Chromatin in Need of a Fix: Phosphorylation of H2AX Connects Chromatin to DNA Repair
2005; Elsevier BV; Volume: 18; Issue: 6 Linguagem: Inglês
10.1016/j.molcel.2005.05.008
ISSN1097-4164
AutoresChristophe Thiriet, Jeffrey J. Hayes,
Tópico(s)DNA and Nucleic Acid Chemistry
ResumoA bevy of recent reports have firmly established a mechanistic link between a histone posttranslational modification associated with DNA double-strand breaks and recruitment of chromatin-modifying activities. These papers show that in addition to providing signals for transcriptional regulation, specific histone "codes" can coordinate and target multiple activities involved in DNA repair. A bevy of recent reports have firmly established a mechanistic link between a histone posttranslational modification associated with DNA double-strand breaks and recruitment of chromatin-modifying activities. These papers show that in addition to providing signals for transcriptional regulation, specific histone "codes" can coordinate and target multiple activities involved in DNA repair. Only one-half of the mass of eukaryotic chromosomes is genomic DNA; the remaining mass is almost entirely made up of a group of proteins known as histones. In addition to packaging and organizing chromosomes, the histones, which comprise the protein core of the basic repeating subunit of chromatin, the nucleosome, have come to be recognized as a true partner in all nuclear processes involving DNA. Indeed, many nuclear signaling pathways ultimately result in posttranslational modification of the four core histone proteins. These modifications either directly alter the stability with which arrays of nucleosomes fold into compact chromatin fibers and higher-order structures or they can serve as recognition codes directing the binding of transacting factors, which in turn elicit changes in the structure and functionality of the chromatin fiber (Strahl and Allis, 2000Strahl B.D. Allis C.D. Nature. 2000; 403: 41-45Crossref PubMed Scopus (6180) Google Scholar, Hansen, 2002Hansen J.C. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 361-392Crossref PubMed Scopus (403) Google Scholar). Thus, histone posttranslational modifications have been found to fulfill critical functions in virtually all nuclear processes that utilize genomic DNA, including the repair of damaged DNA (Peterson and Cote, 2004Peterson C.L. Cote J. Genes Dev. 2004; 18: 602-616Crossref PubMed Scopus (234) Google Scholar). Processes that result in damage to the genetic material and its repair have been the focus of extensive research efforts for nearly half a century. Despite outstanding advances in understanding the many factors involved in multiple distinct repair processes and the checkpoint signals that stall cell cycle progression until the repair is completed (for review see Sancar et al., 2004Sancar A. Lindsey-Boltz L.A. Unsal-Kacmaz K. Linn S. Annu. Rev. Biochem. 2004; 73: 39-85Crossref PubMed Scopus (2328) Google Scholar), a critical question for the field remains: how do eukaryotic cells sense and accommodate DNA repair in a chromatin context? Recent results suggest that, indeed, posttranslational modifications of histone proteins play a central role. For example, 53BP1, a human ortholog of the S. cerevisiae Rad9p, is a protein known to be involved in signaling the occurrence of DNA damage to the cell cycle checkpoint system (Huyen et al., 2004Huyen Y. Zgheib O. Ditullio Jr., R.A. Gorgoulis V.G. Zacharatos P. Petty T.J. Sheston E.A. Mellert H.S. Stavridi E.S. Halazonetis T.D. Nature. 2004; 432: 406-411Crossref PubMed Scopus (682) Google Scholar). Interestingly, 53BP1 has been shown to specifically recognize and bind to histone H3 methylated at K79, and although this modification is not induced upon damage, its availability for binding may be increased by damage-dependent distortions in chromatin structure (Huyen et al., 2004Huyen Y. Zgheib O. Ditullio Jr., R.A. Gorgoulis V.G. Zacharatos P. Petty T.J. Sheston E.A. Mellert H.S. Stavridi E.S. Halazonetis T.D. Nature. 2004; 432: 406-411Crossref PubMed Scopus (682) Google Scholar). Likewise, evidence suggests that Crb2, an S. pombe ortholog of 53BP1, is recruited to sites of DNA damage in chromatin by exposure of a methylated lysine at position 20 in histone H4 (Sanders et al., 2004Sanders S.L. Portoso M. Mata J. Bahler J. Allshire R.C. Kouzarides T. Cell. 2004; 119: 603-614Abstract Full Text Full Text PDF PubMed Scopus (433) Google Scholar). Although direct interactions of both 53BP1 and Crb2 with their respective histone targets need to be confirmed in native chromatin, these examples highlight the potential roles of histone posttranslational modifications in DNA damage responses (for review, see Peterson and Cote, 2004Peterson C.L. Cote J. Genes Dev. 2004; 18: 602-616Crossref PubMed Scopus (234) Google Scholar). Perhaps the clearest example of a direct relationship between the cell's response to DNA damage and histone posttranslational modifications is the phosphorylation of a subset of histone H2A in response to DNA double-strand breaks (DSBs) (Bassing and Alt, 2004Bassing C.H. Alt F.W. DNA Repair (Amst.). 2004; 3: 781-796Crossref PubMed Scopus (238) Google Scholar, Fernandez-Capetillo et al., 2004Fernandez-Capetillo O. Lee A. Nussenzweig M. Nussenzweig A. DNA Repair (Amst.). 2004; 3: 959-967Crossref PubMed Scopus (759) Google Scholar). Such breaks can arise from mistakes during DNA replication, from external agents such as ionizing radiation, or during genomic rearrangements in immune cells. If left unrepaired, DSBs could result in the loss of entire centromere-distal chromosomal regions or deleterious chromosomal rearrangements. Such genetic alterations can be disastrous for the organism and form the basis of many types of cancer and other diseases. Cells generally employ two mechanisms to repair the break and rejoin the ends: nonhomologous end joining (NHEJ), used at any cell cycle stage, and homologous recombination (HR), which is more specific to S/G2 phase (Fernandez-Capetillo et al., 2004Fernandez-Capetillo O. Lee A. Nussenzweig M. Nussenzweig A. DNA Repair (Amst.). 2004; 3: 959-967Crossref PubMed Scopus (759) Google Scholar). Although the former may result in the loss of genetic information, HR generally uses information contained in the sister chromatid or in some cases the homologous chromosome as a template to restore the chromosome to its original state. As originally shown by Bonner and colleagues, upon formation of DSBs, a subset of the core histone H2A is phosphorylated near the C terminus of the protein in nucleosomes in the vicinity of the break point (Redon et al., 2002Redon C. Pilch D. Rogakou E. Sedelnikova O. Newrock K. Bonner W. Curr. Opin. Genet. Dev. 2002; 12: 162-169Crossref PubMed Scopus (586) Google Scholar and references therein). This modification occurs specifically on a variant of H2A in most species, including humans, known as H2AX, which is defined by a four residue C-terminal sequence (S-Q-E/D-L/Y). Phosphorylation occurs on the serine four residues from the C terminus to generate a phosphorylated form known as γ-H2AX (Redon et al., 2002Redon C. Pilch D. Rogakou E. Sedelnikova O. Newrock K. Bonner W. Curr. Opin. Genet. Dev. 2002; 12: 162-169Crossref PubMed Scopus (586) Google Scholar). In mammals, this variant constitutes ∼10% of the total H2A; in yeast, however, H2AX constitutes ∼90% of the total H2A. Most H2As, including H2AX, are unique in having a C-terminal "tail" domain in addition to the N-terminal tail domain characteristic of all core histones. This tail projects out toward the "front" of the nucleosome, toward the dyad, and contacts the linker DNA entering and exiting the nucleosome. The site of phosphorylation in γ-H2AX lies at the end of the C-terminal tail domain and thus is expected to be relatively accessible to diffusible factors, depending on the position of this tail within the compacted chromatin fiber (Hansen, 2002Hansen J.C. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 361-392Crossref PubMed Scopus (403) Google Scholar and references therein). Indeed, the finding that only about 10% of H2AX is actually phosphorylated in foci arising from damage (Redon et al., 2002Redon C. Pilch D. Rogakou E. Sedelnikova O. Newrock K. Bonner W. Curr. Opin. Genet. Dev. 2002; 12: 162-169Crossref PubMed Scopus (586) Google Scholar) may be related to the relative orientation and accessibility of this tail domain within higher-order chromatin structures. However, it is important to note that the availability of the H2AX C-terminal phosphorylation site in higher-order chromatin structures has not been determined, raising the possibility that damage-dependent changes in chromatin structure may be required for formation of γ-H2AX. Formation of γ-H2AX occurs rapidly after damage and appears to be primarily due to ataxia telangiectasia-mutated (ATM) and ATR (ATM-related) kinases in mammalian cells (or the Tel1/Mec1 kinases in yeast), both of which localize to the site of DNA DSBs minutes after damage (Bassing and Alt, 2004Bassing C.H. Alt F.W. DNA Repair (Amst.). 2004; 3: 781-796Crossref PubMed Scopus (238) Google Scholar). Although not absolutely required for repair, loss of γ-H2AX reduces the cell's ability to cope with DSBs. Formation of γ-H2AX in the vicinity of the DSB triggers the accumulation of many components involved in DNA repair, generating "nuclear foci," including proteins involved in cell cycle checkpoint activation (Fernandez-Capetillo et al., 2004Fernandez-Capetillo O. Lee A. Nussenzweig M. Nussenzweig A. DNA Repair (Amst.). 2004; 3: 959-967Crossref PubMed Scopus (759) Google Scholar). Indeed, evidence for a direct interaction between γ-H2AX and several factors involved in DSB repair and checkpoint signaling has been provided (Bassing and Alt, 2004Bassing C.H. Alt F.W. DNA Repair (Amst.). 2004; 3: 781-796Crossref PubMed Scopus (238) Google Scholar, Fernandez-Capetillo et al., 2004Fernandez-Capetillo O. Lee A. Nussenzweig M. Nussenzweig A. DNA Repair (Amst.). 2004; 3: 959-967Crossref PubMed Scopus (759) Google Scholar, Peterson and Cote, 2004Peterson C.L. Cote J. Genes Dev. 2004; 18: 602-616Crossref PubMed Scopus (234) Google Scholar). For example, NBS1, a component of the MRN complex involved in the initial sensing of DSBs and activation of ATM during S/G2 phase (see below), directly binds γ-H2AX via its FHA/BRCT (forkhead-associated/BRCA1 C-terminal) domain and may contribute to tethering broken chromosomal ends together, preventing aberrant translocations (Bassing and Alt, 2004Bassing C.H. Alt F.W. DNA Repair (Amst.). 2004; 3: 781-796Crossref PubMed Scopus (238) Google Scholar). Other repair proteins, including MCD1/NFBD1 and 53BP1, also harbor FHA/BRCT domains and appear to specifically interact with the phosphorylated C terminus of H2AX in repair foci (references can be found in Bassing and Alt, 2004Bassing C.H. Alt F.W. DNA Repair (Amst.). 2004; 3: 781-796Crossref PubMed Scopus (238) Google Scholar and Peterson and Cote, 2004Peterson C.L. Cote J. Genes Dev. 2004; 18: 602-616Crossref PubMed Scopus (234) Google Scholar). Given the large domain of γ-H2AX formation and its apparent role in recruitment of factors directly involved in DNA repair, one might assume that γ-H2AX may direct the binding of factors involved in chromatin remodeling to facilitate repair. However, a mechanistic connection between γ-H2AX formation and chromatin-modifying activities had been lacking until the recent barrage of papers. A common theme to nearly all these papers is the utilization of a model DSB system in yeast cells to investigate factors localizing to the vicinity of the break point. In these cells, the HO endonuclease normally cuts the yeast chromosome near the MAT locus to initiate a recombination event, leading to a switch in mating type. This ensures a supply of opposite mating types, which fuse to produce diploid cells. The HO system has been coopted by yeast geneticists to study repair of DSBs; in cells in which the DNA sequences normally used for homologous recombination in mating type switching have been deleted, artificial induction of HO results in a persistent DSB in a large fraction of the cells. By using the inducible HO system, several groups demonstrated that that yeast H2A (referred to as H2AX in this review for consistency with higher organisms) is rapidly phosphorylated at serine 129 in the immediate vicinity of the DSB (Morrison et al., 2004Morrison A.J. Highland J. Krogan N.J. Arbel-Eden A. Greenblatt J.F. Haber J.E. Shen X. Cell. 2004; 119: 767-775Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, Shroff et al., 2004Shroff R. Arbel-Eden A. Pilch D. Ira G. Bonner W.M. Petrini J.H. Haber J.E. Lichten M. Curr. Biol. 2004; 14: 1703-1711Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar, Unal et al., 2004Unal E. Arbel-Eden A. Sattler U. Shroff R. Lichten M. Haber J.E. Koshland D. Mol. Cell. 2004; 16: 991-1002Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar), consistent with previous studies in mammalian and other cell types showing accumulation of phosphorylated H2AX in the vicinity of DSB breaks (Redon et al., 2002Redon C. Pilch D. Rogakou E. Sedelnikova O. Newrock K. Bonner W. Curr. Opin. Genet. Dev. 2002; 12: 162-169Crossref PubMed Scopus (586) Google Scholar). A broader examination by Shroff et al., 2004Shroff R. Arbel-Eden A. Pilch D. Ira G. Bonner W.M. Petrini J.H. Haber J.E. Lichten M. Curr. Biol. 2004; 14: 1703-1711Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar shows that this phosphorylation extends ∼25 kb to either side of the DSB but is curiously absent from sequences immediately adjacent (1–2 kb) to the break. The absence of detectable γ-H2AX in immediate proximity to the DSB might be caused by the loss of nucleosomes or a steric impediment to epitope detection due to the recruitment of other repair factors to the DSB (see above). To investigate a possible connection between damage-induced H2AX phosphorylation and repair, Downs et al., 2004Downs J.A. Allard S. Jobin-Robitaille O. Javaheri A. Auger A. Bouchard N. Kron S.J. Jackson S.P. Cote J. Mol. Cell. 2004; 16: 979-990Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar fractionated yeast whole-cell extracts to find proteins that specifically bound to a phosphorylated peptide containing the relevant portion of H2A, but not to an unphosphorylated peptide. A subsequent mass spec analysis turned up the protein Eaf3, a member of the NuA4 histone acetyltransferase (HAT) complex, which contains the catalytic subunit Esa1p. This complex and the Esa1 HAT activity were previously known to play a role in DSB repair (see references within Downs et al., 2004Downs J.A. Allard S. Jobin-Robitaille O. Javaheri A. Auger A. Bouchard N. Kron S.J. Jackson S.P. Cote J. Mol. Cell. 2004; 16: 979-990Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Binding experiments with individual components of the NuA4 complex pointed to the actin-related protein Arp4 as providing specificity for binding the phosphorylated (γ-H2AX) peptide, a result substantiated by the observation that NuA4 binding is lost in an Arp4 mutant (Downs et al., 2004Downs J.A. Allard S. Jobin-Robitaille O. Javaheri A. Auger A. Bouchard N. Kron S.J. Jackson S.P. Cote J. Mol. Cell. 2004; 16: 979-990Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Interestingly, HAT activity copurified with phosphopeptide bound complexes in an Arp4-dependent manner. Combining the HO-mediated DSB system with chromatin immunoprecipitation experiments to identify factors bound to chromatin in the vicinity of the DSB, Downs et al. show that NuA4 accumulates in the vicinity of DSB up to 10 kb away. Arp4p is also a component of the INO80 and SWR1 chromatin remodeling complexes involved in transcriptional regulation and replication-independent assembly of the yeast histone variant H2AZ (Htz1) into chromatin, respectively (Shen et al., 2003Shen X. Ranallo R. Choi E. Wu C. Mol. Cell. 2003; 12: 147-155Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, Mizuguchi et al., 2004Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (928) Google Scholar). Genetic evidence indicates that both of these complexes are involved in DNA repair and that a functional interaction exists between Htz1 and NuA4 activities in DSB repair (Downs et al., 2004Downs J.A. Allard S. Jobin-Robitaille O. Javaheri A. Auger A. Bouchard N. Kron S.J. Jackson S.P. Cote J. Mol. Cell. 2004; 16: 979-990Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar and references therein). Indeed, as discussed below, the INO80 complex and possibly the SWR1 complex appear to be targeted to chromatin in the vicinity of the DSB, although factors other than Arp4p may play a more primary role in targeting of these complexes to γ-H2AX (Downs et al., 2004Downs J.A. Allard S. Jobin-Robitaille O. Javaheri A. Auger A. Bouchard N. Kron S.J. Jackson S.P. Cote J. Mol. Cell. 2004; 16: 979-990Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar, Morrison et al., 2004Morrison A.J. Highland J. Krogan N.J. Arbel-Eden A. Greenblatt J.F. Haber J.E. Shen X. Cell. 2004; 119: 767-775Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, van Attikum et al., 2004van Attikum H. Fritsch O. Hohn B. Gasser S.M. Cell. 2004; 119: 777-788Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). Taking another tack, the Shen and Gasser groups (Morrison et al., 2004Morrison A.J. Highland J. Krogan N.J. Arbel-Eden A. Greenblatt J.F. Haber J.E. Shen X. Cell. 2004; 119: 767-775Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, van Attikum et al., 2004van Attikum H. Fritsch O. Hohn B. Gasser S.M. Cell. 2004; 119: 777-788Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar) began by investigating the molecular basis for the role of the yeast INO80 complex in DNA repair. This complex contains helicases exhibiting sequence homology to RuvB, a protein involved in DNA recombination and repair in bacteria, and as mentioned above, cells harboring mutations in INO80 subunits exhibit sensitivity to multiple DNA damage agents, including ionizing radiation (Shen et al., 2003Shen X. Ranallo R. Choi E. Wu C. Mol. Cell. 2003; 12: 147-155Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). Both groups provide evidence that this sensitivity does not arise from a defect in the transcriptional response to damage, as one might suspect from a mutation in a classical chromatin remodeling complex. Rather, by using the HO endonuclease system, they find, similar to the NuA4 complex, the INO80 complex is directly recruited to the vicinity of the DSB by γ-H2AX, as also observed by Downs et al., 2004Downs J.A. Allard S. Jobin-Robitaille O. Javaheri A. Auger A. Bouchard N. Kron S.J. Jackson S.P. Cote J. Mol. Cell. 2004; 16: 979-990Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar. However, there appears to be a small temporal lag before INO80 recruitment; although γ-H2AX can be detected within 15 min of induction of the HO endonuclease, the INO80 complex is not detected until 30 min after induction (Morrison et al., 2004Morrison A.J. Highland J. Krogan N.J. Arbel-Eden A. Greenblatt J.F. Haber J.E. Shen X. Cell. 2004; 119: 767-775Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar, van Attikum et al., 2004van Attikum H. Fritsch O. Hohn B. Gasser S.M. Cell. 2004; 119: 777-788Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). Although all core histones cofractionate with the INO80 complex, γ-H2AX association is dramatically increased after treatment of the yeast cells with a DNA damage agent (Morrison et al., 2004Morrison A.J. Highland J. Krogan N.J. Arbel-Eden A. Greenblatt J.F. Haber J.E. Shen X. Cell. 2004; 119: 767-775Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). Interestingly, despite the fact that this complex contains an Arp4 subunit, recruitment of INO80 appears to be due to a specific interaction between γ-H2AX and the Nhp10p subunit within the INO80 complex. Indeed, Morrison et al. show that loss of the actin, Arp4p, Arp5p, or Arp8p subunits from the complex did not appear to affect the amount of γ-H2AX associated with INO80 in the pull-downs, whereas loss of the Nhp10 subunit (and the les3 subunit) resulted in a reduction of γ-H2AX coimmunoprecipitation. Morrison et al. substantiate the idea that Nhp10p mediates a specific interaction with γ-H2AX and the INO80 complex by showing that recruitment of Ino80p to the site of DSB is compromised in Nhp10 mutant cells. Of note, despite the fact that Nhp10p contains an HMG box, a domain usually associated with binding DNA, DNA does not seem to be required for the γ-H2AX interaction. Moreover, although loss of Nhp10p does not reduce the chromatin remodeling activity of the INO80 complex (Shen et al., 2003Shen X. Ranallo R. Choi E. Wu C. Mol. Cell. 2003; 12: 147-155Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar), deletion of NHP10 exhibits genetic interactions with components of the RAD52 pathway, suggesting that repair-specific functions of INO80 have been abrogated and require recruitment of this complex via the Nhp10p subunit (Morrison et al., 2004Morrison A.J. Highland J. Krogan N.J. Arbel-Eden A. Greenblatt J.F. Haber J.E. Shen X. Cell. 2004; 119: 767-775Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). What is the actual function of the INO80 complex once recruited? In an attempt to shed light on this question, van Attikum et al. monitored a subsequent step in processing of the DSB, generation of single-stranded DNA ends at the break. Interestingly, they found inefficient conversion of double-stranded to single-stranded DNA at the DSB in cells harboring mutations in the INO80 subunit Arp8 or a nonphosphorylatable H2AX (van Attikum et al., 2004van Attikum H. Fritsch O. Hohn B. Gasser S.M. Cell. 2004; 119: 777-788Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar). This result, combined with the observations that loss of the Arp8 subunit severely compromises chromatin remodeling activity of the INO80 complex (Shen et al., 2003Shen X. Ranallo R. Choi E. Wu C. Mol. Cell. 2003; 12: 147-155Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) and that recruitment of the INO80 to the DSB was only slightly affected by the arp8 mutant, points to the chromatin remodeling activity of the INO80 complex as being required for generation of the single-stranded DNA. However, the helicase activity of Rvb1/2 in the Ino80-Rvb1/2 subcomplex has not been investigated and cannot be ruled out as playing a role in facilitating subsequent processing of the DSB. The Arp4 homolog in mammals, Baf53, is present in the Tip60, SWI-SNF, and other ATP-dependent chromatin remodeling complexes, suggesting that these complexes might also be targeted to DSBs in higher organisms. Indeed, DSBs accumulate upon inactivation of the human TIP60 complex (Kusch et al., 2004Kusch T. Florens L. Macdonald W.H. Swanson S.K. Glaser R.L. Yates 3rd, J.R. Abmayr S.M. Washburn M.P. Workman J.L. Science. 2004; 306: 2084-2087Crossref PubMed Scopus (525) Google Scholar). Kusch et al. provide evidence that the Drosophila Tip60 complex (which harbors both NuA4 and SWR1 activities) plays a role in DSB repair by removing phospho-H2Av from chromatin near damage sites in a reaction that involves H2Av acetylation. Note that Drosophila H2Av contains the phosphorylatable H2AX C terminus fused to the H2AZ globular and N-terminal regions, demonstrating that H2AX function can be associated with the major H2A (yeast), a minor H2A component that resembles the major H2A (mammals), or the distinct, conserved H2AZ variant (Drosophila). These workers purified the dTip60 complex, which contains all 16 subunits found in the human complex, including the Tip60 HAT and the Domino ATPase, the ortholog of the yeast Swr1p ATPase. Moreover, mass spec analysis showed that the TAP-tag-purified dTIP60 complex copurified with peptides from the H2Av and H2B, but not other histones. To examine the potential activity of the complex in vitro, Kusch et al. assembled H2Av nucleosomes containing a serine to glutamate substitution to mimic "H2AX" phosphorylation at position 137 in this protein. Surprisingly, they find that the dTip60 complex induces an ATP- and acetyl CoA-dependent transfer of H2Av-FLAG into nucleosomes when arrays contained H2AvS137E, but not when the arrays contained "unphosphorylated" H2Av. H2AvS137E within the arrays was also found to be the preferential target of acetylation by the Tip60 HAT within the complex, depositing the acetyl group predominantly at K5 within H2Av. Thus, the dTip60 complex exhibits specificity similar to that of the INO80 and NuA4 complexes. Kusch et al. also provide evidence that recruitment of the dTip60 complex to DSBs results in acetylation of phospho-H2Av and removal of this protein from chromatin in vivo. Specifically, when irradiated Drosophila S2 cells are treated with RNAi to knock down Tip60 levels, they observe a delay in removal of H2Av phosphorylation and a loss of transient acetylation of H2Av, presumably due to a reduction in the exchange activity of the Tip60 complex and not due to an overall delay in lesion repair. Although the ability of Tip60 to effect histone exchange remains to be demonstrated in vivo, these data point to an interesting role for Tip60 and related complexes in DSB repair—to remove the phosphorylated H2Av (and γ-H2AX in humans) from chromatin when it is no longer required, suggesting the continued presence of this signal might hinder progression of the repair process. Note that, in addition to histone exchange, these data do not rule out the possibility that the Tip60 complex mobilizes nucleosomes or generates remodeled, noncanonical nucleosome structures in the vicinity of the DSB. Moreover, the phosphorylated H2Av/H2B dimer evicted from chromatin may play a role in signaling subsequent steps in the repair process and suggests the existence of a phosphatase specific for phosphorylated H2Av in the free-dimer form. Given that HR repair of DSBs usually requires the interaction of sister chromatids, it is perhaps not surprising that large scaffolding molecules known to hold sisters together, known as cohesins, play a critical role in DSB repair (Sjogren and Nasmyth, 2001Sjogren C. Nasmyth K. Curr. Biol. 2001; 11: 991-995Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar). Indeed, compromised cohesin function sensitizes cells to ionizing radiation, and cohesin is recruited to sites of DNA damage (Strom et al., 2004Strom L. Lindroos H.B. Shirahige K. Sjogren C. Mol. Cell. 2004; 16: 1003-1015Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, Unal et al., 2004Unal E. Arbel-Eden A. Sattler U. Shroff R. Lichten M. Haber J.E. Koshland D. Mol. Cell. 2004; 16: 991-1002Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). However, as with the work described above, a beautiful mechanistic connection between repair and cohesin recruitment has now been provided. By using the yeast HO-endonuclease system coupled with ChIP assays, Unal et al. and Strom et al. demonstrate that cohesin is loaded in the vicinity of the DSB, extending about 50 kb to either side of the break (Strom et al., 2004Strom L. Lindroos H.B. Shirahige K. Sjogren C. Mol. Cell. 2004; 16: 1003-1015Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, Unal et al., 2004Unal E. Arbel-Eden A. Sattler U. Shroff R. Lichten M. Haber J.E. Koshland D. Mol. Cell. 2004; 16: 991-1002Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). Such loading is detectable about 45 min after DSB induction, suggesting that loading depends on more immediate responses to generation of the break; indeed, phosphorylation of H2AX by the Mec1/Tel1 kinases was found to be required for cohesin loading. By using conditional mutants, these authors demonstrate that factors involved in loading cohesin during S phase are also required for cohesin accumulation at DSBs outside of S, indicating that cohesin is loaded de novo in response to such damage, rather than accumulation being due to rearrangement of S phase-loaded cohesins. These results imply that S phase cohesin, loaded every 10–15 kb along the genome during replication is not sufficient to allow for efficient repair of DSBs, suggesting that greater local cohesion of sister chromatids is required or perhaps the complex plays some other role in repair (Strom et al., 2004Strom L. Lindroos H.B. Shirahige K. Sjogren C. Mol. Cell. 2004; 16: 1003-1015Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar). To address this question, Strom et al. used an elegant combination of temperature-sensitive cohesin mutants and an inducible expression system to show that cohesins loaded in response to DSBs outside of S phase do indeed provide chromatid cohesion, supporting the idea that holding chromatids closely together greatly facilitates DSB repair by HR. Indeed, direct evidence that γ-H2AX plays a key role in sister-chromatid HR has recently been provided (Xie et al., 2004Xie A. Puget N. Shim I. Odate S. Jarzyna I. Bassing C.H. Alt F.W. Scully R. Mol. Cell. 2004; 16: 1017-1025Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Xie et al. used an elegant assay in mouse ES cells to assay HR repair between sister chromatids in H2AX+/+, +/−, and −/− cells, showing that a 4-fold decline in HR was detected in cells devoid of H2AX. Interestingly, incorporation of vectors expressing H2AX into H2AX−/− cells demonstrated that a the phosphorylatable serine at position 139 is required for optimal HR and that replacement of the serine with aspartic or glutamic acid, potentially mimicking phosphorylated serine, as observed in the experiments of Kusch et al., 2004Kusch T. Florens L. Macdonald W.H. Swanson S.K. Glaser R.L. Yates 3rd, J.R. Abmayr S.M. Washburn M.P. Workman J.L. Science. 2004; 306: 2084-2087Crossref PubMed Scopus (525) Google Scholar (see above), did not support efficient repair. Further, molecular analysis of the genomic repair targets indicated that H2AX phosphorylation in mammalian cells can direct utilization of a homologous recombination pathway for repair of DSB, whereas, in the absence of phosphorylatable H2AX, a more error-prone single-strand annealing pathway is employed (Xie et al., 2004Xie A. Puget N. Shim I. Odate S. Jarzyna I. Bassing C.H. Alt F.W. Scully R. Mol. Cell. 2004; 16: 1017-1025Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Although the exact mechanism by which γ-H2AX stimulates HR is unclear, these authors suggest that γ-H2AX functions in processes "parallel" to actual enzymology of the recombination reaction, perhaps by facilitating productive interaction between sister chromatids (Xie et al., 2004Xie A. Puget N. Shim I. Odate S. Jarzyna I. Bassing C.H. Alt F.W. Scully R. Mol. Cell. 2004; 16: 1017-1025Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). This supposition is strongly supported by the finding that γ-H2AX spreading is a perquisite for cohesion loading in yeast cells (Strom et al., 2004Strom L. Lindroos H.B. Shirahige K. Sjogren C. Mol. Cell. 2004; 16: 1003-1015Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, Unal et al., 2004Unal E. Arbel-Eden A. Sattler U. Shroff R. Lichten M. Haber J.E. Koshland D. Mol. Cell. 2004; 16: 991-1002Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar) and suggests a chromatin-based mechanism of alignment. These studies highlight the complex succession of steps and paths leading to repair of DSBs and a central role of γ-H2AX in coordinating the recruitment of critical activities. As depicted in Figure 1, γ-H2AX appears very early in response to a DSB, likely as a result of the activities the ATM/ATR (Mec1/Tel1 in yeast) kinases but can be phosphorylated by other activities, including the DNA-stimulated protein kinase catalytic subunit (DNA-PKcs), which is recruited to the DSB by the Ku70/80 DNA end binding complex (for review see Bassing and Alt, 2004Bassing C.H. Alt F.W. DNA Repair (Amst.). 2004; 3: 781-796Crossref PubMed Scopus (238) Google Scholar). The relative contribution of each of these kinases to γ-H2AX formation varies according to cell cycle stage and damage type (Bassing and Alt, 2004Bassing C.H. Alt F.W. DNA Repair (Amst.). 2004; 3: 781-796Crossref PubMed Scopus (238) Google Scholar). Clearly, although the Ku70-80-DNA-PK and the Mre11-Rad50-NBS1 (MRN) complexes likely play key roles in the detection of the DSB and recruitment of ATM/ATR (Mec1/Tel1) kinases, the exact mechanisms for DSB detection and activation of the ATM/ATR kinases remain to be completely elucidated and may involve overlapping functions of the NHEJ and HR activities (Bassing and Alt, 2004Bassing C.H. Alt F.W. DNA Repair (Amst.). 2004; 3: 781-796Crossref PubMed Scopus (238) Google Scholar). After initial phosphorylation in the vicinity of the DSB, the γ-H2AX domain ultimately spreads to ±50 kb from the DSB and marks the chromatin for subsequent recruitment of activities that elicit cell cycle checkpoint responses, remodel nucleosomes, and acetylate histones and provide for sister chromosome cohesion and alignment (Strom et al., 2004Strom L. Lindroos H.B. Shirahige K. Sjogren C. Mol. Cell. 2004; 16: 1003-1015Abstract Full Text Full Text PDF PubMed Scopus (390) Google Scholar, Unal et al., 2004Unal E. Arbel-Eden A. Sattler U. Shroff R. Lichten M. Haber J.E. Koshland D. Mol. Cell. 2004; 16: 991-1002Abstract Full Text Full Text PDF PubMed Scopus (441) Google Scholar). Interestingly, the first chromatin remodeling activity known to be recruited by γ-H2AX is the NuA4 HAT, perhaps via a direct interaction between the Arp4 subunit in this complex and γ-H2AX. After a short lag period, γ-H2AX then apparently directs the recruitment of the INO80 complex and perhaps the SWR1 complex, which contain ATPase subunits and are capable of catalyzing nucleosome translocation or histone exchange, respectively. The lag between recruitment of the NuA4 complex and the INO80-SWR1 complexes, combined with data showing a reduction in the ability to ChIP Rvb1p in esa1 mutant cells, suggests that NuA4 acetylation of H4 may help recruit INO80-SWR1 (Downs et al., 2004Downs J.A. Allard S. Jobin-Robitaille O. Javaheri A. Auger A. Bouchard N. Kron S.J. Jackson S.P. Cote J. Mol. Cell. 2004; 16: 979-990Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). This would perhaps account for the fact that although both the NuA4 and INO80 complexes contain Arp4, Morrison et al., 2004Morrison A.J. Highland J. Krogan N.J. Arbel-Eden A. Greenblatt J.F. Haber J.E. Shen X. Cell. 2004; 119: 767-775Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar provide evidence that the Nhp10 subunit of INO80 is actually responsible for specific association with γ-H2AX. Thus, both Arp4p and Nhp10p may play important roles in binding chromatin containing γ-H2AX in the NuA4 and INO80 complexes, respectively, whereas recruitment of NuA4 HAT activity is required for subsequent binding of INO80 (Downs et al., 2004Downs J.A. Allard S. Jobin-Robitaille O. Javaheri A. Auger A. Bouchard N. Kron S.J. Jackson S.P. Cote J. Mol. Cell. 2004; 16: 979-990Abstract Full Text Full Text PDF PubMed Scopus (450) Google Scholar). Moreover, targeted acetylation by the NuA4 complex may stimulate SWR1 recruitment via the (acetylated lysine binding) bromodomain subunit Bdf1 within this complex. As demonstrated by van Attikum et al., 2004van Attikum H. Fritsch O. Hohn B. Gasser S.M. Cell. 2004; 119: 777-788Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, remodeling by the INO80 complex may facilitate formation of nucleosome-free, single-stranded regions in preparation for HR. Although γ-H2AX appears to be absent from the regions 1–2 kb immediately adjacent to the DSB, diffusion of INO80 from adjacent regions may be sufficient for this purpose. The γ-H2AX domain also facilitates loading of cohesin around the DSB, possibly to provide for juxtaposition of the sister chromatid for HR. Although the cohesin domain and the γ-H2AX domain span similar territories and loading of cohesin depends on γ-H2AX, it is at present unclear how γ-H2AX directs binding of cohesin or the cohesin loading machinery. It also is not clear whether the apparent exclusion of γ-H2AX from the region around the DSB is related to nucleosome eviction, epitope masking by recruited repair factors or due to the intentional recruitment of chromatin-based activities to regions distal to the break, or how γ-H2AX spreads along the chromosome or what limits the spreading. It is known that both NuA4 acetylation and γ-H2AX phosphorylation are transient, suggesting that subsequent steps in repair require resetting the chromatin structure to the naïve state. Alternatively, replacement of γ-H2AX could result from targeted nucleosome disruption during repair. Although the role of Tip60 complex in catalyzing histone exchange in vivo remains to be verified, the results of Kusch et al. suggest an elegant mechanism for a process whereby γ-H2AX (phospho-H2Av) directs the binding, acetylation, and histone exchange activity of the Tip60 complex in Drosophila cells. It also remains to be determined if ATP-dependent histone exchange occurs in yeast cells, but it is noteworthy that such exchange involving the yeast H2AZ variant has been demonstrated for the SWR1 complex (Mizuguchi et al., 2004Mizuguchi G. Shen X. Landry J. Wu W.H. Sen S. Wu C. Science. 2004; 303: 343-348Crossref PubMed Scopus (928) Google Scholar). Perhaps exchange of γ-H2AX is mediated by a related complex in this organism. It is important to note that the yeast studies highlighted in this review all employed an induced endogenous HO endonuclease system and specific genomic deletions of homologous sequences to allow generation of a persistent DSB in a large fraction of the cells. A question then remains as to whether the factors observed to be recruited to this break would be recruited in the same fashion and to the same extent to a "natural" DSB undergoing normal repair at a physiological rate. For example, a majority of DSB repair in yeast is complete in ∼1 hr (Sjogren and Nasmyth, 2001Sjogren C. Nasmyth K. Curr. Biol. 2001; 11: 991-995Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar), whereas accumulation of the INO80 complex near the persistent DSB peaks 2–4 hr after induction of the break (van Attikum et al., 2004van Attikum H. Fritsch O. Hohn B. Gasser S.M. Cell. 2004; 119: 777-788Abstract Full Text Full Text PDF PubMed Scopus (484) Google Scholar, Morrison et al., 2004Morrison A.J. Highland J. Krogan N.J. Arbel-Eden A. Greenblatt J.F. Haber J.E. Shen X. Cell. 2004; 119: 767-775Abstract Full Text Full Text PDF PubMed Scopus (451) Google Scholar). In addition, because the precise mode of DNA repair is dependent upon cell cycle phase (see above) and most of the studies employed asynchronously growing populations of cells, some of the observations may be attributable to multiple distinct repair processes. Future work will be required to determine if, for example, NuA4, INO80, and SWR1 recruitment are all required for NHEJ and HR or whether the requirement for each of these activities is dependent on the phase of the cell cycle. Finally, it is important to note that chromatin remodeling complexes may also contribute to alterations in higher-order chromatin structures in preparation for repair. Indeed reorganization of higher order chromatin structures may be related to the rather large chromatin domains observed to contain γ-H2AX and cohesins. The effect of some SIN mutations and histone acetylation on folding of nucleosome arrays into chromatin fibers (Hansen, 2002Hansen J.C. Annu. Rev. Biophys. Biomol. Struct. 2002; 31: 361-392Crossref PubMed Scopus (403) Google Scholar) raises the possibility that the 30 nm chromatin fiber or higher-order structures need to be decondensed to allow proper orientation of the underlying DNA for HR or NHEJ.
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