Coordinated response of mammalian Rad51 and Rad52 to DNA damage

Biochemical analysis has shown that mammalian Rad51 and Rad52 interact and synergize in DNA recombination reactions in vitro, but these proteins have not been shown to function together in response to DNA damage in vivo. By analysis of murine cells expressing murine Rad52 tagged with green fluorescent protein (GFP)–Rad52, we now show that DNA damage causes Rad51 and GFP–Rad52 to colocalize in distinct nuclear foci. Cells expressing GFP–Rad52 show both increased survival and an increased number of Rad51 foci, raising the possibility that Rad52 is limiting for repair. These observations provide evidence of coordinated function of Rad51 and Rad52 in vivo and support the hypothesis that Rad52 plays an important role in the DNA damage response in mammalian cells.     

Xrcc3 is recruited to DNA double strand breaks early and independent of Rad51

Rad51-mediated homologous recombination (HR) is essential for maintenance of genome integrity. The Xrcc3 protein functions in HR DNA repair, and studies suggest it has multiple roles at different stages in this pathway. Defects in vertebrate XRCC3 result in elevated levels of spontaneous and DNA damage-induced chromosomal abnormalities, as well as increased sensitivity to DNA damaging agents. Formation of DNA damaged-induced nuclear Rad51 foci requires Xrcc3 and the other Rad51 paralog proteins (Rad51B, Rad51C, Rad51D, Xrcc2), thus supporting a model in which an early function of Xrcc3 involves promoting assembly of active Rad51 repair complexes. However, it is not known whether Xrcc3 or other Rad51 paralog proteins accumulate at DNA breaks, and if they do whether their stable association with breaks requires Rad51. Here we report for the first time that Xrcc3 forms distinct foci in human cells and that nuclear Xrcc3 begins to localize at sites of DNA damage within 10 min after radiation treatment. RNAi-mediated knock down of Rad51 has no effect on the DNA damage-induced localization of Xrcc3 to DNA breaks. Our data are consistent with a model in which Xrcc3 associates directly with DNA breaks independent of Rad51, and subsequently facilitates formation of the Rad51 nucleoprotein filament.     

Nuclear matrix binding site in the Rad51 recombinase

It has been shown that the key homologous recombination protein Rad51accumulates in DNA damage‐induced nuclear foci that are attached to the nuclear matrix. In the present communication we attempted to find whether Rad51 contains a functional domain responsible for nuclear matrix binding. By alignments of the sequences encoding nuclear matrix targeting signals of human nuclear matrix binding proteins with the whole length human Rad51sequence a putative nuclear matrix targeting signal was identified. To prove that it is responsible for the nuclear matrix association of Rad51 18 base pairs encoding a cluster of hydrophobic amino acids in the human Rad51 Flag‐tagged gene were deleted. The formation of damage‐induced Rad51 foci and their association with the nuclear matrix were monitored in HeLa cells transfected with the wild‐type and the mutated Rad51gene after treatment with mitomycin C. The results showed that while the wild‐type protein formed Rad51 foci attached to the nuclear matrix, the mutated Rad51 failed to form DNA damage‐induced nuclear foci. The loss of foci formation activity of the mutated protein was not due to impaired ability to bind double‐stranded DNA in an ATP‐dependant way in vitro and to bind chromatin in vivo. These data suggest that the assembly of Rad51 into nuclear foci is assisted by association with the nuclear matrix, which may support the spatial organization of the process of repair by homologous recombination. J. Cell. Physiol. 219: 202–208, 2009. © 2008 Wiley‐Liss, Inc.     

PARP Inhibition Sensitizes to Low Dose-Rate Radiation TMPRSS2-ERG Fusion Gene-Expressing and PTEN-Deficient Prostate Cancer Cells

Exposure to genotoxic agents, such as irradiation produces DNA damage, the toxicity of which is augmented when the DNA repair is impaired. Poly (ADP-ribose) polymerase (PARP) inhibitors were found to be “synthetic lethal” in cells deficient in BRCA1 and BRCA2 that impair homologous recombination. However, since many tumors, including prostate cancer (PCa) rarely have on such mutations, there is considerable interest in finding alternative determinants of PARP inhibitor sensitivity. We evaluated the effectiveness of radiation in combination with the PARP inhibitor, rucaparib in PCa cells. The combination index for clonogenic survival following radiation and rucaparib treatments revealed synergistic interactions in a panel of PCa cell lines, being strongest for LNCaP and VCaP cells that express ETS gene fusion proteins. These findings correlated with synergistic interactions for senescence activation, as indicated by β--galactosidase staining. Absence of PTEN and presence of ETS gene fusion thus facilitated activation of senescence, which contributed to decreased clonogenic survival. Increased radiosensitivity in the presence of rucaparib was associated with persistent DNA breaks, as determined by χ-H2AX, p53BP1, and Rad51 foci. VCaP cells, which harbor the TMPRSS2-ERG gene fusion and PC3 cells that stably express a similar construct (fusion III) showed enhanced sensitivity towards rucaparib, which, in turn, increased the radiation response to a similar extent as the DNA-PKcs inhibitor NU7441. Rucaparib radiosensitized PCa cells, with a clear benefit of low dose-rate radiation (LDR) administered over a longer period of time that caused enhanced DNA damage. LDR mimicking brachytherapy, which is used successfully in the clinic, was most effective when combined with rucaparib by inducing persistent DNA damage and senescence, leading to decreased clonogenic survival. This combination was most effective in the presence of the TMPRSS2-ERG and in the absence of PTEN, indicating clinical potential for brachytherapy in patients with intermediate and high risk PCa.     

Ionizing radiation-induced foci formation of mammalian Rad51 and Rad54 depends on the Rad51 paralogs, but not on Rad52

Homologous recombination is of major importance for the prevention of genomic instability during chromosome duplication and repair of DNA damage, especially double-strand breaks. Biochemical experiments have revealed that during the process of homologous recombination the RAD52 group proteins, including Rad51, Rad52 and Rad54, are involved in an essential step: formation of a joint molecule between the broken DNA and the intact repair template. Accessory proteins for this reaction include the Rad51 paralogs and BRCA2. The significance of homologous recombination for the cell is underscored by the evolutionary conservation of the Rad51, Rad52 and Rad54 proteins from yeast to humans. Upon treatment of cells with ionizing radiation, the RAD52 group proteins accumulate at the sites of DNA damage into so-called foci. For the yeast Saccharomyces cerevisiae, foci formation of Rad51 and Rad54 is abrogated in the absence of Rad52, while Rad51 foci formation does occur in the absence of the Rad51 paralog Rad55. By contrast, we show here that in mammalian cells, Rad52 is not required for foci formation of Rad51 and Rad54. Furthermore, radiation-induced foci formation of Rad51 and Rad54 is impaired in all Rad51 paralog and BRCA2 mutant cell lines tested, while Rad52 foci formation is not influenced by a mutation in any of these recombination proteins. Despite their evolutionary conservation and biochemical similarities, S. cerevisiae and mammalian Rad52 appear to differentially contribute to the DNA-damage response.     

Inhibition of homologous recombination with vorinostat synergistically enhances ganciclovir cytotoxicity

The nucleoside analog ganciclovir (GCV) elicits cytotoxicity in tumor cells via a novel mechanism in which drug incorporation into DNA produces minimal disruption of replication, but numerous DNA double strand breaks occur during the second S-phase after drug exposure. We propose that homologous recombination (HR), a major repair pathway for DNA double strand breaks, can prevent GCV-induced DNA damage, and that inhibition of HR will enhance cytotoxicity with GCV. Survival after GCV treatment in cells expressing a herpes simplex virus thymidine kinase was strongly dependent on HR (>14-fold decrease in IC₅₀ in HR-deficient vs. HR-proficient CHO cells). In a homologous recombination reporter assay, the histone deacetylase inhibitor, suberoylanilide hydroxamic acid (SAHA; vorinostat), decreased HR repair events up to 85%. SAHA plus GCV produced synergistic cytotoxicity in U251tk human glioblastoma cells. Elucidation of the synergistic mechanism demonstrated that SAHA produced a concentration-dependent decrease in the HR proteins Rad51 and CtIP. GCV alone produced numerous Rad51 foci, demonstrating activation of HR. However, the addition of SAHA blocked GCV-induced Rad51 foci formation completely and increased γH2AX, a marker of DNA double strand breaks. SAHA plus GCV also produced synergistic cytotoxicity in HR-proficient CHO cells, but the combination was antagonistic or additive in HR-deficient CHO cells. Collectively, these data demonstrate that HR promotes survival with GCV and compromise of HR by SAHA results in synergistic cytotoxicity, revealing a new mechanism for enhancing anticancer activity with GCV.     

ATR-Chk1 signaling pathway and homologous recombinational repair protect cells from 5-fluorouracil cytotoxicity

5-Fluorouracil (5-FU) has long been a mainstay antimetabolite chemotherapeutic drug for the treatment of major solid tumors, particularly colorectal cancer. 5-FU is processed intracellularly to yield active metabolites that compromise RNA and DNA metabolism. However, the mechanisms responsible for its cytotoxicity are not fully understood. From the phenotypic analysis of mutant chicken B lymphoma DT40 cells, we found that homologous recombinational repair (HRR), involving Rad54 and BRCA2, and the ATR-Chk1 signaling pathway, involving Rad9 and Rad17, significantly contribute to 5-FU tolerance. 5-FU induced γH2AX nuclear foci, which were colocalized with the key HRR factor Rad51, but not with DNA double-strand breaks (DSBs), in a dose-dependent manner as cells accumulated in the S phase. Inhibition of Chk1 kinase by UCN-01 increased 5-FU-induced γH2AX and enhanced 5-FU cytotoxicity not only in wild-type cells but also in Rad54- or BRCA2-deficient cells, suggesting that HRR and Chk1 kinase have non-overlapping roles in 5-FU tolerance. 5-FU-induced Chk1 phosphorylation was significantly impaired in Rad9- or Rad17-deficient cells, and severe γH2AX nuclear foci and DSBs were formed, which was followed by apoptosis. Finally, inhibition of Chk1 kinase by UCN-01 increased 5-FU-induced γH2AX nuclear foci and enhanced 5-FU cytotoxicity in Rad9- or Rad17-deficient cells. These results suggest that Rad9- and Rad17-independent activation of the ATR-Chk1 signaling pathway also significantly contributes to 5-FU tolerance.     

Sequestration of Mammalian Rad51-Recombination Protein into Micronuclei

The mammalian Rad51 protein is involved in homologous recombination and in DNA damage repair. Its nuclear distribution after DNA damage is highly dynamic, and distinct foci of Rad51 protein, distributed throughout the nuclear volume, are induced within a few hours after γ irradiation; these foci then coalesce into larger clusters. Rad51-positive cells do not undergo DNA replication. Rad51 foci colocalize with both replication protein A and sites of unscheduled DNA repair synthesis and may represent a nuclear domain for recombinational DNA repair. By 24 h postirradiation, most foci are sequestered into micronuclei or assembled into Rad51-coated DNA fibers. These micronuclei and DNA fibers display genome fragmentation typical of apoptotic cell death. Other repair proteins, such as Rad52 and Gadd45, are not eliminated from the nucleus. DNA double strand breaks in repair-deficient cells or induced by the clastogen etoposide are also accompanied by the sequestering of Rad51 protein before cell death. The spindle poison colcemid causes cell cycle arrest and Rad51-foci formation without directly damaging DNA. Collectively, these observations suggest that mammalian Rad51 protein associates with damaged DNA and/or with DNA that is temporarily or irreversibly unable to replicate and these foci may subsequently be eliminated from the nucleus.     

Inducibility of nuclear Rad51 foci after DNA damage distinguishes all Fanconi anemia complementation groups from D1/BRCA2

Fanconi anemia (FA) is a cancer susceptibility disorder characterized by chromosomal instability and hypersensitivity to DNA cross-linking agents. So far 11 complementation groups have been identified, from which only FA-D1/BRCA2 and FA-J are defective downstream of the central FANCD2 protein as cells from these groups are capable of monoubiquitinating FANCD2. In this study we show that cells derived from patients from the new complementation groups, FA-I, FA-J and FA-L are all proficient in DNA damage induced Rad51 foci formation, making the cells from FA-D1/BRCA2 patients that are defective in this process the sole exception. Although FA-B patient HSC230 was previously reported to also have biallelic BRCA2 mutations, we found normal Rad51 foci formation in cells from this patient, consistent with the recent identification of an X-linked gene being mutated in four unrelated FA-B patients. Thus, our data show that none of the FA proteins, except BRCA2, are required to sequester Rad51 into nuclear foci. Since cells from the FA-D1 and FA-J patient groups are both able to monoubiquitinate FANCD2, the "Rad51 foci phenotype" provides a convenient assay to distinguish between these two groups. Our results suggest that FANCJ and FANCD1/BRCA2 are part of the integrated FANC/BRCA DNA damage response pathway or, alternatively, that they represent sub-pathways in which only FANCD1/BRCA2 is directly connected to the process of homologous recombination.     

Chk1 inhibition in p53-deficient cell lines drives rapid chromosome fragmentation followed by caspase-independent cell death

Activation of Checkpoint kinase 1 (Chk1) following DNA damage mediates cell cycle arrest to prevent cells with damaged DNA from entering mitosis. Here we provide a high-resolution analysis of cells as they undergo S- and G₂-checkpoint bypass in response to Chk1 inhibition with the selective Chk1 inhibitor GNE-783. Within 4–8 h of Chk1 inhibition following gemcitabine induced DNA damage, cells with both sub-4N and 4N DNA content prematurely enter mitosis. Coincident with premature transition into mitosis, levels of DNA damage dramatically increase and chromosomes condense and attempt to align along the metaphase plate. Despite an attempt to congress at the metaphase plate, chromosomes rapidly fragment and lose connection to the spindle microtubules. Gemcitabine mediated DNA damage promotes the formation of Rad51 foci; however, while Chk1 inhibition does not disrupt Rad51 foci that are formed in response to gemcitabine, these foci are lost as cells progress into mitosis. Premature entry into mitosis requires the Aurora, Cdk1/2 and Plk1 kinases and even though caspase-2 and -3 are activated upon mitotic exit, they are not required for cell death. Interestingly, p53, but not p21, deficiency enables checkpoint bypass and chemo-potentiation. Finally, we uncover a differential role for the Wee-1 checkpoint kinase in response to DNA damage, as Wee-1, but not Chk1, plays a more prominent role in the maintenance of S- and G₂-checkpoints in p53 proficient cells.     

Interaction of human rad51 recombination protein with single-stranded DNA binding protein, RPA.

Replication protein A (RPA), a heterotrimeric single-stranded DNA binding protein, is required for recombination, and stimulates homologous pairing and DNA strand exchange promoted in vitro by human recombination protein HsRad51. Co-immunoprecipitation revealed that purified RPA interacts physically with HsRad51, as well as with HsDmc1, the homolog that is expressed specifically in meiosis. The interaction with HsRad51 was mediated by the 70 kDa subunit of RPA, and according to experiments with deletion mutants, this interaction required amino acid residues 169-326. In exponentially growing mammalian cells, 22% of nuclei showed foci of RPA protein and 1-2% showed foci of Rad51. After gamma-irradiation, the percentage of cells with RPA foci increased to approximately 50%, and those with Rad51 foci to 30%. All of the cells with foci of Rad51 had foci of RPA, and in those cells the two proteins co-localized in a high fraction of foci. The interactions of human RPA with Rad51, replication proteins and DNA are suited to the linking of recombination to replication.     

Spontaneous homologous recombination is decreased in Rad51C-deficient hamster cells

The Chinese hamster cell mutant, CL-V4B that is mutated in the Rad51 paralog gene, Rad51C (RAD51L2), has been described to exhibit increased sensitivity to DNA cross-linking agents, genomic instability, and an impaired Rad51 foci formation in response to DNA damage. To directly examine an effect of the Rad51C protein on homologous recombination (HR) in mammalian cells, we compared the frequencies and rates of spontaneous HR in CL-V4B cells and in parental wildtype V79B cells, using a recombination reporter plasmid in host cell reactivation assays. Our results demonstrate that HR is reduced but not abolished in the CL-V4B mutant. We thus, provide direct evidence for a role of mammalian Rad51C in HR processes. The reduced HR events described here help to explain the deficient phenotypes observed in Rad51C mutants and support an accessory role of Rad51C in Rad51-mediated recombination.     

Activation of the S phase DNA damage checkpoint by mitomycin C

We have studied the rate of DNA synthesis, cell cycle distribution, formation of gamma-H2AX, and Rad51 nuclear foci and association of Rad51 with the nuclear matrix after treatment of HeLa cells with the interstrand crosslinking agent mitomycin C (MMC) in the presence of the kinase inhibitors caffeine and wortmannin. The results showed that MMC treatment arrested the cells in S-phase and induced the appearance of gamma-H2AX and Rad51 nuclear foci, accompanied with a sequestering of Rad51 to the nuclear matrix. These effects were abrogated by caffeine, which inhibits the Ataxia-telangiectasia mutated (ATM) and ATM- and Rad3-related (ATR) kinases. However, wortmannin at a concentration that inhibits ATM, but not ATR did not affect cell cycle progression, damage-induced phosphorylation of H2AX and Rad51 foci formation, and association with the nuclear matrix, suggesting that the S-phase arrest induced by MMC is ATR-dependent. These findings were confirmed by experiments with ATR-deficient and AT cells. They indicate that the DNA damage ATR-dependent S-phase checkpoint pathway may regulate the spatiotemporal organization of the process of repair of interstrand crosslinks.     

Caffeine inhibits gene conversion by displacing Rad51 from ssDNA

Efficient repair of chromosomal double-strand breaks (DSBs) by homologous recombination relies on the formation of a Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends. This filament facilitates the search for a homologous donor sequence and promotes strand invasion. Recently caffeine treatment has been shown to prevent gene targeting in mammalian cells by increasing non-productive Rad51 interactions between the DSB and random regions of the genome. Here we show that caffeine treatment prevents gene conversion in yeast, independently of its inhibition of the Mec1^ATR/Tel1^ATM-dependent DNA damage response or caffeine''s inhibition of 5′ to 3′ resection of DSB ends. Caffeine treatment results in a dosage-dependent eviction of Rad51 from ssDNA. Gene conversion is impaired even at low concentrations of caffeine, where there is no discernible dismantling of the Rad51 filament. Loss of the Rad51 filament integrity is independent of Srs2''s Rad51 filament dismantling activity or Rad51''s ATPase activity and does not depend on non-specific Rad51 binding to undamaged double-stranded DNA. Caffeine treatment had similar effects on irradiated HeLa cells, promoting loss of previously assembled Rad51 foci. We conclude that caffeine treatment can disrupt gene conversion by disrupting Rad51 filaments.     

Targeting ATR in vivo using the novel inhibitor VE-822 results in selective sensitization of pancreatic tumors to radiation

Combined radiochemotherapy is the currently used therapy for locally advanced pancreatic ductal adenocarcinoma (PDAC), but normal tissue toxicity limits its application. Here we test the hypothesis that inhibition of ATR (ATM-Rad3-related) could increase the sensitivity of the cancer cells to radiation or chemotherapy without affecting normal cells. We tested VE-822, an ATR inhibitor, for in vitro and in vivo radiosensitization. Chk1 phosphorylation was used to indicate ATR activity, γH2AX and 53BP1 foci as evidence of DNA damage and Rad51 foci for homologous recombination activity. Sensitivity to radiation (XRT) and gemcitabine was measured with clonogenic assays in vitro and tumor growth delay in vivo. Murine intestinal damage was evaluated after abdominal XRT. VE-822 inhibited ATR in vitro and in vivo. VE-822 decreased maintenance of cell-cycle checkpoints, increased persistent DNA damage and decreased homologous recombination in irradiated cancer cells. VE-822 decreased survival of pancreatic cancer cells but not normal cells in response to XRT or gemcitabine. VE-822 markedly prolonged growth delay of pancreatic cancer xenografts after XRT and gemcitabine-based chemoradiation without augmenting normal cell or tissue toxicity. These findings support ATR inhibition as a promising new approach to improve the therapeutic ration of radiochemotherapy for patients with PDAC.     

Rad51 accumulation at sites of DNA damage and in postreplicative chromatin

Rad51, a eukaryotic RecA homologue, plays a central role in homologous recombinational repair of DNA double-strand breaks (DSBs) in yeast and is conserved from yeast to human. Rad51 shows punctuate nuclear localization in human cells, called Rad51 foci, typically during the S phase (Tashiro, S., N. Kotomura, A. Shinohara, K. Tanaka, K. Ueda, and N. Kamada. 1996. Oncogene. 12:2165-2170). However, the topological relationships that exist in human S phase nuclei between Rad51 foci and damaged chromatin have not been studied thus far. Here, we report on ultraviolet microirradiation experiments of small nuclear areas and on whole cell ultraviolet C (UVC) irradiation experiments performed with a human fibroblast cell line. Before UV irradiation, nuclear DNA was sensitized by the incorporation of halogenated thymidine analogues. These experiments demonstrate the redistribution of Rad51 to the selectively damaged, labeled chromatin. Rad51 recruitment takes place from Rad51 foci scattered throughout the nucleus of nonirradiated cells in S phase. We also demonstrate the preferential association of Rad51 foci with postreplicative chromatin in contrast to replicating chromatin using a double labeling procedure with halogenated thymidine analogues. This finding supports a role of Rad51 in recombinational repair processes of DNA damage present in postreplicative chromatin.     

Poly (ADP-ribose) polymerase inhibition synergizes with the NF-κB inhibitor DHMEQ to kill hepatocellular carcinoma cells

Poly (ADP-ribose) polymerase (PARP) enzymes play a key role in the cellular machinery responsible for DNA repair. Dehydroxymethylepoxyquinomicin (DHMEQ), a new inhibitor of NF-κB, induces oxidative stress and DNA damage. The effects of DHMEQ in combination with Olaparib (PARP inhibitor) were studied on hepatocellular carcinoma (HCC) cells. The DHMEQ–Olaparib combination synergistically inhibited cell viability, cell proliferation and colony formation of Hep3B, but had additive effects on Huh7 cells. The synergistic effects of the combination correlated with increased apoptosis, caspase 3/7 activity and PARP cleavage. There was an induction of an endoplasmic reticulum (ER) stress response with significant up-regulation of CHOP and TRB3 genes and splicing of XBP1 mRNA in Hep3B cells but not in Huh7 cells. Silencing of the TRB3 mRNA in Hep3B cells reversed the reduction in viability caused by DHMEQ–Olaparib treatment, while depletion of unspliced XBP1 mRNA in DHMEQ–Olaparib-treated Huh7 cells reduced viability. ROS production was increased after DHMEQ–Olaparib treatment of Hep3B, which caused DNA damage by an accumulation of γH2AX, increased AKT phosphorylation and reduced cell viability. The combination reduced Rad51 nuclear foci in Hep3B cells (not Huh7 cells), and silencing of Rad51 enhanced sensitivity of Huh7 cells to the DHMEQ–Olaparib combination. Knockdown of AKT in Hep3B cells restored the number of Rad51 nuclear foci after DHMEQ–Olaparib treatment. In summary, the DHMEQ–Olaparib combination induced ROS production, which killed HCC cells via DNA damage that could not be repaired by Rad51.SummaryPARPs and NF-κB are frequently deregulated in HCC. The DHMEQ–Olaparib combination exerted synergistic anti-tumour effects on HCC cells through ROS production via DNA damage that could not be repaired by Rad51. This suggested that the DHMEQ–Olaparib combination could be used to treat tumours that were resistant to Olaparib treatment.     

Poly(ADP-ribose) polymerase (PARP-1) has a controlling role in homologous recombination

Cells with non-functional poly(ADP-ribose) polymerase (PARP-1) show increased levels of sister chromatid exchange, suggesting a hyper recombination phenotype in these cells. To further investigate the involvement of PARP-1 in homologous recombination (HR) we investigated how PARP-1 affects nuclear HR sites (Rad51 foci) and HR repair of an endonuclease-induced DNA double-strand break (DSB). Several proteins involved in HR localise to Rad51 foci and HR-deficient cells fail to form Rad51 foci in response to DNA damage. Here, we show that PARP-1 mainly does not localise to Rad51 foci and that Rad51 foci form in PARP-1^–/– cells, also in response to hydroxyurea. Furthermore, we show that homology directed repair following induction of a site-specific DSB is normal in PARP-1-inhibited cells. In contrast, inhibition or loss of PARP-1 increases spontaneous Rad51 foci formation, confirming a hyper recombination phenotype in these cells. Our data suggest that PARP-1 controls DNA damage recognised by HR and that it is not involved in executing HR as such.     

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.     

ASCIZ regulates lesion-specific Rad51 focus formation and apoptosis after methylating DNA damage

Nuclear Rad51 focus formation is required for homology-directed repair of DNA double-strand breaks (DSBs), but its regulation in response to non-DSB lesions is poorly understood. Here we report a novel human SQ/TQ cluster domain-containing protein termed ASCIZ that forms Rad51-containing foci in response to base-modifying DNA methylating agents but not in response to DSB-inducing agents. ASCIZ foci seem to form prior to Rad51 recruitment, and an ASCIZ core domain can concentrate Rad51 in focus-like structures independently of DNA damage. ASCIZ depletion dramatically increases apoptosis after methylating DNA damage and impairs Rad51 focus formation in response to methylating agents but not after ionizing radiation. ASCIZ focus formation and increased apoptosis in ASCIZ-depleted cells depend on the mismatch repair protein MLH1. Interestingly, ASCIZ foci form efficiently during G1 phase, when sister chromatids are unavailable as recombination templates. We propose that ASCIZ acts as a lesion-specific focus scaffold in a Rad51-dependent pathway that resolves cytotoxic repair intermediates, most likely single-stranded DNA gaps, resulting from MLH1-dependent processing of base lesions.