Replication-dependent DNA Double-strand Breaks Research Articles

Switch-like phosphorylation of WRN integrates end-resection with RAD51 metabolism at collapsed replication forks.

Replication-dependent DNA double-strand breaks are harmful lesions preferentially repaired by homologous recombination (HR), a process that requires processing of DNA ends to allow RAD51-mediated strand invasion. End resection and subsequent repair are two intertwined processes, but the mechanism underlying their execution is still poorly appreciated. The WRN helicase is one of the crucial factors for end resection and is instrumental in selecting the proper repair pathway. Here, we reveal that ordered phosphorylation of WRN by the CDK1, ATM and ATR kinases defines a complex regulatory layer essential for correct long-range end resection, connecting it to repair by HR. We establish that long-range end resection requires an ATM-dependent phosphorylation of WRN at Ser1058 and that phosphorylation at Ser1141, together with dephosphorylation at the CDK1 site Ser1133, is needed for the proper metabolism of RAD51 foci and RAD51-dependent repair. Collectively, our findings suggest that regulation of WRN by multiple kinases functions as a molecular switch to allow timely execution of end resection and repair at replication-dependent DNA double-strand breaks.

A novel cell-cycle-regulated interaction of the Bloom syndrome helicase BLM with Mcm6 controls replication-linked processes.

The Bloom syndrome DNA helicase BLM contributes to chromosome stability through its roles in double-strand break repair by homologous recombination and DNA replication fork restart during the replication stress response. Loss of BLM activity leads to Bloom syndrome, which is characterized by extraordinary cancer risk and small stature. Here, we have analyzed the composition of the BLM complex during unperturbed S-phase and identified a direct physical interaction with the Mcm6 subunit of the minichromosome maintenance (MCM) complex. Using distinct binding sites, BLM interacts with the N-terminal domain of Mcm6 in G1 phase and switches to the C-terminal Cdt1-binding domain of Mcm6 in S-phase, with a third site playing a role for Mcm6 binding after DNA damage. Disruption of Mcm6-binding to BLM in S-phase leads to supra-normal DNA replication speed in unperturbed cells, and the helicase activity of BLM is required for this increased replication speed. Upon disruption of BLM/Mcm6 interaction, repair of replication-dependent DNA double-strand breaks is delayed and cells become hypersensitive to DNA damage and replication stress. Our findings reveal that BLM not only plays a role in the response to DNA damage and replication stress, but that its physical interaction with Mcm6 is required in unperturbed cells, most notably in S-phase as a negative regulator of replication speed.

Dianhydrogalactitol induces replication-dependent DNA damage in tumor cells preferentially resolved by homologous recombination

1,2:5,6-Dianhydrogalactitol (DAG) is a bifunctional DNA-targeting agent causing N7-guanine alkylation and inter-strand DNA crosslinks currently in clinical trial for treatment of glioblastoma. While preclinical studies and clinical trials have demonstrated antitumor activity of DAG in a variety of malignancies, understanding the molecular mechanisms underlying DAG-induced cytotoxicity is essential for proper clinical qualification. Using non-small cell lung cancer (NSCLC) as a model system, we show that DAG-induced cytotoxicity materializes when cells enter S phase with unrepaired N7-guanine DNA crosslinks. In S phase, DAG-mediated DNA crosslink lesions translated into replication-dependent DNA double-strand breaks (DSBs) that subsequently triggered irreversible cell cycle arrest and loss of viability. DAG-treated NSCLC cells attempt to repair the DSBs by homologous recombination (HR) and inhibition of the HR repair pathway sensitized NSCLC cells to DAG-induced DNA damage. Accordingly, our work describes a molecular mechanism behind N7-guanine crosslink-induced cytotoxicity in cancer cells and provides a rationale for using DAG analogs to treat HR-deficient tumors.

Abstract B15: Dissecting the molecular mechanism of dianhydrogalactitol (VAL-083) activity in cancer treatment

Abstract Introduction: Dianhydrogalactitol (VAL-083) is a bi-functional alkylating agent causing N7-guanine-methylation and inter-strand DNA crosslinks. In China, VAL-083 is approved to use in the chemotherapeutic treatment of lung cancer and chronic myelogenous leukemia. In the United States, VAL-083 is currently undergoing investigation as a new therapy in the treatment of temozolomide refractory glioblastoma (GBM). VAL-083 is a small water-soluble molecule that readily crosses blood-brain-barrier and accumulates in the tumor tissues in brain, making it a good candidate for targeting brain malignancies, such as GBM and medulloblastoma. Historical data from preclinical studies and clinical trials sponsored by the US National Cancer Institute (NCI) support anti-neoplastic effects of VAL-083 in a variety of cancer types in addition to GBM, including lung cancer, leukemia, cervical cancer, and ovarian cancer. However, the detailed molecular mechanisms mediating VAL-083 sensitivity or resistance in cancer cells is still unclear. Therefore, we investigated the distinct mechanism of action of VAL-083 in different cancer cell lines. Methods: VAL-083 cytotoxicity was evaluated in a panel of human non-small cell lung cancer (NSCLC) cell lines (A549, H2122, H1792, and H23) and prostate cancer cell lines (PC3 and LNCaP) by crystal violet assays. Cell cycle analysis and DNA damage response were investigated by propidium iodide (PI) and immunofluorescent (IF) staining. Western blot and IF staining analyses were employed to elucidate the DNA repair mechanism involved in VAL-083-treated cancer cells. Results: In this study, we report new insights into VAL-083's mechanisms of action by showing that VAL-083 induces irreversible cell-cycle arrest and cell death caused by replication-dependent DNA double-strand breaks (DSBs). In all the cancer cells tested, VAL-083 showed broad cytotoxicity with an IC50 range of 3.1 - 25.7 μM. In lung cancer (H2122, H1792, and A549) and prostate cancer (PC3 and LNCaP) cell lines, VAL-083 treatment caused irreversible cell cycle arrest at S/G2 phase as measured by PI and IF staining in synchronized cells, indicating that VAL-083-induced inter-strand crosslinks result in more difficult to repair DNA lesions during replication, including DSBs. Western blot and IF analyses of DNA repair markers were employed to investigate the DNA damage response induced by VAL-083 in cancer cells. The S/G2 phase cell cycle arrest and the increased γH2A.X (an indication of DSB lesions) expression persisted for 48-72 h after treatment with VAL-083, indicating prolonged unrepaired DNA lesions caused by VAL-083. VAL-083 pulse-treatment led to persistent phosphorylation of DSB sensors ataxia telangiectasia mutated (ATM), single-strand DNA-binding replication protein A (RPA32), and H2A.X. Furthermore, Western blot analyses also demonstrated activation of the downstream effectors of ATM and ataxia telangiectasia and Rad3-related protein (ATR) kinases, Chk2 (T68) and Chk1 (S317 and S345). These results suggest that VAL-083-induced persistent and irreversible DNA damage activated the homologous recombination DNA repair signaling pathway in the panel of cancer cells studied. Conclusions: VAL-083 displayed broad anti-neoplastic activity in different lung and prostate cancer cells through the replication-dependent DSBs. Elucidation of the molecular mechanisms underlying VAL-083 cytotoxicity provides guidance for improved treatment strategies for cancer patients with VAL-083 in either single or combination regimens. Citation Format: Beibei Zhai, Anne Steino, Jeffrey Bacha, Dennis Brown, Mads Daugaard. Dissecting the molecular mechanism of dianhydrogalactitol (VAL-083) activity in cancer treatment [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr B15.

A Mutation in the FHA Domain ofCoprinus cinereusNbs1 Leads to Spo11-Independent Meiotic Recombination and Chromosome Segregation

Nbs1, a core component of the Mre11-Rad50-Nbs1 complex, plays an essential role in the cellular response to DNA double-strand breaks (DSBs) and poorly understood roles in meiosis. We used the basidiomycete Coprinus cinereus to examine the meiotic roles of Nbs1. We identified the C. cinereus nbs1 gene and demonstrated that it corresponds to a complementation group previously known as rad3. One allele, nbs1-2, harbors a point mutation in the Nbs1 FHA domain and has a mild spore viability defect, increased frequency of meiosis I nondisjunction, and an altered crossover distribution. The nbs1-2 strain enters meiosis with increased levels of phosphorylated H2AX, which we hypothesize represent unrepaired DSBs formed during premeiotic replication. In nbs1-2, there is no apparent induction of Spo11-dependent DSBs during prophase. We propose that replication-dependent DSBs, resulting from defective replication fork protection and processing by the Mre11-Rad50-Nbs1 complex, are competent to form meiotic crossovers in C. cinereus, and that these crossovers lead to high levels of faithful chromosome segregation. In addition, although crossover distribution is altered in nbs1-2, the majority of crossovers were found in subtelomeric regions, as in wild-type. Therefore, the location of crossovers in C. cinereus is maintained when DSBs are induced via a Spo11-independent mechanism.

Oxidative Stress and Replication-Independent DNA Breakage Induced by Arsenic in Saccharomyces cerevisiae

Arsenic is a well-established human carcinogen of poorly understood mechanism of genotoxicity. It is generally accepted that arsenic acts indirectly by generating oxidative DNA damage that can be converted to replication-dependent DNA double-strand breaks (DSBs), as well as by interfering with DNA repair pathways and DNA methylation. Here we show that in budding yeast arsenic also causes replication and transcription-independent DSBs in all phases of the cell cycle, suggesting a direct genotoxic mode of arsenic action. This is accompanied by DNA damage checkpoint activation resulting in cell cycle delays in S and G2/M phases in wild type cells. In G1 phase, arsenic activates DNA damage response only in the absence of the Yku70–Yku80 complex which normally binds to DNA ends and inhibits resection of DSBs. This strongly indicates that DSBs are produced by arsenic in G1 but DNA ends are protected by Yku70–Yku80 and thus invisible for the checkpoint response. Arsenic-induced DSBs are processed by homologous recombination (HR), as shown by Rfa1 and Rad52 nuclear foci formation and requirement of HR proteins for cell survival during arsenic exposure. We show further that arsenic greatly sensitizes yeast to phleomycin as simultaneous treatment results in profound accumulation of DSBs. Importantly, we observed a similar response in fission yeast Schizosaccharomyces pombe, suggesting that the mechanisms of As(III) genotoxicity may be conserved in other organisms.

XPF-ERCC1 participates in the Fanconi anemia pathway of cross-link repair.

Interstrand cross-links (ICLs) prevent DNA strand separation and, therefore, transcription and replication, making them extremely cytotoxic. The precise mechanism by which ICLs are removed from mammalian genomes largely remains elusive. Genetic evidence implicates ATR, the Fanconi anemia proteins, proteins required for homologous recombination, translesion synthesis, and at least two endonucleases, MUS81-EME1 and XPF-ERCC1. ICLs cause replication-dependent DNA double-strand breaks (DSBs), and MUS81-EME1 facilitates DSB formation. The subsequent repair of these DSBs occurs via homologous recombination after the ICL is unhooked by XPF-ERCC1. Here, we examined the effect of the loss of either nuclease on FANCD2 monoubiquitination to determine if the nucleolytic processing of ICLs is required for the activation of the Fanconi anemia pathway. FANCD2 was monoubiquitinated in Mus81(-/-), Ercc1(-/-), and XPF-deficient human, mouse, and hamster cells exposed to cross-linking agents. However, the monoubiquitinated form of FANCD2 persisted longer in XPF-ERCC1-deficient cells than in wild-type cells. Moreover, the levels of chromatin-bound FANCD2 were dramatically reduced and the number of ICL-induced FANCD2 foci significantly lower in XPF-ERCC1-deficient cells. These data demonstrate that the unhooking of an ICL by XPF-ERCC1 is necessary for the stable localization of FANCD2 to the chromatin and subsequent homologous recombination-mediated DSB repair.

Transcriptional profiling of the age-related response to genotoxic stress points to differential DNA damage response with age

The p53 DNA damage response attenuated with age and we have evaluated downstream factors in the DNA damage response. In old animals p21 protein accumulates in the whole cell fraction but significantly declines in the nucleus, which may alter cell cycle and apoptotic programs in response to DNA damage. We evaluated the transcriptional response to DNA damage in young and old and find 2692 genes are differentially regulated in old compared to young in response to oxidative stress ( p < 0.005). As anticipated, the transcriptional profile of young mice is consistent with DNA damage induced cell cycle arrest while the profile of old mice is consistent with cell cycle progression in the presence of DNA damage, suggesting the potential for catastrophic accumulation of DNA damage at the replication fork. Unique sets of DNA repair genes are induced in response to damage in old and young, suggesting the types of damage accumulating differs between young and old. The DNA repair genes upregulated in old animals point to accumulation of replication-dependent DNA double strand breaks (DSB). Expression data is consistent with loss of apoptosis following DNA damage in old animals. These data suggest DNA damage responses differ greatly in young and old animals.

Ataxia telangiectasia mutated activation by transcription‐ and topoisomerase I‐induced DNA double‐strand breaks

Ataxia telangiectasia mutated (ATM), the deficiency of which causes a severe neurodegenerative disease, is a crucial mediator for the DNA damage response (DDR). As neurons have high rates of transcription that require topoisomerase I (TOP1), we investigated whether TOP1 cleavage complexes (TOP1cc)-which are potent transcription-blocking lesions-also produce transcription-dependent DNA double-strand breaks (DSBs) with ATM activation. We show the induction of DSBs and DDR activation in post-mitotic primary neurons and lymphocytes treated with camptothecin, with the induction of nuclear DDR foci containing activated ATM, gamma-H2AX (phosphorylated histone H2AX), activated CHK2 (checkpoint kinase 2), MDC1 (mediator of DNA damage checkpoint 1) and 53BP1 (p53 binding protein 1). The DSB-ATM-DDR pathway was suppressed by inhibiting transcription and gamma-H2AX signals were reduced by RNase H1 transfection, which removes transcription-mediated R-loops. Thus, we propose that Top1cc produce transcription arrests with R-loop formation and generate DSBs that activate ATM in post-mitotic cells.

BRCA2-dependent homologous recombination is required for repair of Arsenite-induced replication lesions in mammalian cells

Arsenic exposure constitutes one of the most widespread environmental carcinogens, and is associated with increased risk of many different types of cancers. Here we report that arsenite (As[III]) can induce both replication-dependent DNA double-strand breaks (DSB) and homologous recombination (HR) at doses as low as 5 µM (0.65 mg/l), which are within the typical doses often found in drinking water in contaminated areas. We show that the production of DSBs is dependent on active replication and is likely to be the result of conversion of a DNA single-strand break (SSB) into a toxic DSB when encountered by a replication fork. We demonstrate that HR is required for the repair of these breaks and show that a functional HR pathway protects against As[III]-induced cytotoxicity. In addition, BRCA2-deficient cells are sensitive to As[III] and we suggest that As[III] could be exploited as a therapy for HR-deficient tumours such as BRCA1 and BRCA2 mutated breast and ovarian cancers.

Homologous recombination and the yKu70/80 complex exert opposite roles in resistance against the killer toxin from Pichia acaciae

The linear plasmid (pPac1-2) encoded killer toxin (PaT) of the yeast Pichia acaciae arrests sensitive Saccharomyces cerevisiae cells in the S-phase of the cell cycle and induces mutations. Here we provide evidence for opposite effects in PaT resistance of homologous recombination (HR) and non-homologous end joining (NHEJ), the two alternative repair mechanisms acting on DNA double strand breaks (DSB). As mutants defective in genes of the RAD52 epistasis group react hypersensitive and cells lacking YKU70 or YKU80 are partially resistant, the yKu70/80 complex facilitates PaT toxicity, whereas HR is antagonistic. In contrast to yku70 and yku80, lif1 mutants, the latter being defective in the ligation step of NHEJ, are PaT sensitive, confining toxicity promoting effects of NHEJ to the DSB end binding Ku proteins. Since rad52 yku80 double mutants display strong hypersensitivity, yku80 mediated resistance depends on HR. Opposite effects of the yKu70/80 complex and HR are consistent with the occurrence of replication dependent (one sided) DSBs in PaT treated cells. Concordantly, two cellular markers signaling DSBs are induced during PaT mediated S-phase arrest, i.e. histone H2A phosphorylation and formation of subnuclear repair foci by GFP tagged recombination protein Rad52. As only moderate chromosome fragmentation could be detected by PFGE, transient occurrence and efficient in vivo repair of PaT induced DSBs is assumed. Consistent with replication dependent DSB formation induced by PaT, we demonstrate a protective function of the RecQ helicase Sgs1 and the structure specific endonuclease Mus81, both of which are considered to be involved in processing and restart of stalled replication forks.

Defective Mre11-dependent Activation of Chk2 by Ataxia Telangiectasia Mutated in Colorectal Carcinoma Cells in Response to Replication-dependent DNA Double Strand Breaks

The Mre11.Rad50.Nbs1 (MRN) complex binds DNA double strand breaks to repair DNA and activate checkpoints. We report MRN deficiency in three of seven colon carcinoma cell lines of the NCI Anticancer Drug Screen. To study the involvement of MRN in replication-mediated DNA double strand breaks, we examined checkpoint responses to camptothecin, which induces replication-mediated DNA double strand breaks after replication forks collide with topoisomerase I cleavage complexes. MRN-deficient cells were deficient for Chk2 activation, whereas Chk1 activation was independent of MRN. Chk2 activation was ataxia telangiectasia mutated (ATM)-dependent and associated with phosphorylation of Mre11 and Nbs1. Mre11 complementation in MRN-deficient HCT116 cells restored Chk2 activation as well as Rad50 and Nbs1 levels. Conversely, Mre11 down-regulation by small interference RNA (siRNA) in HT29 cells inhibited Chk2 activation and down-regulated Nbs1 and Rad50. Proteasome inhibition also restored Rad50 and Nbs1 levels in HCT116 cells suggesting that Mre11 stabilizes Rad50 and Nbs1. Chk2 activation was also defective in three of four MRN-proficient colorectal cell lines because of low Chk2 levels. Thus, six of seven colon carcinoma cell lines from the NCI Anticancer Drug Screen are functionally Chk2-deficient in response to replication-mediated DNA double strand breaks. We propose that Mre11 stabilizes Nbs1 and Rad50 and that MRN activates Chk2 downstream from ATM in response to replication-mediated DNA double strand breaks. Chk2 deficiency in HCT116 is associated with defective S-phase checkpoint, prolonged G2 arrest, and hypersensitivity to camptothecin. The high frequency of MRN and Chk2 deficiencies may contribute to genomic instability and therapeutic response to camptothecins in colorectal cancers.

ATR-Chk1 Axis Protects BCR/ABL Leukemia Cells from the Lethal Effect of DNA Double-Strand Breaks

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and MMC, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assay. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. Elevated levels of drug-induced DSBs in leukemia cells were associated with higher activity of ATR kinase, and enhanced phosphorylation of histone H2AX on serine 139 (γ-H2AX). γ-H2AX eventually started to disappear in BCR/ABL cells, while continued to increase in parental cells. In addition, the expression and ATR-mediated phosphorylation of Chk1 kinase on serine 345 were often more abundant in BCR/ABL-positive leukemia cells than normal counterparts after genotoxic treatment. Inhibition of ATR kinase by caffeine but not Chk1 kinase by indolocarbazole inhibitor, SB218078 sensitized BCR/ABL leukemia cells to MMC in a short-term survival assay. Nevertheless, both caffeine and SB218078 enhanced the genotoxic effect of MMC in a long-term clonogenic assay. This effect was associated with the abrogation of transient accumulation of leukemia cells in S and G2/M cell cycle phases after drug treatment. In conclusion, ATR - Chk1 axis was strongly activated in BCR/ABL-positive cells and contributed to the resistance to DNA cross-linking agents causing numerous replication-dependent DSBs.

ATR-Chk1 Axis Is Activated, but the Function of Chk1 Is Disrupted in BCR/ABL Leukemia Cells Responding to DNA Damage.

BCR/ABL-positive leukemia cells accumulated more replication-dependent DNA double-strand breaks (DSBs) than normal counterparts after treatment with cisplatin and mitomycin C, as assessed by pulse field gel electrophoresis (PFGE) and neutral comet assays. In addition, leukemia cells could repair these lesions more efficiently than normal cells and eventually survive genotoxic treatment. BCR/ABL leukemia cells displayed higher activity of ATR kinase, which is stimulated by replication-dependent DSBs. ATR phosphorylates histone H2AX on serine 139 (γ-H2AX) on megabase-length fragments near DSB sites. In accordance with PFGE and comet assay, BCR/ABL-positive leukemia cells accumulated more γ-H2AX protein and γ-H2AX nuclear foci than normal counterparts during 6h after genotoxic treatment. γ-H2AX started to disappear in BCR/ABL cells, while continued to increase in parental cells. Altogether, these results support the hypothesis that higher kinase activity of ATR in BCR/ABL leukemia cells results from their response to elevated levels of drug-induced replication-dependent DSBs, and that more efficient repair is associated with better survival. More abundant expression and prolonged phosphorylation on serine 345 of the Chk1 kinase, an important ATR kinase downstream effector and cell cycle regulator, was detected in leukemia cell lines and CML patient cells after genotoxic treatment. Interestingly, inhibition of ATR kinase by caffeine, but not Chk1 kinase by indolocarbazole inhibitor SB-218078 sensitized BCR/ABL leukemia cells to mitomycin C. In conclusion, although ATR - Chk1 axis appeared strongly activated in BCR/ABL leukemia cells responding to DNA cross-linking agents causing numerous replication-dependent DSBs, its function in drug resistance may be disassembled downstream of the Chk1 kinase.

RB signaling prevents replication-dependent DNA double-strand breaks following genotoxic insult.

Cell cycle checkpoints induced by DNA damage play an integral role in preservation of genomic stability by allowing cells to limit the propagation of deleterious mutations. The retinoblastoma tumor suppressor (RB) is crucial for the maintenance of the DNA damage checkpoint function because it elicits cell cycle arrest in response to a variety of genotoxic stresses. Although sporadic loss of RB is characteristic of most cancers and results in the bypass of the DNA damage checkpoint, the consequence of RB loss upon chemotherapeutic responsiveness has been largely uninvestigated. Here, we employed a conditional knockout approach to ablate RB in adult fibroblasts. This system enabled us to examine the DNA damage response of adult cells following acute RB deletion. Using this system, we demonstrated that loss of RB disrupted the DNA damage checkpoint elicited by either cisplatin or camptothecin exposure. Strikingly, this bypass was not associated with enhanced repair, but rather the accumulation of phosphorylated H2AX (gammaH2AX) foci, which indicate DNA double-strand breaks. The formation of gammaH2AX foci was due to ongoing replication following chemotherapeutic treatment in the RB-deficient cells. Additionally, peak gammaH2AX accumulation occurred in S-phase cells undergoing DNA replication in the presence of damage, and these gammaH2AX foci co-localized with replication foci. These results demonstrate that acute RB loss abrogates DNA damage-induced cell cycle arrest to induce gammaH2AX foci formation. Thus, secondary genetic lesions induced by RB loss have implications for the chemotherapeutic response and the development of genetic instability.

Phosphorylation of Histone H2AX and Activation of Mre11, Rad50, and Nbs1 in Response to Replication-dependent DNA Double-strand Breaks Induced by Mammalian DNA Topoisomerase I Cleavage Complexes

DNA double-strand breaks originating from diverse causes in eukaryotic cells are accompanied by the formation of phosphorylated H2AX (gammaH2AX) foci. Here we show that gammaH2AX formation is also a cellular response to topoisomerase I cleavage complexes known to induce DNA double-strand breaks during replication. In HCT116 human carcinoma cells exposed to the topoisomerase I inhibitor camptothecin, the resulting gammaH2AX formation can be prevented with the phosphatidylinositol 3-OH kinase-related kinase inhibitor wortmannin; however, in contrast to ionizing radiation, only camptothecin-induced gammaH2AX formation can be prevented with the DNA replication inhibitor aphidicolin and enhanced with the checkpoint abrogator 7-hydroxystaurosporine. This gammaH2AX formation is suppressed in ATR (ataxia telangiectasia and Rad3-related) deficient cells and markedly decreased in DNA-dependent protein kinase-deficient cells but is not abrogated in ataxia telangiectasia cells, indicating that ATR and DNA-dependent protein kinase are the kinases primarily involved in gammaH2AX formation at the sites of replication-mediated DNA double-strand breaks. Mre11- and Nbs1-deficient cells are still able to form gammaH2AX. However, H2AX-/- mouse embryonic fibroblasts exposed to camptothecin fail to form Mre11, Rad50, and Nbs1 foci and are hypersensitive to camptothecin. These results demonstrate a conserved gammaH2AX response for double-strand breaks induced by replication fork collision. gammaH2AX foci are required for recruiting repair and checkpoint protein complexes to the replication break sites.