Báo cáo khoa học: Histone H2A phosphorylation in DNA double-strand break repair pot

These include covalent modifications of histone proteins, ATP-dependent chromatin remodelling, and the exchange of histone variants into and out of chro-matin.. One of the best-characteri

Histone H2A phosphorylation in DNA double-strand break repair

Elinor R Foster and Jessica A Downs

Department of Biochemistry, Cambridge University, UK

DNA repair must, by definition, occur within the

con-text of chromatin The most basic unit of chromatin is

the nucleosome, consisting of two copies of each of

four core histones around which DNA is wrapped in

two left-handed superhelical turns [1] This level of

DNA compaction, often referred to as ‘beads on a

string’, can be folded into numerous higher-order

lev-els of chromatin condensation [2] The shift towards

more condensed structures is facilitated by the

pres-ence of the linker histone [2] This packaging of DNA,

while essential for compressing a very long, highly

neg-atively charged molecule into a relneg-atively small space,

is also inhibitory to processes that require the

mani-pulation of DNA, such as replication, transcription,

and repair It is therefore not surprising that histones

and proteins that modulate chromatin structure are

integral players in these processes

The four core histones that make up the nucleosome

structure are H2A, H2B, H3, and H4 Each of these

proteins contains a histone fold domain, which is

cen-tral to the nucleosome core structure In addition, the

histones all have a flexible N-terminal domain that protrudes from the nucleosome core particle Histones H2A and H2B are unique in having significant sequence on the C-terminal side of the histone fold The C-terminal domain of H2B forms an a-helix, and lies along the side of the nucleosome Interestingly, the H2A C-terminal domain, like the histone N-terminal domains, is partly flexible and protrudes from the nucleosome core (Fig 1) It is located at the point of the nucleosome where the linker DNA enters and exits the structure, and this is the area to which the linker histones bind

There are a number of mechanisms by which chro-matin structure and composition can be manipulated

to facilitate events such as DNA replication and repair These include covalent modifications of histone proteins, ATP-dependent chromatin remodelling, and the exchange of histone variants into and out of chro-matin Notably, these processes are sometimes inter-related, for example, the replacement of H2A with the H2AZ variant (described in more detail below) is

Keywords

chromatin; DNA repair; H2AX; histone H2A

Correspondence

J A Downs, Department of Biochemistry,

Cambridge University, 80 Tennis Court

Road, Cambridge CB2 1GA, UK

Fax: +44 1223 766 002

Tel: +44 1223 333 663

E-mail: jad32@mole.bio.cam.ac.uk

(Received 11 February 2005, revised 4 April

2005, accepted 28 April 2005)

doi:10.1111/j.1742-4658.2005.04741.x

DNA repair must take place within the context of chromatin, and it is therefore not surprising that many aspects of both chromatin components and proteins that modify chromatin have been implicated in this process One of the best-characterized chromatin modification events in DNA-dam-age responses is the phosphorylation of the SQ motif found in histone H2A or the H2AX histone variant in higher eukaryotes This modification

is an early response to the induction of DNA damage, and occurs in a wide range of eukaryotic organisms, suggesting an important conserved func-tion One function that histone modifications can have is to provide a unique binding site for interacting factors Here, we review the proteins and protein complexes that have been identified as H2AS129ph (budding yeast) or H2AXS139ph (human) binding partners and discuss the implica-tions of these interacimplica-tions

Abbreviations

ph, phosphorylation; PIKK, phosphatidylinositol 3-kinase-like kinase.

catalyzed by an ATP-dependent chromatin remodelling

complex Histones are heavily modified by

post-trans-lational modifications in vivo, and the known

modifica-tions include acetylation of lysine residues, methylation

of lysine and arginine residues, ubiquitination and

sumoylation of lysines, and phosphorylation of serine

and threonine residues These modifications can alter

the charge of the histone proteins and affect the ability

to effectively condense DNA, or they can create or

remove binding interfaces for chromatin-associated

proteins (reviewed in [3])

Proteins and protein complexes that use the energy

of ATP hydrolysis to alter chromatin structure are

recognizable by their sequence similarity of the

cata-lytic subunit to the founding member: Swi2⁄ Snf2 The

exact consequence of this ATP-dependent alteration is

variable, and can include the exchange of all histones

from one DNA molecule to another, the sliding of the

histone octamer to a new position, alteration of

his-tone–DNA interactions, removal of H2A–H2B dimers,

and exchange of histones in the nucleosome, including

swapping core histones for histone variants [4,5]

The majority of human histone genes are clustered

in the genome, and their transcription is tightly linked

to replication Histone variants are found outside of these gene clusters and transcriptionally regulated in a replication-independent manner The incorporation of histone variants into chromatin can have profound structural and physiological consequences [6] Interest-ingly, the histone H2A family has the largest number

of described variants, including H2AX [6]

Here, we focus on the role of H2A in DNA-damage responses As discussed in more detail below, this phe-nomenon touches on all three mechanisms of chromatin modulation First, the crucial event in H2A DNA-dam-age responses is the phosphorylation of an SQ motif in the C-terminal tail This appears to function, at least

in part, to recruit chromatin remodelling complexes to sites of DNA damage Finally, this motif is present on a histone variant, H2AX, in higher eukaryotes

Phosphorylation of the SQ motif

of histone H2A or H2AX

In higher eukaryotes, the H2AX variants are charac-terized by a longer C-terminal tail that has an SQ motif followed by two additional amino-acid residues before the stop codon (Fig 2) This SQ motif,

Fig 1 The nucleosome core particle from budding yeast [54] seen from two different angles (A and B) The histone proteins are in the centre of the structure, and the DNA is wrapped around the proteins in two left-handed superhelical turns H2A, yellow and green; H2B, H3 and H4, grey The two H2A C-termini of the H2A tails are indicated with arrows and are in the region where the DNA enters and exits the structure The entire C-terminal tails were not solved in the crystal structure; one H2A molecule ends with H2AT126 (yellow) and the other with H2AK121 (green) Consequently, none

of the C-terminal serine residues discussed

in the text (highlighted in Fig 2) are present

in the solved structure.

Fig 2 Alignment of H2A and H2AX C-terminal sequences downstream of the histone fold from the indicated organisms Budding yeast H2AS122 and analogous residues from other organisms are highlighted in green Budding yeast H2AT126 and the H2AX upstream SQ ⁄ TQ motifs, which may or may not be functioning analogously in DNA-damage responses, are highlighted in blue The major DNA-damage phos-phorylation SQE motif present in the budding yeast core H2A (S129), mammalian H2AX (S139), and Drosophila H2Av (S137) is highlighted in red.

H2AXS139, has been shown to be phosphorylated in

response to DNA damage [7] Here, we will use the

recently proposed unifying nomenclature for histone

modifications [8], but note that this phosphorylation

event is often referred to as c-H2AX in the literature

As alluded to previously, this variant is a single-copy

gene that is outside of the histone gene cluster, and its

transcription is regulated differently from that of the

core histone H2A genes [9,10] In lower eukaryotes,

there is an SQ motif present in H2A proteins in the

same position relative to the stop codon (Fig 2), and

this motif is also phosphorylated in response to DNA

damage [11–13] However, the H2A tails are not

exten-ded, and the genes encoding these proteins are linked

to replication and make up the majority of histone

H2A in the cell, like the core H2A genes in higher

euk-aryotes, suggesting that these are more closely related

to core H2A than H2AX variants Moreover, an SQ

motif exists in the same position of the H2AZ variant

in Drosophila (Fig 2) and is also phosphorylated in

response to DNA damage [14] Together, these results

suggest that the phosphorylation of the SQ motif plays

a role in DNA-damage responses regardless of which

histone variant it is found on The invariant

character-istic appears to be the position of the SQ motif relative

to the end of the protein, and it would be interesting

to see whether this is crucial for function in vivo

The SQ motif is a good consensus target site for

the phosphatidylinositol 3-kinase-like kinases (PIKKs),

and members of this family of kinases have been

impli-cated in DNA-damage responses throughout the

euk-aryotes [15] Not surprisingly, it has been shown that

the PIKK family members Mec1 and Tel1 in budding

yeast [11,12,16] and ATM, ATR and DNA-PK in

higher eukaryotes [17–20] are responsible for

phos-phorylation of this motif in response to DNA damage

In the systems studied, the phosphorylation takes

place very rapidly after DNA damage, within minutes

of exposure to c-irradiation [7,12] In mammalian cells,

the phosphorylation is detected in the vicinity of the

DNA lesions by immunofluorescence in combination

with ‘laser scissors’ [17,21], and is present in megabase

chromatin domains [21] By chromatin

immuno-precipitation approaches, it was found that

phosphory-lation of the budding yeast H2A SQ motif (H2AS129)

occurs in cis on the DNA extending from an induced

double-strand break in both directions [16,22] covering

regions of 50–100 kb of chromatin [23] Notably, the

levels of phosphorylation immediately adjacent to the

DNA break are not particularly high [16,22], which

rai-ses the possibility that phosphorylation does not occur

to a great extent on these nucleosomes However, it is

equally plausible that the antibody is not recognizing

the epitope in the nucleosomes immediately adjacent to the break because of occluding proteins or additional H2A tail modifications Indeed, there is evidence that budding yeast H2A residues S122 and T126 are both phosphorylated and important for DNA-damage responses [25,26], making them potential culprits for loss of H2AS129ph recognition by the antibody Never-theless, these results are consistent with a direct role in facilitating repair at the site of the break, in contrast with an indirect role in the appropriate transcriptional regulation of genes necessary for survival after DNA damage [11]

As discussed above, the consequences of covalent modifications of histone proteins such as phosphoryla-tion include a direct structural effect on the ability of chromatin to fold as well as the creation or removal

of binding interfaces for interacting proteins Although there is some evidence that phosphorylation of H2A(X) can affect chromatin structure in vivo [11,27], there are no biochemical data to indicate whether this

is a direct effect In contrast, numerous H2AS129ph and H2AXS139ph binding partners have been reported

in the literature Therefore, we will focus on these potential binding partners and their activities in the DNA-damage response

Proteins that interact with H2AS129ph and H2AXS139ph

NuA4 The NuA4 histone acetyltransferase complex was iden-tified in an effort to identify proteins that bound spe-cifically to the budding yeast H2A tail when the SQ motif was phosphorylated [22] The studies made use

of a synthetic peptide to demonstrate a direct inter-action with NuA4 in vitro Furthermore, NuA4 was found to be directly associated with chromatin in the vicinity of an induced DNA break by chromatin immunoprecipitation assays [22,28]

Ino80 With the use of recombinant subunits of NuA4, the actin-related protein Arp4 was found to directly inter-act with the H2AS129ph peptide This protein is also

a component of the Ino80 and SwrC (see below) ATP-dependent chromatin remodelling complexes Like NuA4, subunits present in the Ino80 complex were also demonstrated to be present at the site of an induced DNA break [22,29,30], but appeared to localize to the area subsequent to the appearance of NuA4 [22] Moreover, this appearance at the DNA

break was impaired in strains with mutations in either

H2A S129 or the Mec1 and Tel1 kinases [29,30]

SwrC

SwrC, the third Arp4-containing complex in budding

yeast, was also found to interact specifically with the

H2AS129ph peptide in vitro, and subunits present in

SwrC were found to localize to the site of an induced

DNA break in vivo [22] Together, these data suggest

that the binding interface created by phosphorylation

of the budding yeast SQ motif is important for the

interaction of all three Arp4-containing complexes at

sites of DNA damage However, as mentioned above,

subunits present in both Ino80 and SwrC were detected

at the site of the DNA break with different kinetics to

NuA4 If the interface between Arp4 and H2AS129ph

was the sole defining recruitment mechanism, the

beha-viour of the three complexes should be comparable As

it is not, these data suggest additional mechanisms for

appropriate recruitment and retention to DNA breaks

At least for Ino80, this may involve the Nhp10 protein,

identified as important for Ino80-binding chromatin

near DNA breaks [30] As the Ino80 complex from an

nhp10 mutant strain contains Arp4 but is no longer

able to bind to soluble H2AS129ph [30], this raises the

possibility that Nhp10 (or Ies3, which is also missing

from the mutant complex) facilitates the interaction

between Arp4 and H2AS129ph

Tip60

In higher eukaryotes, homologues of subunits found in

NuA4, Ino80 and SwrC exist, but do not form three

distinct homologous complexes Instead, homologues

of a subset of these complexes are found together in

the Tip60 complex, which contains both HAT activity

and ATP-dependent chromatin remodelling activity

(reviewed in [31]) In a study from Workman and

col-leagues [32], Tip60 was shown to preferentially

acety-late chromatin in which the SQ-motif-containing

H2Av variant contained a phosphomimetic glutamic

acid residue in place of the serine residue

(H2AvS137E) Once the H2AvS137E-containing

nucleo-somes were acetylated, the Tip60 complex was found

to remodel the chromatin and remove the

‘phosphoryl-ated’ H2Av–H2B dimer and replace it with a fresh

H2Av–H2B dimer [32] These results are consistent

with the order of appearance at a DNA break in

bud-ding yeast of NuA4 first [22], which then acetylates

chromatin in the vicinity [28], and subsequently Ino80

and⁄ or SwrC are detectably present [22]

Cohesin Although there is no evidence for a direct interaction between phosphorylated H2A and cohesin, a recent report demonstrated that cohesin is localized to regions surrounding an induced DNA break in a man-ner that is dependent on the presence of a phosphory-latable H2A SQ motif in budding yeast [23] Like the complexes described above, there is a detectable delay between the appearance of H2AS129ph and the appearance of cohesin Unlike the complexes described above, however, cohesin appears to localize to a much broader region of chromatin, more closely matching the pattern seen with H2AS129ph The wild-type pat-tern of appearance of cohesin over these regions depends not only on H2A phosphorylation, but also

on Mre11 and Rad53 As H2A phosphorylation is nor-mal in the absence of both Mre11 and Rad53 [11,16], this suggests that, even if there is a direct interaction between cohesin and H2AS129ph, there are additional requirements for recruitment and⁄ or retention at the site of DNA damage

The M⁄ R ⁄ N complex The mammalian Mre11⁄ Rad50 ⁄ Nbs1 (M ⁄ R ⁄ N) com-plex (Mre11⁄ Rad50 ⁄ Xrs2 in budding yeast) accumu-lates into foci after exposure to ionizing radiation, and this was shown to be dependent on the phosphoryla-tion of H2AX S139 [17,33] In support of a role for phosphorylation of H2AX in recruitment of this com-plex to sites of DNA damage, a direct interaction between Nbs1 and phosphorylated, but not unphos-phorylated, H2AX was demonstrated in vitro [34] However, a subsequent study demonstrated that the initial appearance of Nbs1 at sites of DNA damage was unaffected by the loss of H2AX [35], suggesting that H2AX phosphorylation is instead specifically important for the subsequent accumulation of this complex Furthermore, the budding yeast Mre11 pro-tein (part of the Mre11⁄ Rad50 ⁄ Xrs2 complex) localizes

to sites of DNA damage normally in the absence of H2A phosphorylation [16] There are a number of possible interpretations of these data One possibility is that the physical interaction between the proteins detected in vitro is not physiologically relevant, and that the accumulation at sites of DNA damage in vivo

is indirectly affected by H2AX phosphorylation-dependent events Alternatively, redundant recruitment mechanisms may exist, and⁄ or the interaction is only important after recruitment for the retention of the proteins

53BP1(Hs)⁄ Rad9(Sc) ⁄ Crb2(Sp)

The mammalian checkpoint protein 53BP1 was found

to bind to H2AXS139ph, but not H2AX [36,37] Like

the M⁄ R ⁄ N complex, 53BP1 foci formation is also

abrogated in the absence of H2AX [33,36,38] The

fis-sion yeast homologue of 53BP1, Crb2, has also been

shown to accumulate at sites of DNA damage, and this

appears to be dependent on the phosphorylation of

H2A [13] The authors also showed a direct interaction

between Crb2 and H2AS129ph in vitro [13] Taken

together, these reports suggest that phosphorylation of

H2A⁄ H2AX recruits 53BP1 (or its homologue) to sites

of damage However, in the study by Celeste et al [35]

described above, the authors also examined 53BP1

behaviour and found that, like Nbs1, the initial

recruit-ment of 53BP1 to sites of DNA damage is normal in

H2AX–⁄ – cells, but that subsequent accumulation is

impaired Again, it is possible that there is no

physiolo-gical role for the direct interaction detected in vitro,

that the interaction is a mechanism for retention, not

recruitment, and⁄ or there are redundant recruitment

mechanisms In support of the existence of redundant

mechanisms, two recent reports suggest that

methyla-tion of H3 K79 in mammals [39] and of H4 K20 in

fission yeast [40] are important for recruitment of

53BP1 and Crb2 recruitment, respectively

Mdc1

The mammalian Mdc1 protein, which has no obvious

homologues in lower eukaryotes, is also able to interact

with H2AXS139ph peptides in vitro [37] Like the

M⁄ R ⁄ N complex and 53BP1, Mdc1 accumulates in

irra-diation-induced foci, and this foci formation is

abro-gated in the absence of H2AX [37] Interestingly, Mdc1

itself is required for the accumulation of the M⁄ R ⁄ N

complex into foci [37,41] Recently, it was shown that

the H2AXS139ph peptide is able to pull-down the

M⁄ R ⁄ N complex from a whole cell extract only when

Mdc1 is present, suggesting that it acts as a bridging

factor and that, at least in these assays, there is no direct

interaction between Nbs1 and H2AXS139ph [42]

Consequences of H2AS129ph ⁄

H2AXS139ph-mediated recruitment

of these protein(s)

Increased malleability of DNA

One obvious consequence of recruiting both HAT

(NuA4, TIP60) and ATP-dependent chromatin

remod-elling activities (Ino80, SwrC, TIP60) is the

reorganiza-tion of chromatin structure at the site of the break To repair a DNA lesion, a number of enzymatic activities are required, including exonuclease, polymerase and ligase activities to name just a few It is conceivable that most, if not all, of the activities required for DNA repair will be inhibited by condensed chromatin struc-tures Indeed, Gasser and colleagues [29] found that the processing of the DNA break is deficient in either H2A S129A or arp8 (an Ino80 subunit) mutant strains Notably, the formation of single-stranded DNA as a DNA repair intermediate is important not only for the repair event, but for the appropriate checkpoint response [43] This may provide an explanation for the checkpoint defects seen under certain circumstances in the absence of H2AS129ph⁄ H2AXS139ph [13,44]

Exposure of other chromatin modifications

In addition to providing a template that is more amen-able to DNA repair protein manipulation, the reorgan-ization of chromatin in the vicinity of a DNA break may expose other chromatin modifications that play a role in DNA-damage responses Specifically, studies in mammalian cells and fission yeast demonstrated a role for methylation events of H3 K79 and H4 K20, respectively [39,40] Intriguingly, both studies found that the methylation events are constitutive and do not appear to change in either quantity or location upon DNA damage Yet, the methylation of these sites appears to be critical for the recruitment of the check-point proteins 53BP1 (mammalian) and Crb2 (fission yeast) to sites of damage in vivo, and the authors of both studies propose that the methylated motifs are

‘uncovered’ in the regions of DNA breaks

One possibility is that recruitment of chromatin-modi-fying activities by H2AS129ph or H2AXS139ph results

in an increased number of exposed methylated motifs after DNA damage If so, the number of Crb2⁄ 53BP1-binding sites would be severely reduced (but probably not abrogated) in the absence of H2A(X) phosphoryla-tion This model would be consistent with the inability,

in the absence of H2AX or the H2A SQ motif, of 53BP1⁄ Crb2 to accumulate into foci [13,36] and to either properly maintain a checkpoint [13] or initiate a check-point in response to very low doses of ionizing radiation where amplification of the signal might be crucial [44]

Facilitation of the removal of phosphorylated H2A(X)

Once the DNA lesion is repaired, it may be deleterious for the cell to maintain phosphorylated H2A or H2AX

in undamaged chromatin For instance, the recruitment

of repair and⁄ or remodelling factors to undamaged

DNA may result in misregulation of gene expression or

the sequestration of repair factors that are needed

else-where Therefore, it seems likely that there is a

phospha-tase that removes the phosphate residue from H2A or

H2AX Another, not mutually exclusive possibility,

however, is that phosphorylated H2A mediates its own

removal from chromatin by recruitment of the SwrC

complex [22] This complex removes H2A–H2B dimers

from nucleosomes and replaces them with Htz1–H2B

di-mers [45] As Htz1 has also been genetically linked to

H2A S129-dependent DNA-damage responses [22], it is

tempting to speculate that SwrC is recruited to sites of

DNA damage and swaps the phospho-H2A for Htz1

Consistent with this is the study in Drosophila, described

above, in which the homologous complex, Tip60,

prefer-entially acts on phospho-mimic-containing nucleosomes

[32] Finally, it is conceivable that the phosphorylated

H2A(X) is targeted for degradation It has recently been

demonstrated in budding yeast that the proteasome is

present at sites of DNA damage, and that this is

neces-sary for appropriate DNA-damage responses [46]

Mediation of sister chromatid cohesion

by loading cohesin

As mentioned above, in budding yeast, cohesin was

found to be associated with DNA after the induction

of a double-strand break in a manner dependent on the

phosphorylation of H2A S129 [23] This recruitment to

damaged DNA appears to be required for appropriate

sister chromatid cohesion and DNA repair [23,24]

Intriguingly, whereas the absence of cohesin results in a

severe inability to survive in the presence of DNA

dam-age, the absence of S129 results in a strain that is only

mildly sensitive to DNA damage relative to the survival

patterns seen with other DNA signalling and repair

protein mutant strains This raises the possibility that,

although cohesin is not detectably present at DNA

breaks by chromatin immunoprecipitation in the

S129A mutant strain, there is in fact enough cohesin

loaded on to the area to facilitate repair This

possibil-ity again highlights the likely existence of redundant,

albeit impaired, recruitment and⁄ or retention

mecha-nisms Alternatively, cohesin may also facilitate the

repair of DNA breaks in a manner that is not

depend-ent on its localization to the sites of damage

Mediation of foci formation⁄ checkpoint

responses

Regardless of whether the M⁄ R ⁄ N complex, 53BP1 ⁄

Rad9⁄ Crb2 or Mdc1 bind directly to the

phosphoryl-ated H2A(X) tail in vivo, it is clear that the accumula-tion of these proteins in DNA-damage-induced foci is dependent on H2A(X) phosphorylation Notably, both the checkpoint defects and the DNA damage sensitiv-ity seen in yeast H2AS129A or mammalian H2AX–⁄ – mutant cells are not as dramatic as the loss of check-point proteins such as Brca1 or ATM [11,12,44] This suggests that the checkpoint proteins may be impaired

in their behaviour, but that they can still function to facilitate DNA-damage responses to a reasonable degree in the absence of H2A(X) phosphorylation

Decreased malleability of chromatin⁄ anchor ends together

After the creation of a double-strand break, it could

be beneficial to ‘lock down’ the two ends by creating a heterochromatic structure over the broken region This would prevent transcription or replication machinery from traversing the site The heterochromatin may also actively facilitate the maintenance of interactions between the two broken ends This would help to pre-vent inappropriate or inefficient repair of the DNA ends

There is some evidence that H2A(X) phosphoryla-tion may help to form such a heterochromatic struc-ture First, condensation of the X and Y chromosomes during male mouse meiosis is defective in H2AX–⁄ – mice [27] In addition, H2B has been shown to be phosphorylated in response to DNA damage (H2BS14ph), and is located in foci with H2AX phos-phorylation [47] However, in the absence of H2AX, H2BS14 phosphorylation does not change by Western blotting, but is no longer visible in foci One interpret-ation of these data proposed by the authors is that, in the absence of H2AX, the broken ends are unable to condense, leaving the H2BS14 phosphorylation signal too diffuse to visualize by immunofluorescence The H2AZ variants have been shown to have altered stability compared with H2A [48], and FRET-based approaches have shown that H2AZ–H2B dimers are more stably attached to the nucleosome than H2A– H2B dimers [49] If phosphorylation of H2A directs its own replacement with Htz1 in nucleosomes, as pro-posed above, it is possible that this results in the cre-ation of a less malleable structure in the region of DNA breaks

Even without the formation of heterochromatic structures, H2AX phosphorylation may help to

‘anchor’ broken ends together By assisting the accu-mulation of 53BP1⁄ Rad9 ⁄ Crb2, and, at least in mam-malian cells, the M⁄ R ⁄ N complex and Mdc1, H2A(X) phosphorylation may assist in the formation of a

pro-tein bridge that prevents the DNA ends from

dissoci-ating [50] In support of this, H2AX-deficient cells

show increased levels of translocation [33,38]

Discussion

A wide range of proteins have been identified as

H2AS129ph or H2AXS139ph binding partners In

many cases, an interaction was demonstrated with the

protein in vitro in addition to evidence pointing to

colocalization in vivo This raises the obvious question

of whether all of these actually bind to H2AS129ph or

H2AXS139ph in vivo

In response to DNA breaks, a great deal of H2A

and H2AX is phosphorylated, and this increases in a

time-dependent and dose-dependent manner [12,16,22]

Numerous potential binding platforms are being

cre-ated, and thus it is reasonable to speculate that a wide

variety of binding partners can be physically

accom-modated Notably, the consequences of H2A or H2AX

phosphorylation listed above are in some cases directly

opposing activities Yet, the very nature of using

cova-lent modifications to create binding interfaces allows

different protein complexes to be relevant under

differ-ent conditions For instance, binding partners

import-ant for the formation of the condensed X and Y

chromosomes [27] may only exist during meiosis or be

otherwise regulated so they do not ‘see’

phosphory-lated H2AX in mitotically dividing cells In addition,

the binding and accumulation of chromatin-modifying

complexes to DNA breaks in yeast was examined in

asynchronous haploid cell cultures It is conceivable

that they are in fact only recruited during a particular

phase of the cell cycle Even the recruitment of

pro-teins that both increase and decrease the malleability

of chromatin at a single double-strand break are not

necessarily mutually exclusive activities, as long as they

can be either physically or temporally separated at the

DNA lesion (discussed in more detail below)

If we are to postulate that all of the factors listed

above are physiologically bound to H2AS129ph or

H2AXS139ph, then one might hypothesize that they

should all show the same timing and location of

bind-ing, which would be dictated directly by the

appear-ance of H2A(X) phosphorylation Clearly, this is not

the case, but one variable that may help to explain this

discrepancy is the existence of other histone

modifica-tions in the vicinity of the break Specifically, in the

yeast H2A tail, two other residues in very close

proxi-mity to S129 (S122 and T126) have been shown to be

phosphorylated in vivo [25], and both of these residues

have been implicated in DNA-damage responses

[25,26] This raises the possibility that there is interplay

between the residues, which dictates additional specific-ity in the associated binding partner For example, H2AS122phS129ph and H2AT126phS129ph may bind

to two mutually exclusive sets of proteins In higher eukaryotes, the analogous residue to budding yeast H2A S122 is phosphorylated on core H2A [51] and the site exists on the H2AX variant as well (Fig 2) More-over, an additional phosphorylation site on H2AX has been identified, which is in the same position as budding yeast T126 relative to the SQ motif (H2AXS136ph; Fig 2) One obvious difference is that the mammalian residue is followed by a glutamine, making it a potential substrate for the PIKK family of kinases, but the yeast residue is not (Fig 2) Neverthe-less, substrates for the PIKK family of kinases that do not conform to the consensus sequence have been identified (for example [52]), so it is possible that bud-ding yeast T126 is a target for Mec1 and⁄ or Tel1 Nev-ertheless, these additional modifications are likely to contribute to the behaviour of the interacting proteins

Of course, modifications of other core histones, such

as H4 acetylation by NuA4, are also likely to be involved in the differential recruitment of factors to DNA lesions Finally, the regulation of the complexes themselves may dictate their differential behaviour at DNA breaks, despite the common existence of their binding platform

Although it is possible to postulate that all of the proteins identified as H2A or H2AX phospho-specific binding partners are physiologically relevant, it is also reasonable to interpret these results more cautiously For example, many of the experiments demonstrating

a direct physical interaction were performed using either synthetic peptides corresponding to the end of the H2A or H2AX tail that were either unmodified or contained a phosphoserine residue in the SQ motif Proteins containing known phosphoserine⁄ threonine binding motifs such as BRCT or FHA domains may bind to some degree to any phosphorylated peptide and be detected when these assays are performed, as the amount of peptide is often in vast excess to the proteins being analyzed In addition, Arp4, the subunit

in NuA4, Ino80 and SwrC implicated in H2AS129ph binding, may have affinity for phosphate moieties as it

is related to the ATP-binding actin protein, although

it did show a preference for the H2AS129ph tail over other phosphoserine-containing peptides [22] In some cases, such as cohesin, no direct interaction was dem-onstrated [23], which leaves open the possibility that there is an indirect dependency on H2A phosphoryla-tion for binding to sites of DNA damage As 53BP1 has been shown to bind both phosphorylated H2AX and methylated H3 in vitro [36,39], it would be

inter-esting to see what the relative affinities for these

sub-strates are

The use of a chromatin template, instead of

syn-thetic peptides, in which H2A or H2AX is

phosphoryl-ated would be extremely informative about how these

proteins interact The use of chromatin templates may

also shed light on an aspect of H2A or H2AX

phos-phorylation that is as yet unclear: the potential direct

effect of phosphorylation on chromatin structure If,

as discussed above, there are multiple phosphorylation

events in the H2A(X) tail after DNA damage, it is

conceivable that this change in charge will affect

higher-order chromatin structure Given the location

of the tail in the vicinity of the linker DNA (Fig 1),

one tempting possibility is that phosphorylation of the

H2A tail may impinge on the behaviour of the linker

histone, which has been shown to be inhibitory to

DNA repair in yeast [53]

Inevitably, additional proteins that can bind to the

phosphorylated SQ motif of H2A and H2AX will be

identified in the future, and it is likely that we will also

find more covalent modifications of histones that

directly impinge on H2A(X) phospho-SQ-dependent

functions Integrating the roles of the known players

and understanding the different conditions and

situa-tions in which they are relevant will be a key step

to elucidating the interplay between chromatin and

chromatin-interacting factors during DNA-damage

responses

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