Replication protein A and gamma-H2AX foci assembly is triggered by cellular response to DNA double-strand breaks

Human replication protein A (RPA p34), a crucial component of diverse DNA excision repair pathways, is implicated in DNA double-strand break (DSB) repair. To evaluate its role in DSB repair, the intranuclear dynamics of RPA was investigated after DNA damage and replication blockage in human cells. Using two different agents [ionizing radiation (IR) and hydroxyurea (HU)] to generate DSBs, we found that RPA relocated into distinct nuclear foci and colocalized with a well-known DSB binding factor, gamma-H2AX, at the sites of DNA damage in a time-dependent manner. Colocalization of RPA and gamma-H2AX foci peaked at 2 h after IR treatment and subsequently declined with increasing postrecovery times. The time course of RPA and gamma-H2AX foci association correlated well with the DSB repair activity detected by a neutral comet assay. A phosphatidylinositol-3 (PI-3) kinase inhibitor, wortmannin, completely abolished both RPA and gamma-H2AX foci formation triggered by IR. Additionally, radiosensitive ataxia telangiectasia (AT) cells harboring mutations in ATM gene product were found to be deficient in RPA and gamma-H2AX colocalization after IR. Transfection of AT cells with ATM cDNA fully restored the association of RPA foci with gamma-H2AX illustrating the requirement of ATM gene product for this process. The exact coincidence of RPA and gamma-H2AX in response to HU specifically in S-phase cells supports their role in DNA replication checkpoint control. Depletion of RPA by small interfering RNA (SiRNA) substantially elevated the frequencies of IR-induced micronuclei (MN) and apoptosis in human cells suggestive of a role for RPA in DSB repair. We propose that RPA in association with gamma-H2AX contributes to both DNA damage checkpoint control and repair in response to strand breaks and stalled replication forks in human cells.     

gamma-H2AX dephosphorylation by protein phosphatase 2A facilitates DNA double-strand break repair

Phosphorylated histone H2AX (gamma-H2AX) forms foci over large chromatin domains surrounding double-stranded DNA breaks (DSB). These foci recruit DSB repair proteins and dissolve during or after repair is completed. How gamma-H2AX is removed from chromatin remains unknown. Here, we show that protein phosphatase 2A (PP2A) is involved in removing gamma-H2AX foci. The PP2A catalytic subunit [PP2A(C)] and gamma-H2AX coimmunoprecipitate and colocalize in DNA damage foci and PP2A dephosphorylates gamma-H2AX in vitro. The recruitment of PP2A(C) to DNA damage foci is H2AX dependent. When PP2A(C) is inhibited or silenced by RNA interference, gamma-H2AX foci persist, DNA repair is inefficient, and cells are hypersensitive to DNA damage. The effect of PP2A on gamma-H2AX levels is independent of ATM, ATR, or DNA-PK activity.     

The structure-specific endonuclease Ercc1-Xpf is required to resolve DNA interstrand cross-link-induced double-strand breaks

Interstrand cross-links (ICLs) are an extremely toxic class of DNA damage incurred during normal metabolism or cancer chemotherapy. ICLs covalently tether both strands of duplex DNA, preventing the strand unwinding that is essential for polymerase access. The mechanism of ICL repair in mammalian cells is poorly understood. However, genetic data implicate the Ercc1-Xpf endonuclease and proteins required for homologous recombination-mediated double-strand break (DSB) repair. To examine the role of Ercc1-Xpf in ICL repair, we monitored the phosphorylation of histone variant H2AX (gamma-H2AX). The phosphoprotein accumulates at DSBs, forming foci that can be detected by immunostaining. Treatment of wild-type cells with mitomycin C (MMC) induced gamma-H2AX foci and increased the amount of DSBs detected by pulsed-field gel electrophoresis. Surprisingly, gamma-H2AX foci were also induced in Ercc1(-/-) cells by MMC treatment. Thus, DSBs occur after cross-link damage via an Ercc1-independent mechanism. Instead, ICL-induced DSB formation required cell cycle progression into S phase, suggesting that DSBs are an intermediate of ICL repair that form during DNA replication. In Ercc1(-/-) cells, MMC-induced gamma-H2AX foci persisted at least 48 h longer than in wild-type cells, demonstrating that Ercc1 is required for the resolution of cross-link-induced DSBs. MMC triggered sister chromatid exchanges in wild-type cells but chromatid fusions in Ercc1(-/-) and Xpf mutant cells, indicating that in their absence, repair of DSBs is prevented. Collectively, these data support a role for Ercc1-Xpf in processing ICL-induced DSBs so that these cytotoxic intermediates can be repaired by homologous recombination.     

A critical role for histone H2AX in recruitment of repair factors to nuclear foci after DNA damage

BACKGROUND: The response of eukaryotic cells to double-strand breaks in genomic DNA includes the sequestration of many factors into nuclear foci. Recently it has been reported that a member of the histone H2A family, H2AX, becomes extensively phosphorylated within 1-3 minutes of DNA damage and forms foci at break sites. RESULTS: In this work, we examine the role of H2AX phosphorylation in focus formation by several repair-related complexes, and investigate what factors may be involved in initiating this response. Using two different methods to create DNA double-strand breaks in human cells, we found that the repair factors Rad50 and Rad51 each colocalized with phosphorylated H2AX (gamma-H2AX) foci after DNA damage. The product of the tumor suppressor gene BRCA1 also colocalized with gamma-H2AX and was recruited to these sites before Rad50 or Rad51. Exposure of cells to the fungal inhibitor wortmannin eliminated focus formation by all repair factors examined, suggesting a role for the phosphoinositide (PI)-3 family of protein kinases in mediating this response. Wortmannin treatment was effective only when it was added early enough to prevent gamma-H2AX formation, indicating that gamma-H2AX is necessary for the recruitment of other factors to the sites of DNA damage. DNA repair-deficient cells exhibit a substantially reduced ability to increase the phosphorylation of H2AX in response to ionizing radiation, consistent with a role for gamma-H2AX in DNA repair. CONCLUSIONS: The pattern of gamma-H2AX foci that is established within a few minutes of DNA damage accounts for the patterns of Rad50, Rad51, and Brca1 foci seen much later during recovery from damage. The evidence presented strongly supports a role for the gamma-H2AX and the PI-3 protein kinase family in focus formation at sites of double-strand breaks and suggests the possibility of a change in chromatin structure accompanying double-strand break repair.     

Distribution and dynamics of chromatin modification induced by a defined DNA double-strand break

BACKGROUND: In response to DNA double-strand breaks (DSBs), eukaryotic cells rapidly phosphorylate histone H2A isoform H2AX at a C-terminal serine (to form gamma-H2AX) and accumulate repair proteins at or near DSBs. To date, these events have been defined primarily at the resolution of light microscopes, and the relationship between gamma-H2AX formation and repair protein recruitment remains to be defined. RESULTS: We report here the first molecular-level characterization of regional chromatin changes that accompany a DSB formed by the HO endonuclease in Saccharomyces cerevisiae. Break induction provoked rapid gamma-H2AX formation and equally rapid recruitment of the Mre11 repair protein. gamma-H2AX formation was efficiently promoted by both Tel1p and Mec1p, the yeast ATM and ATR homologs; in G1-arrested cells, most gamma-H2AX formation was dependent on Tel1 and Mre11. gamma-H2AX formed in a large (ca. 50 kb) region surrounding the DSB. Remarkably, very little gamma-H2AX could be detected in chromatin within 1-2 kb of the break. In contrast, this region contains almost all the Mre11p and other repair proteins that bind as a result of the break. CONCLUSIONS: Both Mec1p and Tel1p can respond to a DSB, with distinct roles for these checkpoint kinases at different phases of the cell cycle. Part of this response involves histone phosphorylation over large chromosomal domains; however, the distinct distributions of gamma-H2AX and repair proteins near DSBs indicate that localization of repair proteins to breaks is not likely to be the main function of this histone modification.     

ATM-dependent DNA damage-independent mitotic phosphorylation of H2AX in normally growing mammalian cells

H2AX is a core histone H2A variant that contains an absolutely conserved serine/glutamine (SQ) motif within an extended carboxy-terminal tail. H2AX phosphorylation at the SQ motif (gamma-H2AX) has been shown to increase dramatically upon exogenously introduced DNA double-strand breaks (DSBs). In this study, we use quantitative in situ approaches to investigate the spatial patterning and cell cycle dynamics of gamma-H2AX in a panel of normally growing (unirradiated) mammalian cell lines and cultures. We provide the first evidence for the existence of two distinct yet highly discernible gamma-H2AX focal populations: a small population of large amorphous foci that colocalize with numerous DNA DSB repair proteins and previously undescribed but much more abundant small foci. These small foci do not recruit proteins involved in DNA DSB repair. Cell cycle analyses reveal unexpected dynamics for gamma-H2AX in unirradiated mammalian cells that include an ATM-dependent phosphorylation that is maximal during M phase. Based upon similarities drawn from other histone posttranslational modifications and previous observations in haplo-insufficient (H2AX-/+) and null mice (H2AX-/-), gamma-H2AX may contribute to the fidelity of the mitotic process, even in the absence of DNA damage, thereby ensuring the faithful transmission of genetic information from one generation to the next.     

Characteristics of gamma-H2AX foci at DNA double-strand breaks sites.

Phosphorylated H2AX (gamma-H2AX) is essential to the efficient recognition and (or) repair of DNA double strand breaks (DSBs), and many molecules, often thousands, of H2AX become rapidly phosphorylated at the site of each nascent DSB. An antibody to gamma-H2AX reveals that this highly amplified process generates nuclear foci. The phosphorylation site is a serine four residues from the C-terminus which has been evolutionarily conserved in organisms from giardia intestinalis to humans. Mice and yeast lacking the conserved serine residue demonstrate a variety of defects in DNA DSB processing. H2AX Delta/Delta mice are smaller, sensitive to ionizing radiation, defective in class switch recombination and spermatogenesis while cells from the mice demonstrate substantially increased numbers of genomic defects. gamma-H2AX foci formation is a sensitive biological dosimeter and presents new and exciting opportunities to understand important biological processes, human diseases, and individual variations in radiation sensitivity. These potentialities demonstrate the importance of understanding the parameters and functions of gamma-H2AX formation.     

Low-dose hyper-radiosensitivity is not caused by a failure to recognize DNA double-strand breaks

One of the earliest cellular responses to radiation-induced DNA damage is the phosphorylation of the histone variant H2AX (gamma-H2AX). gamma-H2AX facilitates the local concentration and focus formation of numerous repair-related proteins within the vicinity of DNA DSBs. Previously, we have shown that low-dose hyper-radiosensitivity (HRS), the excessive sensitivity of mammalian cells to very low doses of ionizing radiation, is a response specific to G(2)-phase cells and is attributed to evasion of an ATM-dependent G(2)-phase cell cycle checkpoint. To further define the mechanism of low-dose hyper-radiosensitivity, we investigated the relationship between the recognition of radiation-induced DNA double-strand breaks as defined by gamma-H2AX staining and the incidence of HRS in three pairs of isogenic cell lines with known differences in radiosensitivity and DNA repair functionality (disparate RAS, ATM or DNA-PKcs status). Marked differences between the six cell lines in cell survival were observed after high-dose exposures (>1 Gy) reflective of the DNA repair capabilities of the individual six cell lines. In contrast, the absence of functional ATM or DNA-PK activity did not affect cell survival outcome below 0.2 Gy, supporting the concept that HRS is a measure of radiation sensitivity in the absence of fully functional repair. No relationship was evident between the initial numbers of DNA DSBs scored immediately after either low- or high-dose radiation exposure with cell survival for any of the cell lines, indicating that the prevalence of HRS is not related to recognition of DNA DSBs. However, residual DNA DSB damage as indicated by the persistence of gamma-H2AX foci 4 h after exposure was significantly correlated with cell survival after exposure to 2 Gy. This observation suggests that the persistence of gamma-H2AX foci could be adopted as a surrogate assay of cellular radiosensitivity to predict clinical radiation responsiveness.     

Gamma-H2AX expression pattern in non-irradiated neonatal mouse germ cells and after low-dose gamma-radiation: relationships between chromatid breaks and DNA double-strand breaks

The DNA double-strand breaks (DSBs) are considered to be the most relevant lesions for the deleterious effects of ionizing radiation exposure. The discovery that the induction of DSBs is rapidly followed by the phosphorylation of H2AX histone at Ser-139, favoring repair protein recruitment or access, opens the possibility for a wide range of research. This phosphorylated histone, named gamma-H2AX, has been shown to form foci in interphase nuclei as well as megabase chromatin domains surrounding the DNA lesion on chromosomes. Using detection of gamma-H2AX on germ cell mitotic chromosomes 2 h after gamma-irradiation, we studied radiation-induced DSBs during the G(2)/M phase of the cell cycle. We show that 1) non-irradiated neonatal germ cells express gamma-H2AX with variable patterns at metaphase, 2) gamma-irradiation induces foci whose number increases in a dose-dependent manner, 3) some foci correspond to visible chromatid breaks or exchanges, 4) sticky chromosomes characterizing cell radiation exposure during mitosis are a consequence of DSBs, and 5) gamma-H2AX remains localized at the sites of the lesions even after end-joining has taken place. This suggests that completion of DSB repair does not necessarily imply disappearance of gamma-H2AX.     

DNA damage-induced RPA focalization is independent of gamma-H2AX and RPA hyper-phosphorylation

Replication protein A (RPA) is the major eukaryotic single stranded DNA binding protein that plays a central role in DNA replication, repair and recombination. Like many DNA repair proteins RPA is heavily phosphorylated (specifically on its 32 kDa subunit) in response to DNA damage. Phosphorylation of many repair proteins has been shown to be important for their recruitment to DNA damage-induced intra-nuclear foci. Further, phosphorylation of H2AX (gamma-H2AX) has been shown to be important for either the recruitment or stable retention of DNA repair proteins to these intra-nuclear foci. We address here the relationship between DNA damage-induced hyper-phosphorylation of RPA and its intra-nuclear focalization, and whether gamma-H2AX is required for RPA's presence at these foci. Using GFP-conjugated RPA, we demonstrate the formation of extraction-resistant RPA foci induced by DNA damage or stalled replication forks. The strong DNA damage-induced RPA foci appear after phosphorylated histone H2AX and Chk1, but earlier than the appearance of hyper-phosphorylated RPA. We demonstrate that while the functions of phosphoinositol-3-kinase-related protein kinases are essential for DNA damage-induced H2AX phosphorylation and RPA hyper-phosphorylation, they are dispensable for the induction of extraction-resistant RPA and RPA foci. Furthermore, in mouse cells genetically devoid of H2AX, DNA damage-induced extraction-resistant RPA appears with the same kinetics as in normal mouse cells. These results demonstrate that neither RPA hyper-phosphorylation nor H2AX are required for the formation in RPA intra-nuclear foci in response to DNA damage/replicational stress and are consistent with a role for RPA as a DNA damage sensor involved in the initial recognition of damaged DNA or blocked replication forks.     

DNA interstrand crosslinks repair in mammalian cells

We studied the formation of double strand breaks (DSBs) as intermediates in the repair of DNA interstrand crosslinks (ICLs) by homologous recombination (HR). The plasmid EGFP-N1 was crosslinked with trioxsalen to give one ICL per plasmid on average. HeLa cells were transfected with the crosslinked plasmids and the ICL repair was monitored by following the restoration of the GFP expression. It was accompanied by gamma-H2AX foci formation suggesting that DSBs were formed during the process. However, the same amount of gamma-H2AX foci was observed when cells were transfected with native plasmid, which indicated that gamma-H2AX foci appearance could not be used to determine the amount of DSBs connected with the ICL repair in this system. For this reason we further monitored the DSB formation by determining the amount of linearized plasmids, since having one crosslink per plasmid on average, any ICL-driven DSB formation would lead to plasmid linearization. Native and crosslinked plasmids were incubated in repair-competent cell-free extracts from G1 and S phase HeLa cells. Although a considerable part of the ICLs was repaired, no linearization of the plasmids was observed in the extracts, which was interpreted that DSBs were not formed as intermediates in the process of ICL repair. In another set of experiments HR-proficient HeLa and HR-deficient irs3 cells were transfected with native and crosslinked plasmids, and 6 h and 12 h later the plasmid DNA was isolated and analyzed by electrophoresis. The same amount of linear plasmid molecules was observed in both cell lines, regardless of whether they were transfected with native or crosslinked pEGFP-N1, which further confirmed that DSB formation was not an obligatory step in the process of ICL repair by HR.     

A PP4-phosphatase complex dephosphorylates gamma-H2AX generated during DNA replication

The histone H2A variant H2AX is rapidly phosphorylated in response to DNA double-stranded breaks to produce gamma-H2AX. gamma-H2AX stabilizes cell-cycle checkpoint proteins and DNA repair factors at the break site. We previously found that the protein phosphatase PP2A is required to resolve gamma-H2AX foci and complete DNA repair after exogenous DNA damage. Here we describe a three-protein PP4 phosphatase complex in mammalian cells, containing PP4C, PP4R2, and PP4R3beta, that specifically dephosphorylates ATR-mediated gamma-H2AX generated during DNA replication. PP4 efficiently dephosphorylates gamma-H2AX within mononucleosomes in vitro and does not directly alter ATR or checkpoint kinase activity, suggesting that PP4 acts directly on gamma-H2AX in cells. When the PP4 complex is silenced, repair of DNA replication-mediated breaks is inefficient, and cells are hypersensitive to DNA replication inhibitors, but not radiomimetic drugs. Therefore, gamma-H2AX elimination at DNA damage foci is required for DNA damage repair, but accomplishing this task involves distinct phosphatases with potentially overlapping roles.     

Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks

Histone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (gamma-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX(-/-) cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that gamma-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.     

DNA double-strand breaks and gamma-H2AX signaling in the testis

Within minutes of the induction of DNA double-strand breaks in somatic cells, histone H2AX becomes phosphorylated at serine 139 and forms gamma-H2AX foci at the sites of damage. These foci then play a role in recruiting DNA repair and damage-response factors and changing chromatin structure to accurately repair the damaged DNA. These gamma-H2AX foci appear in response to irradiation and genotoxic stress and during V(D)J recombination and meiotic recombination. Independent of irradiation, gamma-H2AX occurs in all intermediate and B spermatogonia and in preleptotene to zygotene spermatocytes. Type A spermatogonia and round spermatids do not exhibit gamma-H2AX foci but show homogeneous nuclear gamma-H2AX staining, whereas in pachytene spermatocytes gamma-H2AX is only present in the sex vesicle. In response to ionizing radiation, gamma-H2AX foci are generated in spermatogonia, spermatocytes, and round spermatids. In irradiated spermatogonia, gamma-H2AX interacts with p53, which induces spermatogonial apoptosis. These events are independent of the DNA-dependent protein kinase (DNA-PK). Irradiation-independent nuclear gamma-H2AX staining in leptotene spermatocytes demonstrates a function for gamma-H2AX during meiosis. gamma-H2AX staining in intermediate and B spermatogonia, preleptotene spermatocytes, and sex vesicles and round spermatids, however, indicates that the function of H2AX phosphorylation during spermatogenesis is not restricted to the formation of gamma-H2AX foci at DNA double-strand breaks.     

Dephosphorylation of histone gamma-H2AX during repair of DNA double-strand breaks in mammalian cells and its inhibition by calyculin A

The induction of DNA double-strand breaks (DSBs) by ionizing radiation in mammalian chromosomes leads to the phosphorylation of Ser-139 in the replacement histone H2AX, but the molecular mechanism(s) of the elimination of phosphorylated H2AX (called gamma-H2AX) from chromatin in the course of DSB repair remains unknown. We showed earlier that gamma-H2AX cannot be replaced by exchange with free H2AX, suggesting the direct dephosphorylation of H2AX in chromatin by a protein phosphatase. Here we studied the dynamics of dephosphorylation of gamma-H2AX in vivo and found that more than 50% was dephosphorylated in 3 h, but a significant amount of gamma-H2AX could be detected even 6 h after the induction of DSBs. At this time, a significant fraction of the gamma-H2AX nuclear foci co-localized with the foci of RAD50 protein that did not co-localize with replication sites. However, gamma-H2AX could be detected in some cells treated with methyl methanesulfonate which accumulated RAD18 protein at stalled replication sites. We also found that calyculin A inhibited early elimination of gamma-H2AX and DSB rejoining in vivo and that protein phosphatase 1 was able to remove phosphate groups from gamma-H2AX-containing chromatin in vitro. Our results confirm the tight association between DSBs and gamma-H2AX and the coupling of its in situ dephosphorylation to DSB repair.     

Heterochromatin is refractory to gamma-H2AX modification in yeast and mammals

Double-strand break (DSB) damage in yeast and mammalian cells induces the rapid ATM (ataxia telangiectasia mutated)/ATR (ataxia telangiectasia and Rad3 related)-dependent phosphorylation of histone H2AX (gamma-H2AX). In budding yeast, a single endonuclease-induced DSB triggers gamma-H2AX modification of 50 kb on either side of the DSB. The extent of gamma-H2AX spreading does not depend on the chromosomal sequences. DNA resection after DSB formation causes the slow, progressive loss of gamma-H2AX from single-stranded DNA and, after several hours, the Mec1 (ATR)-dependent spreading of gamma-H2AX to more distant regions. Heterochromatic sequences are only weakly modified upon insertion of a 3-kb silent HMR locus into a gamma-H2AX-covered region. The presence of heterochromatin does not stop the phosphorylation of chromatin more distant from the DSB. In mouse embryo fibroblasts, gamma-H2AX distribution shows that gamma-H2AX foci increase in size as chromatin becomes more accessible. In yeast, we see a high level of constitutive gamma-H2AX in telomere regions in the absence of any exogenous DNA damage, suggesting that yeast chromosome ends are transiently detected as DSBs.     

Quantitative analysis reveals asynchronous and more than DSB-associated histone H2AX phosphorylation after exposure to ionizing radiation

Rapid phosphorylation of histone H2AX after exposure of cells to ionizing radiation occurs at DSB sites and extends to a region including as much as 30 Mbp of chromatin to form visible microscopic structures called gamma-H2AX foci. Although the kinetics of total cellular histone H2AX phosphorylation after irradiation has been characterized, we still know little about the phosphorylation kinetics of individual gamma-H2AX foci. In addition, there are hundreds of smaller gamma-H2AX foci that are not associated with DNA double-strand breaks. We refer to these sites as DSB-unrelated gamma-H2AX foci. By using indirect immunofluorescence microscopy, deconvolution and three-dimensional image analysis, we established an objective method to quantitatively analyze each gamma-H2AX focus as well as to discriminate DSB-related gamma-H2AX foci from DSB-unrelated gamma-H2AX foci. Using this method, we found that histone H2AX phosphorylation at different DSB sites was asynchronous after exposure to ionizing radiation. This may reflect the heterogeneous characteristic of free DNA ends that are generated under these conditions. In addition, we found that increased histone H2AX phosphorylation also occurred outside of DSB sites after exposure to ionizing radiation. The function of this DSB-unassociated phosphorylation is not known.     

DNA polymerase eta reduces the gamma-H2AX response to psoralen interstrand crosslinks in human cells

DNA interstrand crosslinks are processed by multiple mechanisms whose relationships to each other are unclear. Xeroderma pigmentosum-variant (XP-V) cells lacking DNA polymerase eta are sensitive to psoralen photoadducts created under conditions favoring crosslink formation, suggesting a role for translesion synthesis in crosslink repair. Because crosslinks can lead to double-strand breaks, we monitored phosphorylated H2AX (gamma-H2AX), which is typically generated near double-strand breaks but also in response to single-stranded DNA, following psoralen photoadduct formation in XP-V fibroblasts to assess whether polymerase eta is involved in processing crosslinks. In contrast to conditions favoring monoadducts, conditions favoring psoralen crosslinks induced gamma-H2AX levels in both XP-V and nucleotide excision repair-deficient XP-A cells relative to control repair-proficient cells; ectopic expression of polymerase eta in XP-V cells normalized the gamma-H2AX response. In response to psoralen crosslinking, gamma-H2AX as well as 53BP1 formed coincident foci that were more numerous and intense in XP-V and XP-A cells than in controls. Psoralen photoadducts induced gamma-H2AX throughout the cell cycle in XP-V cells. These results indicate that polymerase eta is important in responding to psoralen crosslinks, and are consistent with a model in which nucleotide excision repair and polymerase eta are involved in processing crosslinks and avoiding gamma-H2AX associated with double-strand breaks and single-stranded DNA in human cells.     

Elimination of radiation-induced gamma-H2AX foci in mammalian nucleus can occur by histone exchange

Double-strand breaks in mammalian DNA lead to rapid phosphorylation of C-terminal serines in histone H2AX (gamma-H2AX) and formation of large nuclear gamma-H2AX foci. After DNA repair these foci disappear, but molecular mechanism of elimination of gamma-H2AX foci remains unclear. H2AX protein can be phosphorylated and dephosphorylated in vitro in the absence of chromatin. Here, we compared global exchange of GFP-H2AX with kinetics of formation and elimination of radiation-induced gamma-H2AX foci. Maximal number of gamma-H2AX foci is observed one hour after irradiation, when approximately 20% of GFP-H2AX is exchanged suggesting that formation of the foci mostly occurs by in situ H2AX phosphorylation. However, slow elimination of gamma-H2AX foci is weakly affected by an inhibitor of protein phosphatases calyculin A which is known as an agent suppressing dephosphorylation of gamma-H2AX. This indicates that elimination of gamma-H2AX foci may be independent of dephosphorylation of H2AX which can occur after its removal from the foci by exchange.