Repression of mutagenesis by Rad51D-mediated homologous recombination. Nucleic Acids Res

Biosciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94551, USA.
Nucleic Acids Research (Impact Factor: 9.11). 02/2006; 34(5):1358-68. DOI: 10.1093/nar/gkl020
Source: PubMed


Homologous recombinational repair (HRR) restores chromatid breaks arising during DNA replication and prevents chromosomal rearrangements that can occur from the misrepair of such breaks. In vertebrates, five Rad51 paralogs are identified that contribute in a nonessential but critical manner to HRR proficiency. We constructed and characterized a knockout of the paralog Rad51D in widely studied CHO cells. The rad51d mutant (clone 51D1) displays sensitivity to a diverse spectrum of induced DNA damage including gamma-rays, ultraviolet (UV)-C radiation, and methyl methanesulfonate (MMS), indicating the broad relevance of HRR to genotoxicity. Spontaneous chromatid breaks/gaps and isochromatid breaks are elevated 3- to 12-fold, but the chromosome number distribution remains unchanged. Most importantly, 51D1 cells exhibit a 12-fold-increased rate of hprt mutation, as well as 4- to 10-fold increased rates of gene amplification at the dhfr and CAD loci, respectively. Xrcc3 irs1SF cells from the same parental CHO line show similarly elevated mutagenesis at these three loci. Collectively, these results confirm the a priori expectation that HRR acts in an error-free manner to repress three classes of genetic alterations (chromosomal aberrations, loss of gene function and increased gene expression), all of which are associated with carcinogenesis.

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    • "HR-deficient mutants often show a mutator phenotype. For example, Hintz et al. (2006) reported that Rad51D mutant cells exhibit a 12-fold increase in the rate of spontaneous Hprt-deficient mutations [25]. If a drug partially inhibits HR and causes an increase in somatic mutation frequencies, the combination of radiation and the drug can result in an increased risk of secondary tumorigenesis. "
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    ABSTRACT: Ionizing radiation induces DNA double-strand breaks (DSBs). Mammalian cells repair DSBs through multiple pathways, and the repair pathway that is utilized may affect cellular radiation sensitivity. In this study, we examined effects on cellular radiosensitivity resulting from functional alterations in homologous recombination (HR). HR was inhibited by overexpression of the forkhead-associated (FHA) domain-mutated NBS1 (G27D/R28D: FHA-2D) protein in HeLa cells or in hamster cells carrying a human X-chromosome. Cells expressing FHA-2D presented partially (but significantly) HR-deficient phenotypes, which were assayed by the reduction of gene conversion frequencies measured with a reporter assay, a decrease in radiation-induced Mre11 foci formation, and hypersensitivity to camptothecin treatments. Interestingly, ectopic expression of FHA-2D did not increase the frequency of radiation-induced somatic mutations at the HPRT locus, suggesting that a partial reduction of HR efficiency has only a slight effect on genomic stability. The expression of FHA-2D rendered the exponentially growing cell population slightly (but significantly) more sensitive to ionizing radiation. This radiosensitization effect due to the expression of FHA-2D was enhanced when the cells were irradiated with split doses delivered at 24-h intervals. Furthermore, enhancement of radiation sensitivity by split dose irradiation was not seen in contact-inhibited G0/G1 populations, even though the cells expressed FHA-2D. These results suggest that the FHA domain of NBS1 might be an effective molecular target that can be used to induce radiosensitization using low molecular weight chemicals, and that partial inhibition of HR might improve the effectiveness of cancer radiotherapy.
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    • "Rad54 knockout mice cells show little or no reduction in spontaneous SCE, but there is a noticeable deficiency in MMC-induced SCE [28,29]. Moreover, some RAD51 paralog mutants show modest reductions in SCE, but isogenic rad51d mutant lines in both chinese hamster ovary and mouse fibroblasts show no decrease in spontaneous SCE [29,30]. Consistent with these studies, we observed that BRCA1 has no obvious role in spontaneous SCE (unpublished data), although BRCA1 promotes replication-stress induced SCE. "
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    ABSTRACT: Germ line mutations in breast cancer gene 1 (BRCA1) predispose women to breast and ovarian cancers. Although BRCA1 is involved in many important biological processes, the function of BRCA1 in homologous recombination (HR) mediated repair is considered one of the major mechanisms contributing to its tumor suppression activity, and the cause of hypersensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors when BRCA1 is defective. Mounting evidence suggests that the mechanism of repairing DNA double strand breaks (DSBs) by HR is different than the mechanism operating when DNA replication is blocked. Although BRCA1 has been recognized as a central component in HR, the precise role of BRCA1 in HR, particularly under replication stress, has remained largely unknown. Given the fact that DNA lesions caused by replication blockages are the primary substrates for HR in mitotic cells, functional analysis of BRCA1 in HR repair in the context of replication stress should benefit our understanding of the molecular mechanisms underlying tumorigenesis associated with BRCA1 deficiencies, as well as the development of therapeutic approaches for cancer patients carrying BRCA1 mutations or reduced BRCA1 expression. This review focuses on the current advances in this setting and also discusses the significance in tumorigenesis and cancer therapy.
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    • "Complementation of Rad51D in deficient cells conferred about 50-fold more resistance to MMC (Figure 4A and Table 2), whereas XRCC3-deficient cells were sensitized more than 160-fold to MMC compared to wild-type cells (Figure 4B and Table 2). The IC50 values in both Rad51D- and XRCC3-deficient cells were close to 0.02 µg/mL (equivalent to 0.06 µmol/L), consistent with the original studies with these cell lines[20],[21]. These results clearly demonstrated that the cell lines were valid for evaluating the functional role of HR proteins in DNA damage repair after nucleoside analogs. "
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