Tails of histones in DNA double-strand break repair

REVIEW

Tails of histones in DNA double-strand break repair

Elizabeth Bilsland and Jessica A.Downs�

Department of Biochemistry, Cambridge University, 80 Tennis Court Road,Cambridge CB2 1GA, UK

DNA double-strand breaks (DSBs) are, arguably, the mostdeleterious form of DNA damage. An increasing body ofevidence points to the inaccurate or inefficient repair ofDSBs as a key step in tumorigenesis. Therefore, it isof great importance to understand the processes by whichDSBs are detected and repaired. Clearly, these events musttake place in the context of chromatin in vivo, and recently,a great deal of progress has been made in understandingthe dynamic and active role that histone proteins and chro-matin modifying activities play in DNA DSB repair. Here,we briefly review some of the most common techniques instudying DNA DSB responses in vivo, and focus on thecontributions of covalent modifications of core histoneproteins to these DNA DSB responses.

Introduction

Within the eukaryotic cell nucleus, genetic information isorganized in a highly conserved structural polymer, the chro-matin, which supports and controls crucial functions of thegenome. The basic unit of chromatin is the nucleosome,which comprises 146 bp of DNA wrapped around eight his-tones, two of each of the four core histone families—H2A,H2B, H3 and H4. Linker histones, termed H1 or H5, associatewith the DNA between individual nucleosomes establishing ahigher level of organization.

This method of packaging DNA allows very long negativelycharged molecules to exist in relatively small 3D space. Forexample, the length of DNA contained in a human cell is ~2 m,yet it occupies a spherical space i.e. in some cases, only 3 mmin diameter. In addition to these powerful properties of con-densation, the composition of chromatin must allow rapid andprecise access to particular regions of the genome during tran-scriptional regulation and accurate partitioning to daughtercells during mitosis. It is not surprising, therefore, that chro-matin is extremely malleable, and that chromatin undergoesdynamic changes, including massive structural reorganization,during many genetic processes, such as DNA replication andcell division, transcription, as well as DNA repair and recom-bination. Furthermore, nucleosomes can transmit epigeneticinformation from one generation to another, having the poten-tial to act as a memory bank of the cell.

The chromatin structure has the potential to influence allgenetic processes and is frequently exploited as means ofgene regulation. The term ‘chromatin remodeling’ has beenused to describe transitions in chromatin structure that include

histone post-translational modifications (described in moredetail below), alterations to the histone variant compositionof nucleosomes, changes in the non-histone protein content ofchromatin and alterations to chromatin structure due to theaction of ATP-dependent chromatin remodeling enzymes[e.g. yeast Swi2, see (1) for review]. In many cases, thesedifferent classes of chromatin alteration are interrelated. Forexample, recent reports indicate that histone dimers can beremoved or exchanged between nucleosomes during the courseof a chromatin remodeling reaction.

Core histone proteins are evolutionarily conserved and con-sist mainly of flexible N-terminal tails protruding outwardfrom the nucleosome, and histone fold-containing domainsthat make up the nucleosome scaffold. In addition, histonesH2A and H2B have C-terminal tails that also protrude outwardfrom the nucleosome structure. These core histone proteins,and in particular, their N-terminal and C-terminal tails, func-tion as acceptors for a variety of post-translational modifica-tions, including acetylation and ubiquitination of lysine (K)residues, phosphorylation of serine (S) and threonine (T) resi-dues and methylation of lysine and arginine (R) residues (Fig-ure 1). Combinations of specific patterns of post-translationalmodifications provide the basis for the ‘histone code’ hypo-thesis (2), in which the interplay between different covalentmodifications determines chromatin functions, by either alter-ing nucleosome conformation or the binding affinities of themodified histones for various proteins. A major challenge inchromatin biology is connecting particular modifications withdistinct biological functions and vice versa.

Although a great deal is known about the role of post-translational modifications, particularly acetylation in tran-scriptional regulation, it is clear that there are manyunanswered questions. Moreover, the role of post-translational modifications in DNA repair and recombinationis far less understood. In this review, we will focus on recentfindings regarding histone modifications that might beinvolved in DNA double-strand break (DSB) repair in euka-ryotes and the tools commonly used to investigate thesehistone modifications.

DNA double strand breaks

We are exposed daily to a vast range of agents capable ofinflicting various types of DNA damage. Of the DNA lesionscreated, probably the most dangerous is the double-strandbreak, as it generates loose ends, which have great potentialto inappropriately recombine with other parts of the genome.Furthermore, inaccurate repair of the lesion could introducemutations in tumor suppressor genes. Clearly, there is signifi-cant tumorigenic potential if DNA DSB activities are impaired.

DNA DSB repair in eukaryotes is mediated primarily by twopathways: homologous recombination [HR; (3)] or non-homo-logous end-joining [NHEJ; (4)]. Both processes are likely to

�To whom correspondence should be addressed. Tel: 101223 333 663; Fax: 101223 766 002; Email: [email protected]

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require chromatin alterations in order to efficiently undergoprocesses, such as strand invasion, branch migration, DNAsynthesis, ligation and recruitment of checkpoint proteins (5).

In addition to the repair activities, cells respond to DNAdamage by instigating a signal transduction cascade that resultsin the arrest of cell-cycle progression and the transcriptionalactivation of DNA damage responsive genes. Members of thePI-3 kinase-like family, including mammalian DNA-PK(DNA-dependent protein kinase), ATM (ataxia telangiectasiamutated protein) and ATR (ataxia telangiectasia relatedprotein), have been implicated as important components ofthese DNA damage responses. While there are no homologsof DNA-PK in lower eukaryotes, clear homologs of ATM andATR exist and are of principal importance to DNA damage-responsive signal transduction pathways. These are Tel1 andMec1 in Saccharomyces cerevisiae and Tel1 and Rad3 inSchizosaccharomyces pombe, respectively.

The repair of DNA must, of course, occur in the context ofchromatin, and therefore, it is reasonable to speculate that one

of the first steps in the DNA repair process is the reorganizationof chromatin structure to allow access and processing by therepair machinery. Interestingly, chromatin remodeling com-plexes, such as Ino80 and Swr1 (S.cerevisiae) and Tip60(Homo sapiens) have been implicated in DNA repair (6).Moreover, chromatin assembly factors, namely Asf1(S.cerevisiae) and CAF-1 (S.cerevisiae and H.sapiens), havealso been implicated in DNA repair. Yeast strains with muta-tions in ASF1 are sensitive to DSBs (7) while CAF-I defectivestrains are sensitive to UV when compared with their wild-typecounterparts (8). Both factors assemble H3 and H4 into nucleo-somes during DNA replication and possibly perform the sametask on newly synthesized repaired DNA. Consistent with thisview, CAF-I is recruited onto DNA after UV irradiation ofhuman cells (8). In addition to chromatin remodeling, post-translational modifications of histones have been implicated inDNA repair activities. It is important to view these modifica-tions in the context of other chromatin activities. For instance,in addition to effects on the repair machinery, histone modi-fications may also function to facilitate chromatin remodelingor assembly activities.

Talking to histones

An astonishing number of biochemical and genetic techniquesare available to study chromatin behavior. The vast majority ofthese approaches have been developed by researchers examin-ing the role of chromatin in the regulation of gene expressionand are both highly applicable and easily adaptable to thestudies on the role of chromatin in DNA repair and recom-bination. Therefore, before summarizing the existing data onpost-translational modifications of histones in DNA repairand recombination, we will briefly review a few of the mostwidespread techniques for investigating chromatin behavior.

Model system

The functions of chromatin compaction and genome mainten-ance are vital to our survival. As such, many proteins involvedin both of these activities have been very strongly conservedthroughout the eukaryotes. The best characterized DNA dam-age dependent phosphorylation event (phosphorylation of theH2A C-terminal tail, described in more detail below) appearsto be conserved from yeast to humans. Moreover, many in vitrostudies of chromatin modulating activities use factors isolatedfrom a variety of organisms [e.g. yeast ATP-dependent chro-matin remodeling factors can be analyzed using chromatincomposed of human histones and sea urchin rDNA sequences(9)], demonstrating extraordinary structural and functional con-servation. Nevertheless, there is variability in the data gener-ated from different labs. It remains to be seen, which of thesedifferences are due to evolutionary variation and which of theseare simply due to the different approaches used in different labs.Therefore, while it is reasonable to extrapolate many funda-mental insights generated in one species to other eukaryoticorganisms, there will inevitably be important differences.

Genetic studies

Historically, the first step in determining gene function is toinactivate or impair the gene and examine the phenotype of theresulting strain. Subsequent analyses, such as complementa-tion, suppression and epistasis studies with other mutations canyield enormous insights into the function of a gene. Unfortu-nately, genes encoding for histones are essential, and therefore,full disruption of the genes is not feasible. Of course,

Yeast

Human

H2A

H2B

H3

H4

H2AX

H2B

H3

H4

Fig. 1. Covalent modifications of histones in DNA DSB responses. Corehistone sequences upstream and downstream of the histone-fold domains(gray circles) are shown. Residues that are covalently modified during DNADSB responses are highlighted in bold with the residue number and themodification type written above (P, phosphorylation; M, methylation;A, acetylation; U, ubiquitination). Note that the initiating methionine isremoved from histone proteins and the numbering of the residues takes thisinto account. The exception to this is histone H2A from yeast, where thenumbering of the residues used in the literature includes the initiatingmethionine, and the residues are given in brackets to indicate this. Sequencesshown are from S.cerevisiae core histones encoded by HTA1 (H2A), HTB1(H2B), HHT1 (H3) and HHF1 (H4) (top panel). In the bottom panel, thesequences from human core histones H2B, H3, H4 and the H2AX histonevariant are shown.

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mutagenesis of specific residues or motifs is possible, and canbe an excellent way to very precisely define the role of aspecific residue in an activity, such as DNA repair. Thisapproach, however, is complicated by the fact that eukaryotestypically have more than one copy of each histone gene—up toas many as 600 in sea urchins and newts. Barring the analysisof dominant mutations, this makes the mutagenesis of histoneschallenging.

Using genetics to analyze histones in higher eukaryotes issomewhat limited. A notable exception to this is the potentialto analyze the roles of non-essential, single-copy histone vari-ants, such as H2AX, discussed in more detail below. In lowereukaryotes, such as the budding yeast, the already powerfulcase of genetic manipulation is enhanced by the fact that thereare only two copies of each core histone.

One common approach to genetic analysis of histones in thebudding yeast is to use strains in which both copies of the corehistone(s) of interest are disrupted and the strains are kept aliveusing one copy of the histone(s) on a plasmid under the controlof the endogenous promoter(s). These strains are viable, andwhen using a plasmid with a selectable marker that can beselected both for and against, allow the wild-type histone plas-mid to be easily swapped with mutagenized histone copies on aplasmid with a different selectable marker. Interestingly, how-ever, there are slight differences in both the promoters and thecoding sequences of the two copies of the core histones, and itis possible that there will be subtle effects of the absence of thesecond copy, particularly when analyzing responses to some-thing as pleiotropic and stressful as ionizing radiation (IR). Tothat end, it is possible to introduce specific mutations into bothgenomic histone genes, which may aid subsequent geneticanalyses.

Once created, the strains can be analyzed for their sensitivityto DNA DSBs to determine whether they are important forDNA damage responses. On account of the myriad of cellularresponses to DNA damage, any implicated motifs or enzymesmay be important for preventing DNA lesions in the first place,regulating the transcription of DNA damage responsive genesor directly facilitating repair at the site of the lesion.

Biochemical studies

A rapid approach to screen for covalent modifications, suchas phosphorylation, methylation, acetylation, sumoylation orubiquitination, occurring in histones is through the use ofantibodies designed to specifically recognize modified resi-dues. This method is commonly used toward several histonemodifications, but unfortunately, it presents some limitations.Covalent modifications of neighboring, non-target residues caninhibit the recognition by antibodies designed against singlemodifications, thus giving an inaccurate reflection of themodified state of the residue of interest. In addition, cross-recognition of covalently modified residues in similar sequencecontexts can be a problem.

Alternatively, some protein modifications can give rise tomobility shifts during polyacrylamide electrophoresis, whichprovides good methods to identify the presence of conditionsthat induce covalent modifications. The use of specializedelectrophoresis approaches, such as 2D and AUT gels, canhelp to resolve differentially modified species when straight-forward SDS--PAGE is insufficient.

In looking to see whether a modification, residue or motif isrequired for the recruitment of DNA repair, recombination orremodeling proteins, one very powerful tool is the chromatin

immunoprecipitation (ChIP) assay. Following the cross-linkingbetween DNA and proteins associated to it, specific proteinscan be immunoprecipitated and the complexes to it bound canbe analyzed. ChIP assays can also be used to determine thetiming and location of histone modifications at DNA lesions byusing antibodies specific to histone modifications.

Generating double strand breaks

The approaches described above are in widespread use in thegene regulation field, and are easily adapted to studies of DNArepair and recombination. However, unlike the studies of adefined promoter or open reading frame, DNA repair andrecombination can occur anywhere in the genome, and sosome special considerations must be made. For example, inorder to perform ChIP assays, the site of the DNA DSB mustbe defined. The mechanisms of generating DNA DSBs areoutlined below.

DNA damaging agents

A rapid approach for generating DNA damage is the use ofmutagenic chemicals or radiation (Table I). These can, inaddition to generating random damage throughout the genomicDNA, also damage various cellular components, which canmake interpretation of phenotypic assays complicated. Never-theless, one major advantage of using mutagenic chemicals orradiation is that they can be applied to any organism, withoutthe need for specialized vectors, expression constructs ortechnology.

DNA DSBs can be created by a variety of agents, includingIR, and radiomimetic chemicals, such as phleomycin. DSBsmay also arise from replication past single-strand breaks(SSBs), processing of other DNA lesions, nuclease activity ortorsional strain. Thus, using a combination of chemicals andcell-cycle analysis can provide great insights into the effects ofmutations on DNA repair and signaling pathways.

Two commonly used methods of creating DNA damagespecifically during S phase are type I topoisomerase inhibitorsand ribonucleotide reductase inhibitors. Type I topoisomerasescan be very effectively inhibited by camptothecin (CPT) or itsanalogs irinotecan and topotecan, and this results in defectiveDNA replication, leading to fork arrest and ultimately S-phasespecific DSBs (10). Hydroxyurea interacts with the free radicalin ribonucleotide reductase, the enzyme responsible for theformation of dNTPs. This leads to lowered dNTP pools, andtherefore, the generation of DNA DSBs specifically in S phaseowing to the collapse of the replication fork (11).

Alkylating agents are electrophiles that add methyl (MMS),ethyl (EMS) and more complicated alkyl groups to nucleic acidbases. Interestingly, N-methyl-N0-nitro-N-nitrosoguanidine(MNNG) in vivo becomes a highly reactive methylatingagent. Nitrogen and sulfur mustards link bases on oppositeDNA strands, creating intra-strand cross-links. N-Methyl-N-nitrosourea is a potent mutagen and carcinogen that reactsdirectly with DNA producing methylated bases. These adductsproduce GC to AT transitions after two rounds of replication.While none of the lesions created as a direct result of exposureto these alkylating chemicals is a DNA DSB, processing of thelesions, particularly MMS and MNNG-induced DNA alkyla-tion, appears to result in the generation of significant levels ofDNA DSBs (11).

Ultraviolet (UV) radiation mainly causes dimerization of therings of adjacent thymines. DSB induction might arise after

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UV treatment during excision repair of closely opposed lesionsor replication through a partially processed lesion (11).

IR, such as X rays or g rays, has been used extensively toinduce DSBs, although DSBs are only a minor component ofthe radiation-induced damage. IR damages DNA either directlyor indirectly through active oxygen species, causing a spec-trum of DNA lesions: SSBs, DSBs, DNA--protein crosslinksand base substitutions. However, studies in mammalian cellsindicate that the DSBs are the most relevant lesions withrespect to biological effects (11), making IR and IR-mimeticchemicals one of the most useful tools in studying DNA DSBresponses.

In summary, most DNA damaging agents have pleiotropiceffects in the cell and with few exceptions, the exact mechan-isms of action, as well as the relative importance of each of thespectrum of changes caused, are not completely clear. There-fore a thorough understanding of the role of a protein in DNArepair might be facilitated by the use of multiple DNA dam-aging agents, ideally also in combination with other approachesfor creating DNA DSBs (described below).

The homothalic endonuclease

The homothalic (HO) endonuclease creates a deliberate singleDSB in the yeast genome at the MAT locus, which is the initialstep in the process of mating type conversion. This DSB isnormally repaired by Rad52-dependent HR with either of thesilent mating loci, HML or HMR, as the source of homologoussequence.

Not surprisingly, this has become a very powerful andwidely used tool to study DNA DSB repair in S.cerevisiae.Sensitivity of a mutant strain to HO endonuclease expressiondirectly implicates DNA DSB responses, as, unlike chemicalexposure, there is no other cellular damage caused. The moststraightforward approach to inducing HO cleavage is by indu-cing HO expression in plate assays. In these assays, the HOendonuclease is under the control of a galactose induciblepromoter and strains are grown on galactose-containing(inducing) plates or glucose-containing (inhibiting) plates.

The consequence of this is continual expression of the HOendonuclease, and the cells are, therefore, constantly exposedto the generation of DSBs, allowing surival under these condi-tions to be a reflection of DNA DSB repair abilities.

To physically monitor the recombination repair of a singleround of HO-induced DSBs, members of the Haber laboratorydeveloped a methodology named in vivo biochemistry [(12)and references therein]. In this methodology, mid-log phaseliquid cultures are exposed to a brief (1 h) pulse of HO endo-nuclease expression; cells can then be harvested at varyingtime points post-expression. The process of mating type con-version can be monitored by Southern blotting, and the orderof association of HR proteins can be analyzed in conjunctionusing ChIP analyses.

In addition to using the HO endonuclease to study DSBrepair by HR, induction of the endonuclease in a strain lackingthe HML or HMR donor regions can be used to assess DNADSB repair by NHEJ. Moreover, the HO recognition andcleavage sequence can be placed in other regions of the gen-ome that have or do not have regions of homology elsewherein the genome in order to probe more precisely the influencesof variables, such as chromatin structure or transcriptionalactivity on DNA repair activities.

The I-SceI endonuclease

I-SceI is a budding yeast mitochondrial endonuclease, whichrecognizes an 18 bp consensus sequences with high specificity.Therefore, it constitutes a powerful tool that has been widelyused in cells from many organisms, including mammals, tostudy DNA DSB repair in vivo. As with the HO endonuclease,it is possible to introduce artificial I-SceI sites at differentchromatin contexts, induce the endonuclease expression froma suitable vector and monitor the chain of events following theintroduction of a localized DSB (13--15).

The EcoRI endonuclease

The EcoRI restriction endonuclease has been adapted foruse in S.cerevisiae (16). Because of the much smaller 6 bp

Table I. DNA damaging agents

Agent Mechanism/damage Reference

Camptothecin/irinotecan/topotecan Topoisomerase inhibitor/DSB at replication fork 10Hydroxy urea or histidine dNTP synthesis inhibitor/DSB due to collapse of replication fork 11Bleomycin/phleomycin Bind to DNA through their N-terminal peptides, and the activated

complex generates free radicals that are responsible for scissionof the DNA chain (DSBs)

11

Ethanemethyl sulfonate Alkylating agent that generates a large number of nitrogen adducts;can generate base substitutions GC to AT and AT to GC

80

N-Methyl-N-nitrosourea Alkylating agent. DNA methylation. Produces GC to AT transitions 11Mitomycin C Bifunctional alkylating agent that can crosslink DNA and is a

potent clastogen80

Methyl methane sulfonate Alkylating agent that causes protein damage, oxidative stress andDNA damage. MMS indirectly causes single-strand damage at lowconcentrations and DSB at high concentrations. Can produce GC to ATtransitions. Most biologically important lesion is the DNA DSB

8081

MNNG Alkylating agent that after processing can result in DSB 11Cis-platin (CDDP) Intra-strand DNA crosslinks. Widely used as cancer chemotherapeutic agent 11Ionizing radiation, such as

X rays or g raysSSBs, DSBs, DNA--protein crosslinks and base substitutions. Most biologically

important lesion is the DSB11

Ultraviolet radiation Base dimers under the processing can result in SSBs or DSBs. Mainlyrepaired by NER

11

4-NQO Inter-strand photoproducts (UV mimetic) 11Reactive oxygen species Produces a vast range of damage, which result in base substitutions,

repaired therefore by BER. Can, however, in some cases form DSBs


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recognition sequence, by using this enzyme, multiple DSBscan be simultaneously generated in the yeast genome, whichmay be more reflective of exposure to higher doses of DNAdamage, and allow the study of cellular responses to numerousDNA lesions. However, unlike HO and I-SceI, EcoRI has a lowcutting frequency and consequently, only ~5% of the cells willhave a cut at a defined chromosome location. This makesassays that measure events at a defined location, such asChIPs and Southern blotting, more problematic since themajority of cells in the population have no DNA lesion at agiven location.

Laser scissors

The laser scissors technique is used to generate DSBs in adefined nuclear domain, which can then be monitored throughoptic techniques to assess the modification or movement offactors around the sites of damage. It makes use of a finelycontrolled laser-dissecting microscope, where UVA-mediatedexcitation of a dye incorporated into the DNA of livingcells introduces DNA DSBs along the path of the laser(17--19). This technique is frequently applied to the observationof DSBs in mammalian cells. However, it is less suitable foruse in lower eukaryotes such as budding yeast, owing to theirminute size.

Most DSB-generating approaches can be used in combina-tion with the monitoring of foci formation, which is the recruit-ment to or modification of factors at the site of a break. Fociare monitored through immunofluorescence studies, and canprovide good clues to the kinetics of factors acting duringthe repair of a lesion.

Unlike EcoRI, I-SceI and HO-induced DSBs, which can beused for both microscopy studies as well as survival assays andmore sensitive biochemical approaches, the laser scissorsapproach can be used only in conjunction with immunofluor-escence. Obviously, in order to be detected in these assays,multiple proteins (or their modifications) need to be amassed ina small region. Only those histone modifications or modifyingenzymes that work directly at the site of the DNA DSB willbe detected. Moreover, responses to damage by a smallnumber of proteins or protein modifications can be missed inall immunofluorescence-based approaches, including laserscissors.

Plasmid repair assay

The plasmid repair assay consists of enzymatically cleaving aplasmid in vitro and introducing this defined linear DNAtemplate into yeast cells. The number of colonies formedwith respect to cells transformed with equivalent amounts ofundigested plasmid DNA gives an indication of the cells torecircularize the plasmid, which is performed by the DNA DSBmachinery (20). By using plasmids that share no significanthomology to the yeast genome in the region of the introducedDNA DSB, this method provides a good indication of DNArepair by NHEJ. Unfortunately, since it is unclear to datewhether the linear plasmid introduced into the host is chromat-inized at the time of repair, caution should be taken whenextrapolating the results obtained in this assay to those expec-ted to take place in a chromatin environment. Nevertheless, atleast some strains lacking histone motifs or histone modifyingenzymes that have defects in the plasmid repair assay are alsosensitive to DNA damaging agents (21--24), suggesting thatthe assay may be physiologically relevant.

H2A

The H2A family

In addition to the histone H2A subfamily that makes up thebulk of mammalian H2A (H2A1 and H2A2, encoded by 11different genes), there are a number of other histone H2Avariants in the human genome, including, but not limited to,H2AX and H2AZ, each encoded by a single gene (25). H2AXrepresents between 2 and 25% of the total cellular H2A,whereas H2AZ appears to account for ~10% (25).

Interestingly, the sequence motifs that define H2AX andH2AZ as separate variants from the bulk H2A species arefound in many eukaryotic organisms, suggestive of a conservedrole. H2AX variants contain a conserved SQ(E/D) motif in theC-terminal tail, and interestingly, this motif appears to besomewhat mobile in evolution. For example, in Drosophila,the histone H2A variant, H2Av, is clearly a member of theH2AZ family, but contains an SQ motif at its C-terminus.Moreover, in S.cerevisiae, the two genes that encode the bulkof cellular H2A, HTA1 and HTA2, also have the SQE motif atthe C-terminus of the protein in common with mammalianH2AX molecules.

Both H2AZ and H2AX have been implicated in DNA dam-age responses (26--28), although H2AZ appears to also beimportant for aspects of gene regulation (25). However, noknown covalent modifications of histone H2AZ have beenreported in conjunction with this activity, so we will focusexclusively on the role of H2AX (and H2A proteins containingX tail motifs) in DNA DSB responses.

H2AX phosphorylation

As mentioned above, H2AX variants possess a highly conservedserine residue, located four amino acids from the C-terminusfollowed by a glutamine, and this motif also exists on the majorreplication linked H2A species in many lower eukaryotes. TheSQ motif of H2AX is rapidly phosphorylated upon DNA dam-age (a modification commonly referred to as g-H2AX) at sitesof DNA DSBs, suggestive of a direct role in the detection,signaling or repair of the lesion itself [(29) and referencestherein]. The presence of phosphorylated H2AX in mammaliancells is now extensively used as a measure of DNA DSBs.However, caution should be used in this interpretation, asseveral recent reports indicate that H2AX phosphorylationcan also occur in response to cellular events that do not resultin DNA DSBs (30,31).

It has subsequently been shown that the C-terminal SQmotifs in numerous eukaryotic organisms are phosphorylatedin response to DNA damage, including S.cerevisiae, S.pombe,Xenopus laevis and Drosophila melanogaster (21,32--36). Inthese studies, phosphorylation was investigated after inductionof DNA damage, but consistent with the recent mammalianH2AX findings, at least one case of H2A phosphorylation ofthe SQ motif has been demonstrated in the absence of DNAlesions (37). Although it has not yet been investigated in greatdepth, phosphorylation of this motif may occur in responseto a variety of cellular stresses, including, but not limited to,DNA DSBs.

The SQ motif found in these histone variants is, in fact, avery good consensus site for the DNA damage dependent PIKKfamily of kinases described earlier (38). Not surprisingly, itwas found that the DNA-damage dependent phosphorylationwas being carried out by the PIKK homologs Mec1 and Tel1 inbudding yeast (21,32), and by ATM, ATR and DNA-PK in


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mammalian cells (18,39,40). There is some evidence from thehigher eukaryotes that the different PIKKs may play slightlydifferent roles in phosphorylating H2AX. For example, Stiffet al. (41) have shown that ATM and DNA-PK functionredundantly to phosphorylated H2AXS139 after exposure to IR(in human, mouse and chicken cells). However, ATM appearsto be the dominant kinase at least in the early periods of post-irradiation. In contrast, ATR was shown to be important forH2AXS139 phosphorylation induced by UV (42).

H2AX phosphorylation and genome stability

The fact that H2A and H2AX phosphorylation of the SQ motifis so highly conserved is strongly suggestive of a central role inDNA damage responses. Consistent with this hypothesis, yeaststrains lacking this motif are sensitive to DNA damagingagents such as phleomycin, MMS and CPT (21,32). Interest-ingly, however, they are not sensitive to other types of DNAdamage, such as UV irradiation or EMS (21). These datasuggest that the SQ motif may be important only for the repairof DNA DSBs. Genetic analyses of the H2A SQ motif withknown DNA DSB repair genes suggest that the H2A SQ motifmay impinge on both HR and NHEJ activities (21). Notably,however, the DNA damage sensitivity of a strain lacking theSQ motif is drastically less than that of a strain lacking genesrequired for either NHEJ or HR. This suggests that appropriateDNA DSB repair responses in yeast are facilitated by, but notdependent on, the H2A SQ motif.

To determine the physiological role of H2AX in mammaliancells, two groups (28,43) produced targeted disruptions ofmouse H2AX (H2AX�=�). Consistent with results obtainedin yeast, H2AX�=� deficient embryonic stem cells were hyper-sensitive to IR; but H2AX was not essential for survival.H2AX�=� mice were, however, growth retarded, and embryofibroblasts from them proliferated poorly in vitro owing to pre-mature senescence. Interestingly, these phenotypes resembledthe ones of mice deficient for Ku80, Ku70 or ATM. As with Kuor ATM deficient murine fibroblasts, H2AX�=� cells exhibitedelevated levels of both spontaneous- and IR-induced genomicinstability. The absence of H2AX impaired DNA repaircaused by IR, probably accounting for an increased radia-tion sensitivity of H2AX�=� mice (28,43). Examining lym-phoid development in H2AX�=� mice, no severe impedimentin V(D)J recombination, which is NHEJ dependent, wasobserved suggesting that H2AX is not required for NHEJ inthat particular context (28). By contrast, male H2AX�=� micewere infertile, as spermatocytes arrested in the pachytene stageof meiosis I and underwent apoptosis, suggestive of a defectin HR (43).

These two groups further investigated the role of H2AX inmammalian DNA DSB responses by examining the pheno-types of the offsprings of H2AX�=1 and p53�=1 mice(27,44). While H2AX�=� mice exhibited only a modest pre-disposition to lymphomas, either haploid or diploid mutationsin H2AX in combination with the absence of the tumor sup-pressor p53, severely predisposed mice to various forms ofcancer. H2AX�=1/p53�=� and H2AX�=�/p53�=� exhibiteddramatic genomic instability, which led to clonal transloca-tions. The finding that haploid insufficiency also results inincreased genomic instability and cancer incidence in theabsence of p53 is particularly intriguing, and indicates thatH2AX expression levels are crucial for its functions. Interest-ingly, human H2AX maps to a genomic region that exhibits‘loss of homology’ in a large number of human cancers.

This region has been proposed to possess an unidentifiedtumor suppressor gene, making H2AX an excellent candidate.

Through the analysis of tumors from H2AX�=1 p53�=� andH2AX�=� p53�=� mice, Celeste et al. and Bassing et al.(27,44) showed that H2AX is required for normal processingof G1 phase DSBs in the context of V(D)J recombination,demonstrating a role for H2AX in NHEJ. Taken together,H2AX, like the H2A SQ motif in yeast, may impinge on bothmajor DNA DSB repair pathways.

H2AX phosphorylation and foci formation

Several factors known to be involved in DNA repair and sig-naling the presence of damage have been shown to accumulatein large nuclear domains (foci) after DNA double-strandbreakage. This response is not yet fully understood but hasbeen suggested to be a visual indication of DNA repair centres.Interestingly, several studies have demonstrated that H2AXS139

phosphorylation is important for foci formation under a vastrange of conditions where DSBs are formed (45).

Following H2AXS139 phosphorylation, the product of theBRCA1 tumor suppressor gene and later, the Rad50 andRad51 repair factors colocalize with phospho-H2AX foci (46).DNA DSBs generated by either a laser scissors apparatus or a137Cs source of IR, show that the initial pattern of phospho-H2AX molecules formed in the nucleus corresponded to Brca1,Rad50 and Rad51 IR-induced foci (IRIF), which appearsubsequently in the recovery. Interference with H2AX phos-phorylation by the use of the PIKK inhibitor wortmannin or theuse of a kinase defective cell line (DNA-PK absent and ATM atextremely low levels), inhibits the initiation of focus formation(46), proving further evidence for the role of H2AX phos-phorylation in the process.

H2AX works with mediator of DNA damage checkpointprotein 1 (MDC1) to promote the recruitment of repair proteinsto the sites of DNA breaks, besides controlling the damage-induced cell-cycle arrest checkpoints (47,48). H2AX is alsoimportant for both MDC1 and 53BP1 damage-induced phos-phorylation; and peptides representing the C-tail of H2AXspecifically recruit MDC1 and 53BP1 proteins in aphosphorylation-dependent manner. Interestingly, depletingcells of MDC1 protein by siRNA, significantly affectedH2AX phosphorylation and phospho-H2AX foci formation,after exposure to both IR and UV (47). Therefore, in responseto DNA damage, MDC1 and H2AX appear to form a complexat sites of DNA lesions and are phosphorylated in a mutuallydependent fashion.

Surprisingly, H2AX is actually dispensable for the initialrecruitment of DNA repair factors to sites of DSB (17).H2AX is important, however, for the retention and the sub-sequent increase in the concentration of repair factors at sitesof DNA damage, which can be visualized as IRIF. Althoughthe physiological function of IRIF is not clear, the concentra-tion of DNA DSB repair and signaling factors in the vicinityof a DSB is thought to facilitate DNA repair and amplify thedamage induced checkpoint signal.

H2A and checkpoints

The PIKK family of kinases is vital to instigating a signaltransduction cascade resulting in the arrest of progressionthrough the cell cycle as well as transcriptional upregulationof a subset of genes. Therefore, the DNA damage sensitivitydetected in strains lacking either the SQ motif or the entireH2AX gene may be the result of impaired signal transduction


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responses. The transcription of well characterized DNA dam-age responsive genes was examined in yeast lacking the H2ASQ motif and was found to be normal (21), suggesting that thisfunction is not affected. Moreover, experiments where buddingyeast lacking a phosphorylatable H2AS129, were treated withthe topoisomerase inhibitor CPT, suggested that H2AS129 phos-phorylation is not involved in the activation of the intra-Scheckpoint but rather in the efficiency of DNA repair (32).H2AS129 phosphorylation has a central and important functionin the S-phase repair of DNA lesions that do not activate theintra S-phase checkpoint (32). Similarly, no detectable G2/Mcheckpoint defect of S.cerevisiae H2AS129stop mutants exposedto MMS was detected (21). Taken together, these data suggestthat the PIKK-dependent signal transduction cascade is unim-paired in the absence of the H2A SQ motif. However, it wasrecently reported that H2A phosphorylation in fission yeast isimportant for prolonged checkpoint arrest in response to IR andbleomycin (34).

Consistent with the results obtained in budding yeast, the IR-induced G1/S and G2/M checkpoint functions in mammaliancells lacking H2AX were found to be intact after high doses.Interestingly, however, at lower doses of IR, H2AX wasrequired for G2 arrest (49). It is possible that the differentresults obtained are due to evolutionary differences betweenthe organisms tested, different mutations in the SQ motif ordifferent assay conditions. In any event, it will be of greatinterest to further explore the potential role of the H2A SQmotif in checkpoint responses.

Chromatin modulation

There are no data available to indicate whether H2AX phos-phorylation will directly impinge on chromatin structure. Nev-ertheless, there is evidence to suggest that chromatin is alteredin vivo in a manner dependent on H2AX. For example, H2AXis required for chromatin condensation and transcriptionalsilencing of the sex chromosomes during spermatogenesis(50). In addition, changing the phosphorylated serine to aglutamine residue in budding yeast results not only in wild-typelevels of survival in the presence of DNA damage but also inless condensed chromatin (21).

Interestingly, phosphorylated yeast H2AS129 but not unphos-phorylated H2A interacts specifically with Arp4, a proteinpresent in the NuA4, SwrC and Ino80 chromatin modifyingcomplexes (51), raising the possibility that H2A phosphoryla-tion may indirectly affect chromatin structure by recruitingproteins which modify the local environment. Indeed, evidenceshows that all three of these complexes are recruited to the sitesof DNA damage in an Arp4-dependent manner (51). Consistentwith these results, two recent reports also found that the Ino80complex is recruited to the sites of DNA damage in a mannerdependent on the phosphorylation status of H2A (52,53),although different conclusions as to the mechanism of recruit-ment were obtained (54).

If different chromatin modifying complexes are recruited todifferent locations depending on the stage of cell cycle, differ-entiation or tissue type, then phosphorylation of H2AX couldmediate both condensation and decondensation of chromatin.This may partially explain the opposing conclusions reachedby studies performed in yeast and mice described above.

Other histone H2A modifications

The phosphorylation of the SQ motif is the most well studiedDNA damage-dependent histone modification. However, there

is evidence that other covalent modifications of histone H2Aproteins will be important for DNA damage responses. First,in addition to H2AX S139 DSB induced phosphorylation,mammalian H2AX is also phosphorylated at S136 upon DSBinduction, although to a lesser extent (25). The significance ofthis, as well as of the responsible kinase is unclear, although areasonable hypothesis would involve the PIKK family ofkinases, as S136 is also followed by a glutamine residue. It isimportant to note that the studies of H2AX knockout mice haveprovided us with enormous insights into the role that the gene isplaying in DNA repair responses, but that they may not bereflective of the exclusive role of H2AX S139 in these events.

Recently, Drosophila H2A has been shown to be phos-phorylated on T119 (55). While the authors did not demon-strate a role for this residue in DNA DSB responses, we haverecently found that mutation of the analogous residue, S122 inS.cerevisiae H2A, results in hypersensitivity to phleomycinand defective sporulation (56). Furthermore, by performing2D gel electrophoresis of steady-state yeast cells labeledwith 32P, Wyatt et al. (24) found that H2A S122 is indeedphosphorylated, along with T126 and S129. Moreover, Wyattet al. found that strains with a threonine to alanine mutation atposition 126 of H2A are hypersensitive to bleomycin.Together, these data suggest that covalent modifications ofother residues in the C-terminal tail of H2A are important forDNA damage responses, and there might be a complex inter-play between the phosphorylation events in their ability tofacilitate survival. Yeast H2A T126 may be the analogous res-idue to the phosphorylated mammalian H2AX S126, although inyeast it is not followed by a glutamine residue, raising thepossibility that it is not a target for the PIKKs. Notably, how-ever, yeast H2A S122 is not a part of the X-type tail andis conserved as a phosphorylatable residue, not only inDrosophila but also throughout eukaryotes, in the majorreplication-linked H2A1 and H2A2 families as well as beingpresent in a number of other H2A variants.

H2B

The N-terminal tail of histone H2B is flexible and protrudes outof the nucleosomes like the other histone N-terminal tails(57,58). In contrast, histone H2B has a C-terminal extensionof similar size to histone H2A, but in structural studies, this‘tail’ forms an a-helix. Nevertheless, the C-terminal a-helixprotrudes from the nucleosome core and is thus accessible forinteractions with DNA, adjacent nucleosomes or regulatoryfactors.

H2B ubiquitination

Histone H2B can be mono-ubiquitinated on the C-terminala-helix at K123; a process dependent on the ubiquitin conju-gating enzymes Rad6 and Bre1 (59--61). This covalent modi-fication regulates H3 methylation and gene silencing in yeast,whereas H3 does not affect H2B ubiquitination (62), demon-strating the intricate interplay between different histone modi-fications. This is the first demonstration of a unidirectional‘trans-tail’ histone modification where a covalent modificationof one histone tail is dependent on a different histone tail (63).

Recent findings indicate that H2B ubiquitination affects H3methylation through the recruitment of the proteosomalATPases Rpt4 and Rpt6 to chromatin (64). Mutations in Rpt6and Rpt4 affect therefore, global levels of dimethylated andtrimethylated H3K4 and H3K79, leading to loss of telomeric


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gene silencing, probably by titrating Sir proteins away fromheterochromatic regions.

Rad6 is important for DNA damage responses and strainswith rad6 mutations are sensitive to UV irradiation. One obvi-ous possibility, therefore, is that H2B ubiquitination is import-ant for mediating DNA repair. In support of this, H2BK123R

mutants are unable to sporulate properly (59). However, unlikerad6 mutant strains, H2BK123R mutant strains are not sensitiveto UV (59).

Intriguingly, the defect in sporulation is not due to aninability to repair DNA DSBs, but due to the inability togenerate them (65). Strains defective in rad6, bre1 orH2BK123 have lower DSB formation during meiosis, yetDSBs ectopically induced by the use of Gal4-Spo11 are normalin these mutant strains. Therefore, the authors speculate thathistone H2B ubiquitination might be important for the recruit-ment and/or stabilization of the DSB forming machineryduring meiosis.

Because the DNA damage is never generated in the firstplace, it is not known whether H2B ubiquitination would alsobe important in mediating DNA repair events after the induc-tion of DSBs during meiosis. It is plausible to speculate that amodification required for a programmed generation of DSBsmight also be involved in facilitating its repair either throughthe rapid recruitments of repair factors to the break site or byanchoring the DNA ends together. Clearly, it will be interestingto elucidate the potential role of H2B ubiquitination and itsdownstream effects on H3 methylation, in DNA damageresponses, discussed in more detail below.

H2B phosphorylation

H2BS14 phosphorylation is associated with chromatin con-densation both in vivo and in vitro. Indeed, Cheung et al. (66)found that H2B is phosphorylated at S14 by the 34 kDaapoptosis-induced H2B kinase (Mst1). Furthermore, a peptidefrom H2B N-tail has the property of self-aggregating whenphosphorylated at S14 (66) and therefore, this modificationcould play a direct role in regulating chromatin condensation,which is a hallmark event during apoptosis. H2BS14 is con-served in chicken, frog, rat, mouse and human (vertebrates) butabsent in fly, worm and yeast. H2BS14 phosphorylation isdetectable 2 h following the induction of the apoptotic process(67), in contrast to H2AXS139 phosphorylation, which isvirtually instant (18).

Interestingly, mammalian H2BS14 is phosphorylated at thesites of DNA DSBs, indicative of a role for H2B as well asH2AX in mediating DNA repair responses at the site of thelesion (67). However, as with H2AXS139 phosphorylation,H2B S14 phosphorylation forms IRIF with a significant delayand at fewer chromatin locations (67). This may indicate thatthe involvement of H2BS14 phosphorylation in DNA damageresponses is a late event, or that this phosphorylation marks asubset of DSBs; e.g. irrepairable DSBs or abnormal chromo-some rearrangements induced by excessive DNA damage.Alternatively, low but physiologically relevant levels ofH2BS14 phosphorylation may be present earlier during theprocess. Consistent with this possibility, H2BS14 phosphoryla-tion was detected as early as 1 min using the laser scissorstechnique instead of IRIF formation. This overlapped well withH2AXS139 phosphorylation kinetics. Therefore, although thedetection of phosphorylated H2BS14 in IRIF is not visible atearly time points, phosphorylation of Ser-14 in H2B occursrapidly at sites of DSBs.

Interestingly, Fernandez-Capetillo et al. (67) found thatH2BS14-P shows a similar staining pattern to that ofH2AXS139-P in mouse spermatocytes, being particularlyenriched in the highly compacted XY chromosome. Thereforeit is possible that phosphorylation of H2BS14 can act in concertwith H2AX phosphorylation to promote appropriate chromatinstructure of the chromosomes in the sex body as well as DNAdamage responses elsewhere in the genome.

H3

In mammalian cells, there is evidence that phosphorylation oftwo residues, S10 and S28 of the histone H3 N-terminal tailmay be important for mediating UV irradiation responses.Utilizing western blotting approaches in combination withantibodies against specific phospho-residues, work fromDong et al. (68) generated a series of articles demonstratingthe importance of mammalian MAPK cascades for the UVB-induced phosphorylation of H3S10 and H3S28. They found thatUVB strongly induced H3 phosphorylation in vivo. Moreover,UVB-induced histone H3S10 phosphorylation was mediatedby ERKs and p38, whereas UVB-induced histone H3S28 phos-phorylation was mediated by MSK1, Erks, p38 and JNKspathways (69,70). Interestingly, there appears to be a couplingbetween mammalian H3S28 phosphorylation and mitotic chro-mosome condensation (70). In contrast, phosphorylation ofH3S10 has been shown to be associated with regions of rela-tively decondensed chromatin in other studies (71). Whetherchanges in condensation occur in response to UV-irradiation,and whether these changes, if any, are mediated by H3 phos-phorylation is not known. Moreover, it is not known whetherthese modifications play any functional role in the repair ofUV-induced DNA lesions.

The role of the budding yeast acetyltransferase Hat1 andhistone H3 in DNA repair was recently investigated, and itwas found that both Hat1 and specific residues in the H3 N-tailplay a role in DNA DSB repair though recombination repair(22). By substituting H3 lysine residues 9, 14, 18, 23 and 27 forarginine in different combinations and observing cell growth inthe presence of MMS, the authors determined that the presenceof a single lysine residue either at position 14 or at position 23was necessary for DNA damage repair at levels close to wildtype. When lysines 14 and 23 were replaced with glutamine(H3K14,23Q), which partially mimics acetylation, they foundthat sensitivity to MMS was decreased, supporting the ideathat acetylation of H3K14 or H3K23 is important for the repairof DNA damage. Combining different H3 lysine mutationswith a hat1 null mutant, they found that triple mutation inlysines 9,18 and 27 (H3K9,18,27R) was sensitive to MMS whenHAT1 was absent. Accordingly, the viability of these mutantstrains was significantly reduced when DSBs were inducedusing EcoRI. Using a plasmid repair assay the authors foundthat hat1D or hat1D H3K9,18,27R had normal NHEJ, whereasH3K14,23R mutants had the plasmid repair capacity reduced to60% of wild-type levels. Inducing the HO endonuclease inplate assays, the authors found a high degree of sensitivity ofH3K14,23R mutants to HO induction, which is indicative of arole of histone H3 lysines 14 and 23 in DNA DSB repair byHR. Furthermore, in this assay the authors found an increasedsensitivity of the H3K14,23R allele to HO induction when com-bined with a hat1D mutant. As expected, the H3K9,18,27R allelein combination with a hat1D mutant was also sensitive toHO induction. Taking advantage of ‘in vivo biochemistry’


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methodologies similar results were obtained, indicating thatmutations affecting both Hat1 and the acetylable lysineresidues in the histone H3 N-tail compromise the repair ofDSBs by HR.

Although not on the histone tail, methylation of histone H3at lysine 79 has recently been implicated in DNA damageresponses (72). Methylation of this residue provides a bindingsite for interaction with the evolutionarily conserved check-point proteins 53BP1 (human) and Rad9 (S.cerevisiae), andthis is necessary for the recruitment of these proteins to thesites of DNA damage (72). Perplexingly, histone H3 is con-stitutively phosphorylated at K79, which is not consistent witha mechanism for regulated recruitment in response to DNAdamage. The authors, therefore, propose that the methylatedresidue is uncovered at the sites of DNA damage, thus present-ing the docking site for repair proteins. One intriguing possi-bility is that this reorganization is dependent on the chromatinmodifying activities brought to DNA DSBs by H2A phos-phorylation (51--53).

H4

Four lysines, at positions 5, 8, 12 and 16, in the N-terminal tailof histone H4 are reversibly acetylated in vivo in all eukaryotes(23). Interestingly, histone acetyl transferases (HATs) associ-ate physically with the Ku70 DNA repair protein and acetyla-tion of Ku70 influences apoptotic responses [for review, see(73)], which suggests that histone acetylation may be importantin DNA damage responses.

Recently, it was shown that yeast strains carrying mutationsin all H4 N-tail lysines (hhf1-10) have a pronounced defect ingenome integrity (23). The hhf1-10 mutant is markedly hyper-sensitive to both CPT and MMS but not even modestly sensi-tive to UV, suggesting the presence of a defect in DNA DSBrepair, but not a global defect in all DNA repair processes.Interestingly the reintroduction of a single lysine, even atectopic locations, is able to rescue hhf1-10 sensitivity to DSBgenerating agents. This rescue was shown to be due to thelysine acetylation in vivo, demonstrating that lysine acetylationis required, either directly or indirectly, for the correct repair ofDNA DSBs. The HAT responsible for this activity is Esa1, anessential protein whose DSB repair role can be separated fromits essential function (23). Esa1 is a subunit of the NuA4 HATcomplex (74).

In plasmid repair assays, hhf1-10 mutants were defective inNHEJ, and unlike the sensitivity to CPT, mutants containingsingle lysines in any position could not rescue this defect (23).One interpretation of these results is that there are at least twodistinct repair pathways that are defective in hhf1-10 mutantstrains: an NHEJ pathway that requires acetylation of morethan one lysine at the H4 N-tail, which has a minor role incellular resistance to CPT or MMS, and a distinct pathway thatrequires acetylation of any one of the H4 N-tail lysines and isthe principal determinant of DSB repair capacity. As the latterpathway is important in the repair of CPT-induced DSB (at thereplication fork) but not DSB caused by IR (random), thispathway was named replication-coupled pathway. To furtherunderstand the replication-coupled pathway, the authors car-ried out a suppressor screen to identify genes whose over-expression restored CPT resistance to hhf1-10 and identifiedArp4 (23). Interestingly, Arp4 is also a component of the NuA4HAT complex, and thus independent lines of investigationimplicated the NuA4 complex in facilitating DNA repair.

As mentioned above, Arp4 is also a component of thebudding yeast ATP-dependent chromatin remodeling com-plexes Ino80 and SwrC. Interestingly, in higher eukaryotes,including Drosophila and mammals, clear homologs of pro-teins present in these complexes exist and are found in theTIP60 complex. This complex contains both HAT activityand ATP-dependent chromatin remodeling activity. The mam-malian TIP60 histone acetylase complex (which contains manycomponents homologous to the yeast NuA4 complex) wasshown to be involved in DNA repair and apoptosis (75). Byinducing DSBs in wild-type or mutated TIP60 carrying cellsand performing pulse-field gel electrophoresis at various timesafter g-irradiation, the authors demonstrated that the TIP60complex is important for the efficient repair of DNA DSBs.Interestingly, it was recently demonstrated that the DrosophiladTip60 complex is crucial for the optimal exchange of phos-phorylated H2Av/H2B heterodimers from sites of DNA dam-age (76). Moreover, this exchange was more efficient onsubstrates that had a phospho-mimicking glutamic acid residueat the SQ motif of H2Av (76), again linking the phosphorylationstatus of H2AX with these chromatin remodeling activities.

In addition, recent data from fission yeast have suggestedthat Set9 methylation of histone H4 at lysine 20 is important forDNA damage responses (77). As with H3 methylation at lysine79 (72), methylation of H4 K20 creates a binding site for theS.pombe checkpoint protein Crb2 (homolog of budding yeastRad9 and human 53BP1) and is likewise critical for the recruit-ment of this protein to sites of DNA damage (77). Also as in H3K79 methylation, the levels of H4 K20 methylation are notregulated in response to DNA damage, and similar mechanismsof exposing the modified region of the histone after DNAdamage are likely to exist.

Conclusions

There is increasing evidence for a role of chromatin in DNAdamage responses, and studies implicating histone modifica-tions and chromatin modulation to genome stability abound.

The number of histone modifications already implicated inDNA DSB repair is clearly enough to provide significantcomplexity in the histone code, although not all of these willnecessarily be located and act at the site of the DNA lesion.Nevertheless, it is clear that there will be more histone modi-fications with roles in DNA DSB repair uncovered as the fieldprogresses. If one considers that in mammalian cells one singleDSB results in the phosphorylation of H2AXS139 as far as 2 Gbfrom the damaged site, the potential for combinations of modi-fications and recruitment of factors is enormous. Adding to thisis the likelihood that histone modification patterns and kineticswill be influenced by variables such as cell-cycle stage, cellularorigin and chromatin context at the site of a break.

Histone motifs and modifications affecting the cellular re-sistance to DNA damaging agents that do not act directly at thesite of the break are of course still of great physiologicalimportance. These can affect the transcription of DNA repairproteins or global chromatin conformation and therefore itssusceptibility to damage. It will be extremely informative toexamine these roles and their interplay with chromatin activ-ities occurring at the sites of DNA damage.

Ultimately, these studies should have significant impact onthe clinical side of DNA damage responses and genomicinstability, including the treatment of tumors. Chemical inhib-itors of chromatin modulation activities have already been


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successfully used in the treatment of tumors (78), although it isnot known whether these affect DNA DSB repair activities. Inaddition, H2AX phosphorylation levels in IR treated tumorsproved to be a good predictor of tumor sensibility to radiother-apy (79), allowing more precision in the choice of radiotherapydoses. Moreover, the authors found that a peptide mimickingthe H2AX C-terminal tail was capable of antagonizing H2AXfunctions and enhanced cell death in irradiated, radioresistanttumor cells. With the identification of DNA damage-dependentchromatin modifying activities, comes the possibility of newtherapeutic or diagnostic approaches.

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Received on December 21, 2004; revised on March 29, 2005;accepted on March 30, 2005


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