 2 H2A/H2B dimers and a (H3/H4)2 tetramer (reviewed in Luger 2003). Variation can be introduced by covalently modifying the histones (usually, but not exclusively, on their protruding N-terminal tails) by a variety of enzymatic activities (including acetylation, methylation, phosphorylation, and ubiquitination) (reviewed in Cosgrove and Wolberger 2005; Jenuwein and Allis 2001; Strahl and Allis 2000). Variant histones can also be deposited at specific sites to replace the major histones assembled during DNA replication (reviewed in Malik and Henikoff 2003). Finally, nucleosomes can be remodeled by a superfamily of ATPases (reviewed in Johnson et al. 2005). All of these activities have been shown to impinge on the repair of damaged DNA (reviewed in Bilsland and Downs 2005; Ehrenhofer-Murray 2004; Moore and Krebs 2004; Peterson and Coˆte´ 2004; Vidanes et al. 2005; Wuebbles and Jones 2004). gH2A(X) creation The earliest mark yet identified at a DSB is the rapid (within minutes) and extensive (more than hundreds of kilobases around a break point) serine phosphorylation of the histone H2A variant H2AX on the carboxy-terminal SQE motif to create gH2AX (Downs et al. 2000; Rogakou et al. 1998) (Fig. 1). This phosphorylation is highly conserved and is observed in unicellular yeast and metazoan cells (reviewed in Foster and Downs 2005; Li et al. 2005; Redon et al. 2002; Thiriet and Hayes 2005). In Saccharomyces cerevi-

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siae, the major isoform H2A contains this SQE motif and the phosphorylated form is termed gH2A. Members of the phosphatidylinositol 3-kinase family mediate this modification in yeast (Mec1 and Tel1) and human cells (ataxia telangiectasia mutated (ATM), AT-related, and DNA-dependent protein kinases) (Burma et al. 2001; Burma and Chen 2004; Downs et al. 2000; Ward and Chen 2001). These kinases also initiate the signaling cascades that activate DNA repair, induce cell-cycle arrest until repair has been performed, and, in higher eukaryotes, trigger apoptosis. DSBs invariably lead to gH2A(X) formation, irrespective of the source of the insult: ionizing radiation (IR) (Rogakou et al. 1998), MMS (Nazarov et al. 2003), bleomycin (Tomilin et al. 2001), laser resection (Rogakou et al. 1999), camptothecin (CPT)-induced replication fork collision (Furuta et al. 2003; Redon et al. 2003), RAG-mediated V(D)J recombination (Chen et al. 2000), immunoglobulin class-switching (Petersen et al. 2001), apoptosis (Mukherjee et al. 2006; Rogakou et al. 2000), or retroviral integration (Daniel et al. 2004). Despite its highly coordinated regulation, the precise role of gH2A(X) in DSB repair is not completely understood. H2AXnull transgenic mice are viable but immunocompromised, IR sensitive, and subject to increased genomic instability (Bassing et al. 2002; Celeste et al. 2002). Schizosaccharomyces pombe cells unable to generate gH2A are hypersensitive to a variety of DSB-inducing chemicals and unable to maintain a cell-cycle checkpoint arrest (Nakamura et al. 2004). In contrast, Saccharomyces cerevisiae containing unphosphorylatable H2A are only mildly sensitive to DSB-inducing genotoxins and appear competent in initiating and maintaining checkpoint arrest (Downs et al. 2000; Keogh et al. 2006a; Redon et al. 2003). The cellular response to a DSB is characterized by the relocalization of many repair proteins to microscopically discernible subnuclear structures, termed repair foci (Lisby et al. 2004). It is generally believed that at the heart of each of these foci is a DSB or a cluster of DSBs under active repair. In Saccharomyces cerevisiae, higher resolution chromatin immunoprecipitation (ChIP) studies at an induced DSB have allowed more detailed analyses (Keogh et al. ¨ nal et al. 2006a; Shroff et al. 2004; Tsukuda et al. 2005; U 2004). While gH2A is present in a broad (&50 kb) region surrounding the DSB, it is reduced or absent from sequences bound by repair proteins within 1–2 kb of the break itself ¨ nal et al. 2004). This gH2A valley is (Shroff et al. 2004; U not caused by nucleosome depletion (Keogh et al. 2006a; Shroff et al. 2004; Tsukuda et al. 2005), replacement with the unphosphorylatable H2A variant H2A.Z (Keogh et al. 2006a), or action of the gH2A phosphatase Pph3 (Keogh et al. 2006a) (see below). Despite these contradictory occupancy patterns, gH2A(X) is reported to mediate the recruitment of numerous DSB-recognition and repair factors to the immediate area of the break, including many chromatin modifiers (Downs et al. 2004; Morrison et al. 2004; van Attikum et al. 2004), DNA checkpoint proteins (FernandezCapetillo et al. 2004; Nakamura et al. 2004), and cohesins ¨ nal et al. 2004). (Bassing and Alt 2004; Shroff et al. 2004; U

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Fig. 1. gH2A and homologous recombination-mediated double-strand break repair (DSBR). (A) Some highlights of HR are shown (Lisby et al. 2004). In this pathway, the ends of the broken chromosome must locate an intact, undamaged homologous template, which will be copied to repair the lesion. HR is mediated by the conserved RAD52 epistasis group of proteins, including Rad51, Rad52, Rad54, Rad55, Rad57, Rad59, and the MRX complex (Mre11–Rad50–Xrs2) (reviewed in Symington 2002). Successful HR requires resection of the 5’ ends of DNA flanking the break by an exonuclease to form a region of ssDNA, bound by replication-protein A (RPA). Rad51, aided by Rad52, Rad54, and Rad55, then displaces RPA to form the presynaptic filament. Following a successful homology search, Rad51 facilitates invasion of the presynaptic filament into the donor template. The sequence is then copied and the broken ends religated (Paˆques and Haber 1999). gH2A is created very soon after DSB induction, and while the modification stretches for >20 kb on either side of the DSB, it is not found within 1–2 kb of the break itself (i). gH2A has been implicated in many downstream events, including recruitment of a collection of chromatin modifiers (ii), and the cohesin complex (iii). Chromatin structure changes dramatically during DSBR, and many nucleosomes are probably lost with the creation of the region of ssDNA (iv). After the strand invasion step, gH2A is displaced from chromatin in an unknown form (although a gH2A/H2B dimer is a likely possibility) by an unidentified activity. The phosphorylated histone is then targeted by the Pph3 phosphatase-containing histone H2A phosphatase complex, and the resulting H2A/H2B is possibly reused (v). If this dephosphorylation is prevented, yeast cells persist in G2/M arrest, even though repair has completed, suggesting that gH2A is used to maintain the checkpoint (Keogh et al. 2006a). (B) Saccharomyces cerevisiae uses the homothallic (HO) endonuclease to switch mating types by creating a DSB in MAT and repairing this lesion by homologous recombination (HR) with donor sequences at the silent mating loci HML or HMR (Haber 1998). Expressing the HO endonuclease from a galactose-inducible promoter and inserting its 24 bp recognition sequence at different locations provides an opportunity to monitor DSB recognition and repair (Paˆques and Haber 1999). (C) The approximate recruitment kinetics of many repair factors has been defined (Lisby et al. 2004; Wolner et al. 2003). Under optimal conditions, repair of a DSB at MAT can be completed in less than 2 h (Keogh et al. 2006a).

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The role of gH2A(X) in recruiting DNA repair factors NuA4 Using a peptide corresponding to gH2A, Downs and colleagues identified an in vitro interaction with Saccharomyces cerevisiae NuA4 (Downs et al. 2004), an essential histone acetyltransferase (HAT) required for the acetylation of histone H4 (Lys 5 (K5), K8, and K12), histone H2A (K7), and histone H2A.Z (K3, K8, K10, and K14) (Allard et al. 1999; Babiarz et al. 2006; Keogh et al. 2006b; Millar et al. 2006; Smith et al. 1998). NuA4 has been proposed to play a direct role in double-strand break repair (DSBR), as strains with compromised catalytic activity are sensitive to genotoxins (Bird et al. 2002). The NuA4 subunit Arp4 (actin-related factor 4) is recruited to an induced DSB (Bird et al. 2002), and binds gH2A in vitro (Downs et al. 2004), although it should be noted that Arp4 is also a subunit of the chromatin remodelers SWR-C and INO80-C (Kobor et al. 2004; Krogan et al. 2004a; Krogan et al. 2003; Mizuguchi et al. 2004; Shen et al. 2000). Since Act1, another shared component of NuA4, SWR-C, and INO80-C, also copurifies with gH2A (our unpublished data), one interesting possibility is that these actin proteins regulate recruitment of the 3 chromatin modifiers to sites of DNA damage (see below). However, although NuA4 is recruited to an induced DSB, it has not been demonstrated that this is gH2A dependent. NuA4-dependent histone H4 acetylation increases rapidly at a DSB (Bird et al. 2002; Downs et al. 2004), and mutating the 4 possible acetylation sites in the H4 N-terminal tail results in hypersensitivity to DNA-damaging agents (Bird et al. 2002). The 4 lysines act redundantly in DSBR as restoration of any one rescues the phenotype. The importance of histone acetylation to DSBR is underscored by the fact that inhibiting the TIP60 complex (TIP60-C), a metazoan homolog of NuA4, impedes HR in Drosophila (Kusch et al. 2004) and human (Murr et al. 2006) cells. In human cells, this can be rescued by a hypotonic shock, which promotes chromatin decondensation and suggests that histone acetylation by TIP60 (or NuA4 by analogy) may function to relax chromatin and allow DSBR factors to access the break site (Murr et al. 2006). Another function for the Drosophila melanogaster TIP60-C has been identified by Workman and colleagues (Kusch et al. 2004). In Drosophila, the histone H2A variant H2Av (an amalgam of the histone variants H2AX and H2AZ not found in yeast or human cells) is phosphorylated at DSBs in a manner analogous to that of H2A(X). dTIP60-C then acetylates nucleosomal gH2Av and exchanges it with unmodified H2Av, events that are catalyzed by 2 subunits of the complex: the Tip60 HAT and the p400/Domino ATPase, respectively. A pathway based on TIP60-C homologs has been proposed to regulate gH2A removal in yeast. Esa1 (the catalytic subunit of NuA4) could acetylate the modified histone, and the ATPase Swr1 (the catalytic subunit of the SWR-C) could then potentially exchange gH2A for H2AZ. The SWR-C does catalyze the exchange of H2A for H2AZ (Kobor et al. 2004; Krogan et al. 2003; Mizuguchi et al. 2004), but this variant does not accumulate at a DSB, at least not in the early stages of break repair (Keogh et al. 2006a).

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INO80-C The ATP-dependent chromatin-remodeling complex INO80-C is recruited to an induced DSB in a gH2A-dependent manner (van Attikum et al. 2004), either via the actinrelated subunits Arp4 (Downs et al. 2004) and Act1 (our unpublished results), or through Nhp10 (Morrison et al. 2004). Deletion of subunits of the complex results in genotoxin sensitivity (Morrison et al. 2004; Shen et al. 2000; van Attikum et al. 2004) and synthetic genetic interactions with components of the HR machinery (Morrison et al. 2004). It has therefore been suggested that INO80-C regulates chromatin structure at a DSB and facilitates processing of the lesion. One possible function of INO80-C is to remove nucleosomes in the immediate neighborhood of a DSB during NHEJ (Tsukuda et al. 2005). This activity is dependent on the DNA damage sensor Mre11 (a component of the yeast MRX complex), and the unique INO80-C subunit Arp8. However, this is not the mechanism that ensures low levels of gH2A close to a DSB (Keogh et al. 2006a; Tsukuda et al. 2005). Significantly, Arp8-dependent nucleosome removal may regulate the loading of Rad51, an obligate step in HRmediated DSB repair. Nucleosome removal, and subsequent loading of Rad51, was shown to be independent of gH2A, suggesting that a pre-existing pool of INO80-C present at the locus (perhaps previously engaged in transcription) is responsible for nucleosome depletion (Keogh et al. 2006a; Tsukuda et al. 2005). This, however, begs the question: what is the function of the INO80-C pool that is recruited to a DSB by gH2A? SWR-C The ATP-dependent chromatin remodeler SWR-C inserts the histone variant H2AZ into chromatin in exchange for H2A (Kobor et al. 2004; Krogan et al. 2003; Mizuguchi et al. 2004). Deletion of H2AZ or members of the SWR-C yields hypersensitivity to drugs that generate DSBs (Downs et al. 2004; Keogh et al. 2006b; Kobor et al. 2004; Krogan et al. 2004a) and results in synthetic growth defects when combined with deletions of known DSBR genes (our unpublished data). SWR-C binds gH2A in peptide pull-down experiments (Morrison et al. 2004), perhaps via the actinrelated subunits Arp4 and Act1 (as above), although it is not clear whether it is specifically recruited to a DSB (Downs et al. 2004). It will be of interest to examine H2AZ and SWR-C recruitment in strains capable of repairing an induced DSB by HR, as opposed to strains forced to repair by NHEJ, as tested previously (Keogh et al. 2006a). If SWR-C is involved in the removal of gH2A from chromatin surrounding a DSB, unique members of the SWR-C may be recruited relatively late, around the time when gH2A levels decrease. Alternatively, if redundant mechanisms exist with other chromatin remodelers, removal of gH2A (or recruitment of H2AZ) may only be detected when one or more of the remodeling machines are disabled. SWI/SNF and RSC At least 2 other ATP-dependent chromatin-remodeling complexes are recruited to an induced DSB in Saccharomyces cerevisiae, although their possible dependence on gH2A is unclear. Mutation in subunits of the SWI/SNF (Snf2 and Snf5) and RSC (Rsc1, Rsc2, and Sth1) complexes cause hy#

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persensitivity to agents that induce DSBs (Bennett et al. 2001; Chai et al. 2005; Shim et al. 2005). Both complexes appear to have distinct roles in HR. SWI/SNF is required preceding or during synapsis, while RSC is involved at a later step after extension of the invading strand, perhaps for the postsynapsis dissociation of the invading DNA from the donor prior to ligation (Chai et al. 2005) (Fig. 1). RSC is likely recruited to a DSB via direct interactions with Mre11 and (or) possibly through yKu70/yKu80 (Shim et al. 2005). The latter association suggests a potential role for RSC in NHEJ, and indeed RSC mutants have defects in this repair pathway (Shim et al. 2005). The means by which SWI/SNF is recruited are unknown, although a damage-specific chromatin modification is certainly an attractive possibility. Even though RSC is implicated in the relatively late postsynapsis step of HR, its early recruitment to a DSB suggests that the remodeler may play an additional early role in DSBR (Chai et al. 2005). RSC has previously been implicated in promoting cohesin between sister chromatids (Baetz et al. 2004). This, combined with the demonstrated role of cohesin in DNA repair (see below), raises the intriguing possibility that RSC may promote the recruitment of DSBdependent cohesin. BRCT-domain proteins BRCT (BRCA1-carboxyl terminus) domains are phosphopeptide-binding motifs. Several proteins containing the BRCT repeat have important roles in DSBR, including BRCA1, in which the repeats mediate interaction with the BACH helicase (Manke et al. 2003). It was recently shown that the BRCT repeats of human MDC1 (mediator of checkpoint signaling protein 1) directly bind gH2AX (Stucki et al. 2005). MDC1 plays an early role in the response to DNA damage and rapidly colocalizes to gH2AX foci. It is also required for the subsequent recruitment of other proteins, including the mammalian MRE11 complex (MRN) and the checkpoint adaptor 53BP1 (Bekker-Jensen et al. 2005). Whether this pathway applies to budding or fission yeast is unclear, as these species do not contain readily apparent sequence homologs of MDC1. The mammalian checkpoint protein 53BP1 also has a tandem BRCT domain, and is capable of binding gH2AX in vitro (Ward et al. 2003). In addition, an Schizosaccharomyces pombe homologue of 53BP1, Crb2, is recruited to a DSB in a gH2A-dependent manner (Nakamura et al. 2004). It is currently unclear whether these interactions are functionally relevant in vivo. 53BP1 also contains a tudor domain that binds methylated histone H3-K79 (H3-K79Me), an association that may mediate 53BP1 recruitment to the DSB (Huyen et al. 2004). A potential budding yeast homolog of 53BP1/Brb2, Rad9, is recruited to DSBs via both gH2A and H3-K79Me in a non-redundant manner (Toh et al. 2006; Wysocki et al. 2005). While efficient Rad9 recruitment to a DSB is dependent on the Rad9- and H2A-kinase Mec1 (Naiki et al. 2004), Saccharomyces cerevisiae lacking gH2A are generally competent in the induction and maintenance of cell-cycle checkpoints (Downs et al. 2004; Keogh et al. 2006a; Redon et al. 2003) (see below). This may support a model in which there are 2 pools of Rad9 involved in DSB repair: a hyperphosphorylated form engaged in early check-

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point signaling off chromatin, and a hypophosporylated form recruited to a subset of DSBs after checkpoint activation, perhaps to promote efficient HR (Toh et al. 2006). Cohesins Cohesin is an evolutionarily conserved 4-protein (Scc1, Scc3, Smc1, and Smc3) complex loaded onto chromosomes during S phase. The complex holds sister chromatids together from their replication at S phase until the metaphase–anaphase transition (Glynn et al. 2004; Nasmyth 2002)). It has recently been shown that cohesin is also loaded onto broken chromosomes in G2 phase (Stro¨m ¨ nal et al. 2004). ChIP experiments in budet al. 2004; U ding yeast show that the cohesin complex spreads over ~100 kb from an induced DSB, but is significantly underrepresented in the 1–2 kb immediately proximal to the ¨ nal et al. 2004). This occupancy is strikingly remiDSB (U niscent of the gH2A pattern (Keogh et al. 2006a; Shroff et ¨ nal et al. 2004), and indeed, it is dependent on gH2A (U al. 2004). This cohesin band tethers the broken chromosome to its template sister chromatid and plays a role during certain types of HR. Thus, it is important for replicative/postreplicative repair using the sister chromatid, but plays no role in the intrachromosomal gene conversion used during Saccharomyces cerevisiae mating-type switch¨ nal et al. 2004). ing (U Recent genetic analyses demonstrate that efficient repair of the checkpoint-blind damage caused by camptothecin (whose efficient repair is gH2A dependent) requires several other proteins involved in the cohesin pathway, including Csm3, Tof1, and Mrc1 (Redon et al. 2006). This study places Tof1 and Csm3 in the same pathway, both downstream of gH2A. It will be interesting to determine whether the role of gH2A in the repair of checkpoint-blind damage is related to its ability to induce the loading of cohesin, i.e., in a situation in which checkpoint signaling has not been activated. If true, it follows that an alternate repair pathway may exist when a DNA damage checkpoint has been engaged. Does this pathway involve the Ctf8/Ctf18/Dcc1 complex, which also plays a role in chromatid cohesion (Redon et al. 2006)?

Removal of gH2A(X) gH2A(X) levels decrease during DSBR, but where does it go? Recent studies have identified 2 related phosphatases that target the phosphorylated histone in budding yeast (Keogh et al. 2006a) and human (Chowdhury et al. 2005) cells. Saccharomyces cerevisiae contain the heterotrimeric HTP-C (histone H2A phosphatase complex), the deletion of which leads to constitutively high gH2A levels in dividing cells. HTP-C subunit deletions are insensitive to many genotoxins, although they display synthetic growth defects in combination with deletions of genes known to be involved DNA damage recognition and repair (Keogh et al. 2006a). gH2A is lost from chromatin during HR-mediated DSB repair at the appearance of a specific intermediate after synapse formation. This suggests that the signal to trigger gH2A loss is not the completion of repair, but the completion of a step that normally leads to repair (Keogh et al. 2006a). It is possible that gH2A is removed specifically at #

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this point so that recruited cohesins (see above) do not interfere with later steps in DSBR. Many lines of evidence suggest that gH2A is dephosphorylated after removal from chromatin. First, in the absence of the Pph3 phosphatase, gH2A is lost from a DSB with kinetics comparable to those of wild-type cells. Second, components of the HTP-C are not effectively recruited to an induced DSB in vivo. Finally, isolating the HTP-C with a protocol designed to predominantly target the soluble cellular fraction results in the copurification of large amounts of histones H2A and H2B (and not H3 and H4). Indeed, isolating histone H2A under the same conditions results in the copurification of known histone chaperones (e.g., Nap1) and the HTP-C (our unpublished data). To complete the cycle, after displacement from the DSB, gH2A is dephosphorylated by the HTP-C and likely reused in chromatin at another location (Keogh et al. 2006a). DNA damage cell-cycle checkpoints are triggered and maintained in gH2A-deficient Saccharomyces cerevisiae (Downs et al. 2004; Keogh et al. 2006a; Redon et al. 2003), but not in Schizosaccharomyces pombe (Nakamura et al. 2004). However, G2/M checkpoint recovery is significantly delayed in Saccharomyces cerevisiae deleted for PPH3, the phosphatase subunit of the HTP-C, even though these cells are fully competent in DSB repair (Keogh et al. 2006a). This is presumably due to the persistence of high gH2A levels, since the kinetics of recovery are comparable to those of wild-type cells when gH2A formation is prevented (H2AS129A). Indeed, these cells (H2A-S129A or pph3
/H2AS129A) escape the G2/M checkpoint slightly faster than wild-type cells, suggesting that the phosphorylation status of H2A is monitored by Saccharomyces cerevisiae for recovery from DNA DSB damage. Pph3, the phosphatase component of the Saccharomyces cerevisiae HTP-C, is a member of the PP2/4/6 family of phosphatases (Cohen et al. 2005; Ronne et al. 1991), and PP2A, a member of this family, regulates gH2A.X levels in human cells (Chowdhury et al. 2005). However, numerous mechanistic differences exist between the 2 phosphatases. While yeast Pph3, which is more similar to PP4c, primarily targets a displaced form of gH2A (Keogh et al. 2006a), human PP2A colocalizes with gH2A.X at DSBs, and experiments suggest that it directly dephosphorylates gH2A.X on chromatin (Chowdhury et al. 2005). Also, while Pph3 is dispensable for efficient DSB repair, PP2A is required. One complication is that PP2A is involved in the regulation of many cellular processes in metazoan cells. In particular, it controls the activation of ATM, a central player in the response to DNA damage and 1 of the kinases that create gH2AX (Goodarzi et al. 2004). However gH2AX still accumulates after inhibiting PP2A in ATM-deficient cells, supporting a model in which the phosphatase regulates the phosphorylated histone directly rather than via an intermediate (Chowdhury et al. 2005). Interestingly, the 2 phosphatases most similar to PP2A in budding yeast, Pph21 and Pph22, are not involved in the dephosphorylation of gH2A (Keogh et al. 2006a). The closest Pph3 homolog, PP4c, is found in a human complex analogous to HTP-C in human and subunits can functionally complement the cisplatin sensitivity of HTP-C deletions in budding yeast (Gingras et al. 2005). It is currently unknown whether this complex has any

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role in gH2AX dynamics (soluble or insoluble) or checkpoint recovery in human cells.

Conclusions If the HTP-C is not involved in the removal of gH2A from chromatin, what is? One attractive model of gH2A removal from a DSB is via ATP-dependent chromatin remodeling combined with histone exchange (see above). Another complex that may play a role in histone removal is the proteasome. The remodeling ATPase subunits of the 19S proteasome are found in the nucleus (Huh et al. 2003; Russell et al. 1999) and play a role in chromatin remodeling during transcription (reviewed in (Collins and Tansey 2006)). The proteasome is also recruited to an induced DSB that cannot be repaired by HR (Krogan et al. 2004b) and certain proteasome mutants are hypersensitive to the genotoxins CPT and hydroxyurea (Funakoshi et al. 2004; Krogan et al. 2004b). A human cullin (E3 ubiquitin ligase), CUL4A, has recently been demonstrated to target human H2A at sites of UV damage (Kapetanaki et al. 2006). The H2A ubiquitination site is conserved in humans and yeast (and in human H2AX), although it has never been demonstrated that yeast contains this modification. The related yeast cullin Rtt101 is also likely to play a role in DNA repair, since deletion is hypersensitive to a wide variety of DSB-inducing genotoxins. The substrate of Rtt101 is currently unknown, but of considerable interest. As the first chromatin modification definitively identified at a DSB, it is not surprising that so much effort has been expended to identify the specific roles of gH2A(X). Even though gH2A(X) is not the only chromatin modification that occurs at DSBs, it is the only mark identified to date that is exclusive to these lesions. We know the kinases responsible for gH2A(X) phosphorylation, how far the modification extends from a DSB, several factors that are recruited to it, and more recently, when and how it is removed and dephosphorylated. Further work will provide more insight into how chromatin impinges on DSBR and how other marks identify the break to aid the recruitment and (or) activation of repair factors.

Acknowledgement J.F. is supported by a Canadian Institutes of Health Research fellowship.

References Allard, S., Utley, R.T., Savard, J., Clarke, A., Grant, P., Brandl, C.J., et al. 1999. NuA4, an essential transcription adaptor/histone H4 acetylase complex containing Esa1p and the ATM-related cofactor Tra1p. EMBO J. 18: 5108–5119. doi:10.1093/emboj/18. 185108. PMID: 10487762. Babiarz, J.E., Halley, J.E., and Rine, J. 2006. Telomeric heterochromatin boundaries require NuA4-dependent acetylation of histone variant H2A.Z in Saccharomyces cerevisiae. Genes Dev. 20: 700–710. doi:10.1101/gad.1386306. PMID: 16543222. Baetz, K.K., Krogan, N.J., Emili, A., Greenblatt, J.F., and Hieter, P. 2004. The ctf13–30/CTF13 genomic haploinsufficiency modifier screen identifies the yeast chromatin remodeling complex RSC, which is required for the establishment of sister chromatid #

2006 NRC Canada

Fillingham et al. cohesion. Mol. Cell. Biol. 24: 1232–1244. doi:10.1128/MCB.24. 3.1232-1244.2003. PMID: 14729968. Bassing, C.H., and Alt, F.W. 2004. H2AX may function as an anchor to hold broken chromosomal DNA ends in close proximity. Cell Cycle, 3: 149–153. PMID: 14712078. Bassing, C.H., Chua, K.F., Sekiguchi, J., Suh, H., Whitlow, S.R., Fleming, J.C., et al. 2002. Increased ionizing radiation sensitivity and genomic instability in the absence of histone H2AX. Proc. Natl. Acad. Sci. U.S.A. 99: 8173–8178. doi:10.1073/pnas. 122228699. PMID: 12034884. Bekker-Jensen, S., Lukas, C., Melander, F., Bartek, J., and Lukas, J. 2005. Dynamic assembly and sustained retention of 53BP1 at the sites of DNA damage are controlled by Mdc1/NFBD1. J. Cell Biol. 170: 201–211. doi:10.1083/jcb.200503043. PMID: 16009723. Bennett, C.B., Lewis, L.K., Karthikeyan, G., Lobachev, K.S., Jin, Y.H., Sterling, J.F., et al. 2001. Genes required for ionizing radiation resistance in yeast. Nat. Genet. 29: 426–434. doi:10. 1038/ng778. PMID: 11726929. Bird, A.W., Yu, D.Y., Pray-Grant, M.G., Qiu, Q., Harmon, K.E., Megee, P.C., et al. 2002. Acetylation of histone H4 by Esa1 is required for DNA double-strand break repair. Nature (London), 419: 411–415. doi:10.1038/nature01035. PMID: 12353039. Bilsland, E., and Downs, J.A. 2005. Tails of histones in DNA double-strand break repair. Mutagenesis, 20: 153–163. doi:10. 1093/mutage/gei031. PMID: 15843385. Burma, S., and Chen, D.J. 2004. Role of DNA-PK in the cellular response to DNA double-strand breaks. DNA Repair (Amsterdam), 3: 909–918. PMID: 15279776. Burma, S., Chen, B.P., Murphy, M., Kurimasa, A., and Chen, D.J. 2001. ATM phosphorylates histone H2AX in response to DNA double-strand breaks. J. Biol. Chem. 276: 42 462 – 42 467. doi:10.1074/jbc.C100466200. PMID: 11571274. Celeste, A., Petersen, S., Romanienko, P.J., Fernandez-Capetillo, O., Chen, H.T., Sedelnikova, O.A., et al. 2002. Genomic instability in mice lacking histone H2AX. Science (Washington, D.C.), 296: 922–927. doi:10.1126/science.1069398. PMID: 11934988. Chai, B., Huang, J., Cairns, B.R., and Laurent, B.C. 2005. Distinct roles for the RSC and Swi/Snf ATP-dependent chromatin remodelers in DNA double-strand break repair. Genes Dev. 19: 1656–1661. doi:10.1101/gad.1273105. PMID: 16024655. Chen, H.T., Bhandoola, A., Difilippantonio, M.J., Zhu, J., Brown, M.J., Tai, X., et al. 2000. Response to RAG-mediated VDJ cleavage by NBS1 and gamma-H2AX. Science (Washington, D.C.), 290: 1962–1965. doi:10.1126/science.290.5498.1962. PMID: 11110662. Chowdhury, D., Keogh, M.-C., Ishii, H., Peterson, C.L., Buratowski, S., and Lieberman, J. 2005. gH2AX dephosporylation by Protein Phosphatase 2A facilitates DNA double-strand break repair. Mol. Cell, 20: 801–809. doi:10.1016/j.molcel.2005.10. 003. PMID: 16310392. Cohen, P.T., Pjilp, A., and Vasquez-Martin, C. 2005. Protein phosphatase 4- from obscurity to vital functions. FEBS Lett. 579: 3278–3286. doi:10.1016/j.febslet.2005.04.070. PMID: 15913612. Collins, G.A., and Tansey, W.P. 2006. The proteasome: a utility tool for transcription? Curr. Opin. Genet. Dev. 16: 197–202. doi:10.1016/j.gde.2006.02.009. PMID: 16503126. Cosgrove, M.S., and Wolberger, C. 2005. How does the histone code work? Biochem. Cell Biol. 83: 468–476. doi:10.1139/o05137. PMID: 16094450. Daley, J.M., Palmbos, P.L., Wu, D., and Wilson, T.E. 2005. Nonhomologous end joining in yeast. Annu. Rev. Genet. 39: 431–451. doi:10.1146/annurev.genet.39.073003.113340. PMID: 16285867. Daniel, R., Ramcharan, J., Rogakou, E., Taganov, K.D., Greger,

565 J.G., Bonner, W.M., et al. 2004. Histone H2AX is phosphorylated at sites of retroviral DNA integration, but is dispensable for post-integration repair. J. Biol. Chem. 279: 45 810 – 45 814. doi:10.1074/jbc.M407886200. PMID: 15308627. Downs, J.A., Allard, S., Jobin-Robitaille, O., Javaheri, A., Auger, A., Bouchard, N., et al. 2004. Binding of chromatin-modifying activities to phosphorylated histone H2A at DNA damage sites. Mol. Cell, 16: 979–990. doi:10.1016/j.molcel.2004.12.003. PMID: 15610740. Downs, J.A., Lowndes, N.F., and Jackson, S.P. 2000. A role for Saccharomyces cerevisiae histone H2A in DNA repair. Nature (London), 408: 1001–1004. PMID: 11140636. Ehrenhofer-Murray, A.E. 2004. Chromatin dynamics at DNA replication, transcription and repair. Eur. J. Biochem. 271: 2335– 2349. doi:10.1111/j.1432-1033.2004.04162.x. PMID: 15182349. Fernandez-Capetillo, O., Allis, C.D., and Nussenzweig, A. 2004. Phosphorylation of histone H2B at DNA double-strand breaks. J. Exp. Med. 199: 1671–1677. doi:10.1084/jem.20032247. PMID: 15197225. Foster, E.R., and Downs, J.A. 2005. Histone H2A phosphorylation in DNA double-strand break repair. FEBS J. 272: 3231–3240. doi:10.1111/j.1742-4658.2005.04741.x. PMID: 15978030. Funakoshi, M., Li, X., Velichutina, I., Hochstrasser, M., and Kobayashi, H. 2004. Sem1, the yeast ortholog of a human BRCA2-binding protein, is a component of the proteasome regulatory particle that enhances proteasome stability. J. Cell Sci. 117: 6447–6454. doi:10.1242/jcs.01575. PMID: 15572408. Furuta, T., Takemura, H., Liao, Z.Y., Aune, G.J., Redon, C., Sedelnikova, O.A., et al. 2003. Phosphorylation of histone H2AX and activation of Mre11, Rad50, and Nbs1 in response to replicationdependent DNA double-strand breaks induced by mammalian DNA topoisomerase I cleavage complexes. J. Biol. Chem. 278: 20 303 – 20 312. doi:10.1074/jbc.M300198200. PMID: 12660252. Gingras, A.C., Caballero, M., Zarske, M., Sanchez, A., Hazbun, T.R., Fields, S., et al. 2005. A novel, evolutionarily conserved protein phosphatase complex involved in cisplatin sensitivity. Mol. Cell. Proteomics, 4: 1725–1740. PMID: 16085932. Glynn, E.F., Megee, P.C., Yu, H.G., Mistrot, C., Unal, E., Koshland, D.E., et al. 2004. Genome-wide mapping of the cohesin complex in the yeast Saccharomyces cerevisiae. PLoS Biol. 2: E259. doi:10.1371/journal.pbio.0020259. PMID: 15309048. Goodarzi, A.A., Jonnalagadda, J.C., Douglas, P., Young, D., Ye, R., Moorhead, G.B., et al. 2004. Autophosphorylation of ataxiatelangiectasia mutated is regulated by protein phosphatase 2A. EMBO J. 23: 4451–4461. doi:10.1038/sj.emboj.7600455. PMID: 15510216. Haber, J.E. 1998. Mating-type gene switching in Saccharomyces cerevisiae. Annu. Rev. Genet. 32: 561–599. doi:10.1146/ annurev.genet.32.1.561. PMID: 9928492. Huh, W.-K., Falvo, J.V., Gerke, L.C., Carroll, A.S., Howson, R.W., Weissman, J.S., and O’Shea, E.K. 2003. Global analysis of protein localization in budding yeast. Nature (London), 425: 686– 691. doi:10.1038/nature02026. PMID: 14562095. Huyen, Y., Zgheib, O., Ditullio, R.A., Jr., Gorgoulis, V.G., Zacharatos, P., Petty, T.J., et al. 2004. Methylated lysine 79 of histone H3 targets 53BP1 to DNA double-strand breaks. Nature (London), 432: 406–411. doi:10.1038/nature03114. PMID: 15525939. Jackson, S.P. 2002. Sensing and repairing DNA double-strand breaks. Carcinogenesis, 23: 687–696. doi:10.1093/carcin/23.5. 687. PMID: 12016139. Jenuwein, T., and Allis, C.D. 2001. Translating the histone code. Science (Washington, D.C.), 293: 1074–1080. doi:10.1126/ science.1063127. PMID: 11498575. #

2006 NRC Canada

566 Johnson, C.N., Adkins, N.L., and Georgel, P. 2005. Chromatin remodeling complexes: ATP-dependent machines in action. Biochem. Cell Biol. 83: 405–417. doi:10.1139/o05-115. PMID: 16094444. Kapetanaki, M.G., Guerrero-Santoro, J., Bisi, D.C., Hsieh, C.L., RapicOtrin, V., and Levine, A.S. 2006. The DDB1–CUL4ADDB2 ubiquitin ligase is deficient in xeroderma pigmentosum group E and targets histone H2A at UV-damaged DNA sites. Proc. Natl. Acad. Sci. U.S.A. 103: 2588–2593. doi:10.1073/pnas. 0511160103. PMID: 16473935. Kegel, A., Sjojstrand, J.O., and Astrom, S.U. 2001. Nej1p, a celltype specific regulator of nonhomologous end joining in yeast. Curr. Biol. 11: 1611–1617. doi:10.1016/S0960-9822(01)004882. PMID: 11676923. Keogh, M.-C., Kim, J.-A., Downey, M., Fillingham, J., Chowdhury, D., Harrison, J.C., et al. 2006a. A phosphatase complex that dephosphorylates gH2AX regulates DNA damage checkpoint recovery. Nature (London), 439: 497–501. doi:10.1038/ nature04384. PMID: 16299494. Keogh, M.-C., Mennella, T.A., Sawa, C., Berthelet, S., Krogan, N.J., Wolek, A., et al. 2006b. The Saccharomyces cerevisiae histone H2A variant Htz1 is acetylated by NuA4. Genes Dev. 20: 660–665. doi:10.1101/gad.1388106. PMID: 16543219. Kobor, M.S., Venkatasubrahmanyam, S., Meneghini, M.D., Gin, J.W., Jennings, J.L., Link, A.J., et al. 2004. A protein complex containing the conserved Swi2/Snf2-related ATPase Swr1p deposits histone variant H2A.Z into euchromatin. PLoS Biol. 2(5): e131. PMID: 15045029. Kolodner, R.D., Putnam, C.D., and Myung, K. 2002. Maintenance of genome stability in Saccharomyces cerevisiae. Science (Washington, D.C.), 297: 552–557. doi:10.1126/science.1075277. PMID: 12142524. Krogan, N.J., Keogh, M.-C., Datta, N., Sawa, C., Ryan, O.W., Ding, H., et al. 2003. A Snf2-Family ATPase complex required for recruitment of the Histone H2A variant Htz1. Mol. Cell, 12: 1565– 1576. doi:10.1016/S1097-2765(03)00497-0. PMID: 14690608. Krogan, N.J., Baetz, K.K., Keogh, M.-C., Datta, N., Sawa, C., Kwok, T.C.Y., et al. 2004a. Regulation of chromosome stability by the histone H2A variant Htz1, the Swr1 chromatin remodeling complex and the histone acetyltransferase NuA4. Proc. Natl. Acad. Sci. U.S.A. 101: 13513–13518. doi:10.1073/pnas. 0405753101. PMID: 15353583. Krogan, N.J., Lam, M.H.Y., Fillingham, J., Keogh, M.-C., Gebbia, M., Li, J., et al. 2004b. Proteasome involvement in the repair of DNA double-strand breaks. Mol. Cell, 16: 1027–1034. doi:10. 1016/j.molcel.2004.11.033. PMID: 15610744. Krogh, B.O., and Symington, L.S. 2004. Recombination proteins in yeast. Annu. Rev. Genet. 38: 233–271. doi:10.1146/annurev. genet.38.072902.091500. PMID: 15568977. Kusch, T., Florens, L., MacDonald, W.H., Swanson, S.K., Glaser, R.L., Yates, J.R., III, et al. 2004. Acetylation by Tip60 is required for selective histone variant exchange at DNA lesions. Science (Washington, D.C.), 306: 2084–2087. doi:10.1126/ science.1103455. PMID: 15528408. Li, A., Eirı´n-Lo´pez, J.M., and Ausio´, J. 2005. H2AX: Tailoring histone H2A for chromatin-dependent genomic integrity. Biochem. Cell Biol. 83: 505–515. doi:10.1139/o05-114. PMID: 16094454. Lindahl, T. 1993. Instability and decay of the primary structure of DNA. Nature (London), 362: 709–715. doi:10.1038/362709a0. PMID: 8469282. Lindahl, T. 2000. Suppression of spontaneous mutageneisis in human cells by DNA base-excision repair. Mutat. Res. 462: 129– 135. PMID: 10767624. Lindahl, T., and Wood, R.D. 1999. Quality control of DNA repair.

Biochem. Cell Biol. Vol. 84, 2006 Science (Washington, D.C.), 286: 1897–1905. doi:10.1126/ science.286.5446.1897. PMID: 10583946. Lisby, M., Barlow, J.H., Burgess, R.C., and Rothstein, R. 2004. Choreography of the DNA damage response: spatiotemporal relationships among checkpoint and repair proteins. Cell, 118: 699–713. doi:10.1016/j.cell.2004.08.015. PMID: 15369670. Luger, K. 2003. Structure and dynamic behavior of nucleosomes. Curr. Opin. Genet. Dev. 13: 127–135. doi:10.1016/S0959437X(03)00026-1. PMID: 12672489. Malik, H.S., and Henikoff, S. 2003. Phylogenomics of the nucleosome. Nat. Struct. Biol. 10: 882–891. doi:10.1038/nsb996. PMID: 14583738. Manke, I.A., Lowery, D.M., Nguyen, A., and Yaffe, M.B. 2003. BRCT repeats as phosphopeptide-binding modules involved in protein targeting. Science (Washington, D.C.), 302: 636–639. doi:10.1126/science.1088877. PMID: 14576432. Millar, C.B., Xu, F., Zhang, K., and Grunstein, M. 2006. Acetylation of H2AZ Lys 14 is associated with genome-wide gene activity in yeast. Genes Dev. 20: 711–722. doi:10.1101/gad. 1395506. PMID: 16543223. Mizuguchi, G., Shen, X., Landry, J., Hu, W.-H., Sen, S., and Wu, C. 2004. ATP-driven exchange of histone H2AZ variant catalyzed by SWR1 chromatin remodeling complex. Science (Washington, D.C.), 303: 343–348. doi:10.1126/science.1090701. PMID: 14645854. Moore, J.K., and Haber, J.E. 1996. Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repiar of double-strand breaks in Saccharomyces cerevisiae. Mol. Cell. Biol. 16: 2164–2173. PMID: 8628283. Moore, J.D., and Krebs, J.E. 2004. Histone modifications and DNA double-strand break repair. Biochem. Cell Biol. 82: 446–452. doi:10.1139/o04-034. PMID: 15284897. Morrison, A.J., Highland, J., Krogan, N.J., Arbel-Eden, A., Greenblatt, J.F., Haber, J.E., and Shen, X. 2004. Ino80 and g-H2AX interaction links ATP-dependent chromatin remodeling to DNA damage repair. Cell, 119: 767–775. doi:10.1016/j.cell.2004.11. 037. PMID: 15607974. Mukherjee, B., Kessinger, C., Kobayashi, J., Chen, B.P.C., Chen, D.J., Chatterjee, A., and Burma, S. 2006. DNA-PK phosphorylates histone H2AX during apoptotic DNA fragmentation in mammalian cells. DNA Repair (Amsterdam), 5: 575–590. PMID: 16567133. Murr, R., Loizou, J.I., Yang, Y.G., Cuenin, C., Li, H., Wang, Z.Q., and Herceg, Z. 2006. Histone acetylation by Trrap-Tip60 modulates loading of repair proteins and repair of DNA double-strand breaks. Nat. Cell Biol. 8: 91–99. doi:10.1038/ncb1343. PMID: 16341205. Naiki, T., Wakayama, T., Nakada, D., Matsumoto, K., and Sugimoto, K. 2004. Association of Rad9 with double-strand breaks through a Mec1-dependent mechanism. Mol. Cell. Biol. 24: 3277–3285. doi:10.1128/MCB.24.8.3277-3285.2004. PMID: 15060150. Nakamura, T.M., Du, L.L., Redon, C., and Russell, P. 2004. Histone H2A phosphorylation controls Crb2 recruitment at DNA breaks, maintains checkpoint arrest, and influences DNA repair in fission yeast. Mol. Cell. Biol. 24: 6215–6230. doi:10.1128/ MCB.24.14.6215-6230.2004. PMID: 15226425. Nasmyth, K. 2002. Segregating sister genomes: the molecular biology of chromosome separation. Science (Washington, D.C.), 297: 559–565. doi:10.1126/science.1074757. PMID: 12142526. Nazarov, I.B., Smirnova, A.N., Krutilina, R.I., Svetlova, M.P., Solovjeva, L.V., Nikiforov, A.A., et al. 2003. Dephosphorylation of histone gamma-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A. Radiat. Res. 160: 309–317. PMID: 12926989. #

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Fillingham et al. Nyberg, K.A., Michelson, R.J., Putnam, C.W., and Weinert, T.A. 2002. Toward maintaining the genome: DNA damage and replication checkpoints. Annu. Rev. Genet. 36: 617–656. doi:10. 1146/annurev.genet.36.060402.113540. PMID: 12429704. Paˆques, F., and Haber, J.E. 1999. Multiple pathways of recombination induced by double strand breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63: 349–404. PMID: 10357855. Petersen, S., Casellas, R., Reina-San-Martin, B., Chen, H.T., Difilippantonio, M.J., Wilson, P.C., et al. 2001. AID is required to initiate Nbs1/gamma-H2AX focus formation and mutations at sites of class switching. Nature (London), 414: 660–665. doi:10. 1038/414660a. PMID: 11740565. Peterson, C.L., and Coˆte´, J. 2004. Cellular machineries for chromosomal DNA repair. Genes Dev. 18: 602–616. doi:10.1101/gad. 1182704. PMID: 15075289. Redon, C., Pilch, D., Rogakou, E., Sedelnikova, O., Newrock, K., and Bonner, W. 2002. Histone H2A variants H2AX and H2AZ. Curr. Opin. Genet. Dev. 12: 162–169. doi:10.1016/S0959-437X(02) 00282-4. PMID: 11893489. Redon, C., Pilch, D.R., Rogakou, E.P., Orr, A.H., Lowndes, N.F., and Bonner, W.M. 2003. Yeast histone H2A serine 129 is essential for the efficient repair of checkpoint-blind DNA damage. EMBO Rep. 4: 678–684. doi:10.1038/sj.embor.embor871. PMID: 12792653. Redon, C., Pilch, D.R., and Bonner, W.M. 2006. Genetic analysis of Saccharomyces cerevisiae H2A serine 129 mutant suggests a functional relationship between H2A and the sister chromatin cohesion partners Csm3-Tof1 for the repair of topoisomerase Iinduced DNA damage. Genetics, 172: 67–76. PMID: 16219777. Rogakou, E.P., Boon, C., Redon, C., and Bonner, W.M. 1999. Megabase chromatin domains involved in DNA double-strand breaks in vivo. J. Cell Biol. 146: 905–916. doi:10.1083/jcb.146. 5.905. PMID: 10477747. Rogakou, E.P., Nieves-Neira, W., Boon, C., Pommier, Y., and Bonner, W.P. 2000. Initiation of DNA fragmentation during apoptosis induces phosphorylation of H2AX histone at Serine 139. J. Biol. Chem. 275: 9390–9395. doi:10.1074/jbc.275.13. 9390. PMID: 10734083. Rogakou, E.P., Pilch, D.R., Orr, A.H., Ivanova, V.S., and Bonner, W.M. 1998. DNA double-strand breaks induce histone H2AX phosphorylation on serine 139. J. Biol. Chem. 273: 5858–5868. doi:10.1074/jbc.273.10.5858. PMID: 9488723. Ronne, H., Carlberg, M., Hu, G.Z., and Nehlin, J.O. 1991. Protein phosphatase 2A in Saccharomyces cerevisiae: effects on cell growth and bud morphogenesis. Mol. Cell. Biol. 11: 4876– 4884. PMID: 1656215. Rouse, J., and Jackson, S.P. 2002. Interfaces between the detection, signaling, and repair of DNA damage. Science (Washington, D.C.), 297: 547–551. doi:10.1126/science.1074740. PMID: 12142523. Rudin, N., and Haber, J.E. 1988. Efficient repair of HO-induced chromosomal breaks in Saccharomyces cerevisiae by recombination between flanking homologous sequences. Mol. Cell. Biol. 8: 3918–3928. PMID: 3065627. Russell, S.J., Steger, K.A., and Johnston, S.A. 1999. Subcellular localization, stoichiometry, and protein levels of 26 S proteasome subunits in yeast. J. Biol. Chem. 274: 21 943 – 21 952. doi:10. 1074/jbc.274.31.21943. PMID: 10419517. Shen, X., Mizuguchi, G., Hamiche, A., and Wu, C. 2000. A chromatin remodeling complex involved in transcription and DNA processing. Nature (London), 406: 541–544. doi:10.1038/35020123. PMID: 10952318. Shim, E.Y., Ma, J.L., Oum, J.H., Yanez, Y., and Lee, S.E. 2005. The yeast chromatin remodeler RSC complex facilitates end joining repair of DNA double-strand breaks. Mol. Cell. Biol. 25:

567 3934–3944. doi:10.1128/MCB.25.10.3934-3944.2005. PMID: 15870268. Shroff, R., Arbel-Eden, A., Pilch, D., Ira, G., Bonner, W.M., Petrini, J.H., et al. 2004. Distribution and dynamics of chromatin modification at a defined DNA double strand break. Curr. Biol. 14: 1703–1711. doi:10.1016/j.cub.2004.09.047. PMID: 15458641. Smith, E.R., Eisen, A., Gu, W., Sattah, M., Pannuti, A., Zhou, J., et al. 1998. ESA1 is a histone acetyltransferase that is essential for growth in yeast. Proc. Natl. Acad. Sci. U.S.A. 95: 3561–3565. doi:10.1073/pnas.95.7.3561. PMID: 9520405. Strahl, B.D., and Allis, C.D. 2000. The language of covalent histone modifications. Nature (London), 403: 41–45. PMID: 10638745. Stro¨m, L., Lindroos, H.B., Shirahige, K., and Sjo¨gren, C. 2004. Postreplicative recruitment of cohesin to double-strand breaks is required for DNA repair. Mol. Cell, 16: 1003–1015. doi:10. 1016/j.molcel.2004.11.026. PMID: 15610742. Stucki, M., Clapperton, J.A., Mohammad, D., Yaffe, M.B., Smerdon, S.J., and Jackson, S.P. 2005. MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks. Cell, 123: 1213–1326. doi:10.1016/j.cell. 2005.09.038. PMID: 16377563. Symington, L.S. 2002. Role of RAD52 epistasis group genes in homologous recombination and double-strand break repair. Microbiol. Mol. Biol. Rev. 66: 630–670. PMID: 12456786. Thiriet, C., and Hayes, J.J. 2005. Chromatin in need of a fix: phosphorylation of H2AX connects chromatin to DNA repair. Mol. Cell, 18: 617–622. doi:10.1016/j.molcel.2005.05.008. PMID: 15949437. Toh, G.W., O’Shaughnessy, A.M., Jimeno, S., Dobbie, I.M., Grenon, M., Maffini, S., et al. 2006. Histone H2A phosphorylation and H3 methylation are required for a novel Rad9 DSB repair function following checkpoint activation. DNA Repair (Amsterdam), 5: 693–703. PMID: 16650810. Tomilin, N.V., Solovjeva, L.V., Svetlova, M.P., Pleskach, N.M., Zalenskaya, I.A., Yau, P.M., and Bradbury, E.M. 2001. Visualization of focal nuclear sites of DNA repair synthesis induced by bleomycin in human cells. Radiat. Res. 156: 347–354. PMID: 11554846. Tsukuda, T., Fleming, A.B., Nickoloff, J.A., and Osley, M.A. 2005. Chromatin remodeling at a DNA double-strand break site in Saccharomyces cerevisiae. Nature (London), 438: 379–383. doi:10.1038/nature04148. PMID: 16292314. ¨ nal, E., Arbel-Eden, A., Sattler, U., Shroff, R., Lichten, M., HaU ber, J.E., and Koshland, D. 2004. DNA damage response pathway uses histone modification to assemble a double-strand break-specific cohesin domain. Mol. Cell, 16: 991–1002. doi:10. 1016/j.molcel.2004.11.027. PMID: 15610741. van Attikum, H., Fritsch, O., Hohn, B., and Gasser, S.M. 2004. Recruitment of the INO80 complex by H2A phosphorylation links ATP-dependent chromatin remodeling with DNA double-strand break repair. Cell, 119: 777–788. PMID: 15607975. Vidanes, G.M., Bonilla, C.Y., and Toczyski, D.P. 2005. Complicated tails: Histone modifications and the DNA damage response. Cell, 121: 973–976. doi:10.1016/j.cell.2005.06.013. PMID: 15989948. Ward, I.M., and Chen, J. 2001. Histone H2AX is phosphorylated in an ATR-dependent manner in response to replicational stress. J. Biol. Chem. 276: 47 759 – 47 762. PMID: 11673449. Ward, I.M., Kinn, M., Jorda, K.G., and Chen, J. 2003. Accumulation of checkpoint protein 53BP1 at DNA breaks involves its binding to phosphorylated histone H2AX. J. Biol. Chem. 278: 19 579 – 19 582. doi:10.1074/jbc.C300117200. PMID: 12697768. Wilson, T.E. 2002. A genomics-based screen for yeast mutants #

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568 with an altered recombination/end-joining repair ratio. Genetics, 162: 677–688. PMID: 12399380. Wolner, B., van Komen, S., Sung, P., and Peterson, C.L. 2003. Recruitment of the recombinational repair machinery to a DNA double-strand break in yeast. Mol. Cell, 12: 221–232. doi:10. 1016/S1097-2765(03)00242-9. PMID: 12887907. Wuebbles, R.D., and Jones, P.L. 2004. DNA repair in a chromatin environment. Cell. Mol. Life Sci. 61: 2148–2153. PMID: 15338044.

Biochem. Cell Biol. Vol. 84, 2006 Wysocki, R., Javaheri, A., Allard, S., Sha, F., Coˆte´, J., and Kron, S.J. 2005. Role of Dot1-dependent histone H3 Methylation in G1 and S phase DNA damage checkpoint functions of Rad9. Mol. Cell. Biol. 25: 8430–8443. doi:10.1128/MCB.25.19.84308443.2005. PMID: 16166626. Zhou, B.B., and Elledge, S.J. 2000. The DNA damage response: putting checkpoints in perspective. Nature (London), 408: 433– 439. PMID: 11100718.

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