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.     

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.     

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.     

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.     

Imaging features that discriminate between foci induced by high- and low-LET radiation in human fibroblasts

In this study, we investigated the formation of radiation-induced foci in normal human fibroblasts exposed to X rays or 130 keV/mum nitrogen ions using antibodies to phosphorylated protein kinase ataxia telangiectasia mutated (ATMp) and histone H2AX (gamma-H2AX). High-content automatic image analysis was used to quantify the immunofluorescence of radiation-induced foci. The size of radiation-induced foci increased for both proteins over a 2-h period after nitrogen-ion irradiation, while the size of radiation-induced foci did not change after exposure to low-LET radiation. The number of radiation-induced ATMp foci showed a more rapid rise and greater frequency after X-ray exposure and was resolved more rapidly such that the frequency of radiation-induced foci decreased by 90% compared to 60% after exposure to high-LET radiation 2 h after 30 cGy. In contrast, the kinetics of radiation-induced gamma-H2AX focus formation was similar for high- and low-LET radiation in that it reached a plateau early and remained constant for up to 2 h. High-resolution 3D images of radiation-induced gamma-H2AX foci and dosimetry computation suggest that multiple double-strand breaks from nitrogen ions are encompassed within large nuclear domains of 4.4 Mbp. Our work shows that the size and frequency of radiation-induced foci vary as a function of radiation quality, dose, time and protein target. Thus, even though double-strand breaks and radiation-induced foci are correlated, the dynamic nature of both contradicts their accepted equivalence for low doses of different radiation qualities.     

γH2AX foci as a measure of DNA damage: a computational approach to automatic analysis

The γH2AX focus assay represents a fast and sensitive approach for the detection of one of the critical types of DNA damage - double-strand breaks (DSB) induced by various cytotoxic agents including ionising radiation. Apart from research applications, the assay has a potential in clinical medicine/pathology, such as assessment of individual radiosensitivity, response to cancer therapies, as well as in biodosimetry. Given that generally there is a direct relationship between numbers of microscopically visualised γH2AX foci and DNA DSB in a cell, the number of foci per nucleus represents the most efficient and informative parameter of the assay. Although computational approaches have been developed for automatic focus counting, the tedious and time consuming manual focus counting still remains the most reliable way due to limitations of computational approaches. We suggest a computational approach and associated software for automatic focus counting that minimises these limitations. Our approach, while using standard image processing algorithms, maximises the automation of identification of nuclei/cells in complex images, offers an efficient way to optimise parameters used in the image analysis and counting procedures, optionally invokes additional procedures to deal with variations in intensity of the signal and background in individual images, and provides automatic batch processing of a series of images. We report results of validation studies that demonstrated correlation of manual focus counting with results obtained using our computational algorithm for mouse jejunum touch prints, mouse tongue sections and human blood lymphocytes as well as radiation dose response of γH2AX focus induction for these biological specimens.     

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.     

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.     

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.     

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.     

Focus formation of DNA repair proteins in normal and repair-deficient cells irradiated with high-LET ions

To investigate the repair of clustered lesions within the DNA/chromatin, the focus formation and persistence of foci of the phosphorylated histone protein H2AX and the repair protein MRE11 were studied in normal cells and in cells lacking DNA-PKcs (M059J) or ATM (GM2052D) after irradiation with high-LET nitrogen ions or low-LET photons. There was a rapid formation of MRE11 and gamma-H2AX foci, and 0.5 h after high-LET irradiation, the number of foci in normal cells correlated well with the number of particle hits per cell nucleus. After 8 h of repair, there were significantly more gamma-H2AX foci than MRE11 foci remaining in the normal cells, independent of radiation quality. The difficulty in repairing clustered breaks was detected as slower rejoining of DSBs (measured by DNA fragmentation analysis), as quantification of the amount of gamma-H2AX over time, and as a larger fraction of repair foci remaining after 24 h in cells irradiated with high- LET ions. These data indicate that clustered lesions are repaired by a pathway involving the same proteins that repair sparsely distributed breaks. Further, for both low- and high- LET radiation, no reduction of the initial number of gamma-H2AX and MRE11 foci was detected in M059J cells up to 21 h after irradiation, which was in accordance with a complete absence of DSB rejoining in these cells. In the GM2052D cells there was also a higher level of foci remaining after 21 h; however, this was not accompanied by unrejoined DSBs, indicating that these foci not only represent DSBs but also may be a sign of persistent problems even when breaks are rejoined.     

Comparison of the induction and disappearance of DNA double strand breaks and gamma-H2AX foci after irradiation of chromosomes in G1-phase or in condensed metaphase cells

The induction and disappearance of DNA double strand breaks (DSBs) after irradiation of G1 and mitotic cells were compared with the gamma-H2AX foci assay and a gel electrophoresis assay. This is to determine whether cell cycle related changes in chromatin structure might influence the gamma-H2AX assay which depends on extensive phosphorylation and dephosphorylation of the H2AX histone variant surrounding DSBs. The disappearance of gamma-H2AX foci after irradiation was much slower for mitotic than for G1 cells. On the other hand, no difference was seen for the gel electrophoresis assay. Our data may suggest the limited accessibility of dephosphorylation enzyme in irradiated metaphase cells or trapped gamma-H2AX in condensed chromatin.     

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.     

Phosphorylation of histone H2AX in radiation-induced micronuclei

DNA double-strand breaks are thought to precede the formation of most radiation-induced micronuclei. Phosphorylation of the histone H2AX is an early indicator of DNA double-strand breaks. Here we studied the phosphorylation status of the histone H2AX in micronuclei after exposure of cultured cells to ionizing radiation or treatment with colchicine. In human astrocytoma SF268 cells, after exposure to gamma radiation, the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei increases. The majority of the gamma-H2AX-positive micronuclei are centromere-negative. The number of gamma-H2AX-positive micronuclei continues to increase even 24 h postirradiation when most gamma-H2AX foci in the main nucleus have disappeared. In contrast, in normal human fibroblasts (BJ), the proportion of gamma-H2AX-positive to gamma-H2AX-negative micronuclei remains constant, and the majority of the centromere-negative cells are gamma-H2AX-negative. Treatment of both cell lines with colchicine results in mostly centromere-positive, gamma-H2AX-negative micronuclei. Immunostaining revealed co-localization of MDC1 and ATM with gamma-H2AX foci in both main nuclei and micronuclei; however, other repair proteins, such as Rad50, 53BP1 and Rad17, that co-localized with gamma-H2AX foci in the main nuclei were not found in the micronuclei. Combination of the micronucleus assay with gamma-H2AX immunostaining provides new insights into the mechanisms of the formation and fate of micronuclei.     

Quantitative detection of (125)IdU-induced DNA double-strand breaks with gamma-H2AX antibody

When mammalian cells are exposed to ionizing radiation and other agents that introduce DSBs into DNA, histone H2AX molecules in megabase chromatin regions adjacent to the breaks become phosphorylated within minutes on a specific serine residue. An antibody to this phosphoserine motif of human H2AX (gamma-H2AX) demonstrates that gamma-H2AX molecules appear in discrete nuclear foci. To establish the quantitative relationship between the number of these foci and the number of DSBs, we took advantage of the ability of (125)I, when incorporated into DNA, to generate one DNA DSB per radioactive disintegration. SF-268 and HT-1080 cell cultures were grown in the presence of (125)IdU and processed immunocytochemically to determine the number of gamma-H2AX foci. The numbers of (125)IdU disintegrations per cell were measured by exposing the same immunocytochemically processed samples to a radiation-sensitive screen with known standards. Under appropriate conditions, the data yielded a direct correlation between the number of (125)I decays and the number of foci per cell, consistent with the assumptions that each (125)I decay yields a DNA DSB and each DNA DSB yields a visible gamma-H2AX focus. Based on these findings, we conclude that gamma-H2AX antibody may form the basis of a sensitive quantitative method for the detection of DNA DSBs in eukaryotic cells.     

Senescence of human fibroblasts after psoralen photoactivation is mediated by ATR kinase and persistent DNA damage foci at telomeres

Cellular senescence is a phenotype that is likely linked with aging. Recent concepts view different forms of senescence as permanently maintained DNA damage responses partially characterized by the presence of senescence-associated DNA damage foci at dysfunctional telomeres. Irradiation of primary human dermal fibroblasts with the photosensitizer 8-methoxypsoralen and ultraviolet A radiation (PUVA) induces senescence. In the present study, we demonstrate that senescence after PUVA depends on DNA interstrand cross-link (ICL) formation that activates ATR kinase. ATR is necessary for the manifestation and maintenance of the senescent phenotype, because depletion of ATR expression before PUVA prevents induction of senescence, and reduction of ATR expression in PUVA-senesced fibroblasts releases cells from growth arrest. We find an ATR-dependent phosphorylation of the histone H2AX (gamma-H2AX). After PUVA, ATR and gamma-H2AX colocalize in multiple nuclear foci. After several days, only few predominantly telomere-localized foci persist and telomeric DNA can be coimmunoprecipitated with ATR from PUVA-senesced fibroblasts. We thus identify ATR as a novel mediator of telomere-dependent senescence in response to ICL induced by photoactivated psoralens.     

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.     

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.     

Megabase chromatin domains involved in DNA double-strand breaks in vivo.

The loss of chromosomal integrity from DNA double-strand breaks introduced into mammalian cells by ionizing radiation results in the specific phosphorylation of histone H2AX on serine residue 139, yielding a specific modified form named gamma-H2AX. An antibody prepared to the unique region of human gamma-H2AX shows that H2AX homologues are phosphorylated not only in irradiated mammalian cells but also in irradiated cells from other species, including Xenopus laevis, Drosophila melanogaster, and Saccharomyces cerevisiae. The antibody reveals that gamma-H2AX appears as discrete nuclear foci within 1 min after exposure of cells to ionizing radiation. The numbers of these foci are comparable to the numbers of induced DNA double-strand breaks. When DNA double-strand breaks are introduced into specific partial nuclear volumes of cells by means of a pulsed microbeam laser, gamma-H2AX foci form at these sites. In mitotic cells from cultures exposed to nonlethal amounts of ionizing radiation, gamma-H2AX foci form band-like structures on chromosome arms and on the end of broken arms. These results offer direct visual confirmation that gamma-H2AX forms en masse at chromosomal sites of DNA double-strand breaks. The results further suggest the possible existence of units of higher order chromatin structure involved in monitoring DNA integrity.