Collaborative roles of γH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair

Sonoda, Eiichiro; Zhao, Guang Yu; Kohzaki, Masaoki; Dhar, Pawan K.; Kikuchi, Koji; Redon, Christophe E.; Pilch, Duane R.; Bonner, William M.; Nakano, Atsushi; Watanabe, Masami; Nakayama, Tatsuo; Takeda, Shunichi; Yasuda, Takami

doi:10.1016/j.dnarep.2006.10.025

Cited by 46 publications

(43 citation statements)

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“…The initial, rapid induction of g-H2AX foci after irradiation (IR) is followed by a reduction in the number of foci over several hours, presumably reflecting the loss of a DNA damage signal following repair (Paull et al, 2000). cMcph1 also localized to À/À (Zachos et al, 2003) and H2AX mutant (Sonoda et al, 2007) DT40 cells and stable transfections by electroporation were performed as described previously (Dodson et al, 2004). For transient transfections, 15 mg of endotoxin-free DNA (Qiagen, Crawley, UK) was introduced into 5 Â 10 6 cells using nucleofection (Amaxa, Cologne, Germany, programme B-23).…”

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confidence: 99%

Distinct BRCT domains in Mcph1/Brit1 mediate ionizing radiation-induced focus formation and centrosomal localization

et al. 2007

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Microcephalin (MCPH1/BRIT1) forms ionizing radiationinduced nuclear foci (IRIF) and is required for DNA damage-responsive S and G 2 -M-phase checkpoints. MCPH1 contains three BRCT domains. Here we report the cloning of chicken Mcph1 (cMcph1) and functional analysis of its individual BRCT domains. Full-length cMcph1 localized to centrosomes throughout the cell cycle and formed IRIF that colocalized with c-H2AX. The tandem C-terminal BRCT2 and BRCT3 domains of cMcph1 were necessary for IRIF formation, while the N-terminal BRCT1 was required for centrosomal localization in irradiated cells. Centrosomal targeting of cMcph1 was independent of ATM, Brca1 or Chk1. cMcph1 formed IRIF in ATM-and Brca1-deficient cells, but not in H2AX-deficient cells. Inability to form cMcph1 IRIF impaired the cellular response to DNA damage. These results suggest that the role of microcephalin in the vertebrate DNA damage response is controlled by interaction of the Cterminal BRCT domains with c-H2AX. The tumour suppressor BRCA1 is involved in numerous activities that maintain genome stability (Starita and Parvin, 2003). Crucial to the functions of BRCA1 are a pair of C-terminal domains, which can recognize phosphopeptides and mediate protein-protein interactions in the response to DNA damage (Manke et al., 2003;Yu et al., 2003). These BRCA1 C-terminal (BRCT) repeats are found in a large superfamily of proteins involved in cellular responses to genotoxic stress (Glover et al., 2004).One member of this BRCT domain-containing superfamily is microcephalin (MCPH1). MCPH1 is encoded by MCPH1/BRIT1 (hereafter MCPH1), a gene mutated in primary microcephaly (OMIM 251200) (Jack- (Rogakou et al., 1999). A recent study (Rai et al., 2006) found that depletion of MCPH1 in human cells blocked formation of IRIF by NBS1, 53BP1, phosphorylated ATM and MDC1, but not g-H2AX. DNA damage-induced chromatin association of several DNA damage response proteins was also blocked by MCPH1 RNAi and a high level of chromosome aberrations was observed. MCPH1 aberrations were frequently detected in breast, ovarian and prostate cancer (Rai et al., 2006). Together, these data suggest an important role for MCPH1 in DNA damage responses and in preventing cellular transformation.We identified the chicken MCPH1 orthologue by database analysis, cloned it by RT-PCR and confirmed the sequence (DDBJ/EMBL/GenBank accession no. DQ788861). Zebrafish Mcph1 sequence was derived by database analysis and these sequences were aligned with known mammalian Mcph1 sequences using ClustalW. As shown in Supplementary Figure S1, all vertebrate sequences analysed showed similar organization, with a highly conserved single N-terminal BRCT domain and a pair of C-terminal BRCT domains, which will be referred to here as BRCT1, BRCT2 and BRCT3, respectively. The conserved BRCT1 and BRCT2-BRCT3 regions are separated by a poorly conserved region that does not contain any distinct structural motifs, as determined by SMART domain search at http://smart.embl-heidelberg.de/. These findings suggest that k...

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confidence: 99%

Distinct BRCT domains in Mcph1/Brit1 mediate ionizing radiation-induced focus formation and centrosomal localization

et al. 2007

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“…Rad51 initiates strand invasion and promotes homologous pairing and strand exchange within a regular nucleoprotein filament during homologous recombination, which predominately takes place in the S/G 2 phases of the cell cycle (33,34). Studies on cells lacking H2AX showed that H2AX is not required for Rad51 focus formation (17,27). A similar cisplatin effect on Rad51 and on g-H2AX foci formation points to an inhibition of common upstream events.…”

Section: Discussionmentioning

confidence: 73%

“…g-H2AX foci are important platforms to concentrate repair proteins [e.g., Nbs, Mre11, Rad50, Mdc1, 53BP1, and ataxia-telangiectasia mutated kinase (ATM)] to double-strand break -flanking chromatin, which are also involved in the homologous recombination process (16). A distinct role of g-H2AX in Rad51 foci formation, a key protein of homologous recombination, has also been shown (17).…”

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confidence: 99%

Long-Term In vivo Effects of Cisplatin on γ-H2AX Foci Signaling in Peripheral Lymphocytes of Tumor Patients After Irradiation

Sak

Grehl

Engelhard

et al. 2009

Clinical Cancer Research

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Purpose: This study determined the effects of cis-diamminedichloroplatinum(II) on radiationinduced foci formation of g-H2AX and Rad51in lymphocytes. Experimental Design: Twenty-eight cancer patients were irradiated for intrathoracic, pelvic, or head and neck tumors and received simultaneous cisplatin containing chemotherapy. The effect of cisplatin on radiation-induced g-H2AX and Rad51 foci as a response to ionizing radiationî nduced DNA double-strand breaks was measured in lymphocytes after in vivo and in vitro radiochemotherapy.The role of DNA-dependent protein kinase and ataxia-telangiectasia mutated kinase in g-H2AX signaling, the consequences of altered g-H2AX foci formation on doublestrand break end joining, was studied. Results: Cisplatin decreased the number of induced g-H2AX foci in lymphocytes after in vivo or in vitro irradiation by 34% F 6% at days 0 to 3 after cisplatin (P < 0.0001) and remained significant until day 6. The variation in this cisplatin effect from patient to patient was larger than the retest error within the same patient (P = 0.01).The cisplatin effect was not accompanied by an inhibition of end joining of double-strand break as analyzed using gel electrophoresis of DNA under neutral conditions. Cisplatin also decreased radiation induced Rad51foci formation in lymphocytes after stimulation of proliferation with phytohemagglutinin by 47% F 6% (P < 0.0001).Conclusion: Cisplatin has long-term effects on the early double-strand break response of g-H2AX and Rad51 foci formation after ionizing radiation. Inhibition of sensing and processing of double-strand break by g-H2AX and Rad51foci formation are important mechanisms by which cisplatin can alter the radiation response.

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“…14 A recent study has identified the Ser-139 phosphorylated form of histone H2AX (γH2AX) has a collaborator of Xrcc3 in the Rad51 foci formation and repair of DSB induced by ionising radiation. 15 γH2AX is a sensitive marker of DSBs, 16 which accumulate following treatment with topoisomerase inhibitors causing DSBs at replication blockage. 17,18 As a consequence of DSBs accumulation, cell death pathways are turned on.…”

Section: Introductionmentioning

confidence: 99%

XRCC3 Depletion Induces Spontaneous DNA Breaks and p53-Dependent Cell Death

Loignon¹,

Amrein²,

Dunn³

et al. 2007

Cell Cycle

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AbbReviAtions DSBDNA double strand break SSB DNA single strand break HRR homologous recombination repair, NHEJ non homologous end joining γH2AX pSer139 H2AX AcKnowLeDgeMentsThis work was supported by the funding from the Weekend to End Breast Cancer-Jewish General Hospital Foundation to R.A. We thank Dr. Lawrence Panasci and Dr. Jean-Yves Masson for their support and advice. Report XRCC3 Depletion Induces Spontaneous DNA Breaks and p53-Dependent Cell Death AbstRActIn vertebrate cells, Xrcc3 initiates the repair of exogenous induced-DNA breaks during S and G 2 /M phases of the cell cycle by homologous recombination. However, much less is known of the role of Xrcc3 in the response to spontaneous DNA breaks. Using a siRNA approach, we show that depletion of XRCC3 inhibits the proliferation of MCF7 breast cancer cells. This inhibition of replication coincides with the accumulation of DNA breaks, as shown by the comet assay. Cell cycle specific analysis of γH2AX expression shows that S and G 2 /M phase cells express the highest fraction of γH2AX positive cells. This is consistent with replication-dependent accumulation of DNA breaks and deficient homologous recombination. While the induction of γH2AX is followed by cell death in parental cells, a p53 knockdown derivative becomes more resistant to XRCC3 depletion-induced death without changes in the levels of γH2AX. These results show that XRCC3 is required for the proliferation of MCF7 cells, and that decrease in its expression leads to the accumulation of DNA breaks and the induction of p53-dependent cell death.

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Collaborative roles of γH2AX and the Rad51 paralog Xrcc3 in homologous recombinational repair

Cited by 46 publications

References 53 publications

Distinct BRCT domains in Mcph1/Brit1 mediate ionizing radiation-induced focus formation and centrosomal localization

Distinct BRCT domains in Mcph1/Brit1 mediate ionizing radiation-induced focus formation and centrosomal localization

Long-Term In vivo Effects of Cisplatin on γ-H2AX Foci Signaling in Peripheral Lymphocytes of Tumor Patients After Irradiation

XRCC3 Depletion Induces Spontaneous DNA Breaks and p53-Dependent Cell Death

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