Brca2 (XRCC11) Deficiency Results in Radioresistant DNA Synthesis and a Higher Frequency of Spontaneous Deletions

Abstract

We show here that the radiosensitive Chinese hamster cell mutant (V-C8) of group XRCC11 is defective in the breast cancer susceptibility gene Brca2. The very complex phenotype of V-C8 cells is complemented by a single human chromosome 13 providing the BRCA2 gene, as well as by the murine Brca2 gene. The Brca2 deficiency in V-C8 cells causes hypersensitivity to various DNA-damaging agents with an extreme sensitivity toward interstrand DNA cross-linking agents. Furthermore, V-C8 cells show radioresistant DNA synthesis after ionizing radiation, suggesting that Brca2 deficiency affects cell cycle checkpoint regulation. In addition, V-C8 cells display tremendous chromosomal instability and a high frequency of abnormal centrosomes. The mutation spectrum at the hprt locus showed that the majority of spontaneous mutations in V-C8 cells are deletions, in contrast to wild-type V79 cells. A mechanistic explanation for the genome instability phenotype of Brca2-deficient cells is provided by the observation that the nuclear localization of the central DNA repair protein in homologous recombination, Rad51, is reduced in V-C8 cells.V-C8 is a Chinese hamster cell mutant that represents the XRCC11 complementation group, among X-ray-sensitive rodent cell mutants, as well as a distinguished group among mitomycin C (MMC)-sensitive rodent cell mutants (for a review, see reference 46). This mutant is extremely sensitive to cross-linking agents, but it also shows an increased sensitivity toward many other DNA-damaging agents, such as methyl methanesulfonate (MMS) and UV light (19,48). This suggests that the XRCC11 gene is involved in a wide-ranging cellular response induced by various types of DNA damage. V-C8 cells display radioresistant DNA synthesis (RDS) after ionizing irradiation (36), which is indicative of a defect in DNA damage recognition or cell cycle checkpoint regulation. However, the high level of spontaneous and cross-link-induced chromosomal aberrations manifested by V-C8 cells (19) indicates a possible defect in DNA repair. Indeed, V-C8 cells have an impaired capacity for repair of DNA double-strand breaks (DSBs) after irradiation (36).In mammalian cells, DSBs are repaired via either nonhomologous end joining (NHEJ) or homologous recombination (HR) (reviewed in reference 14). Genetic complementation studies have determined that V-C8 cells are not defective in DNA-PKcs, Ku80, or Xrcc4, key components of NHEJ, nor in Xrcc2 or Xrcc3, proteins involved in HR, and that this mutant represents a separate complementation group, XRCC11 (36,46). When compared to cell lines defective in the above-mentioned proteins, the overall phenotype of V-C8 cells more closely resembles those of the Xrcc2-and Xrcc3-defective hamster cell lines irs1 and irs1SF, respectively (8,12,16). In common with V-C8, irs1 and irs1SF exhibit an extreme sensitivity to cross-linking agents that is not observed in cell lines defective in NHEJ proteins. This favors the hypothesis that the XRCC11 gene defective in V-C8 cells might be involved in HR. A key playe...

Minding the gap: The underground functions of BRCA1 and BRCA2 at stalled replication forks

“…This review has not touched upon the contributions of BRCA1 and BRCA2 to checkpoint functions [150][151][152], transcriptional regulation [153,154], gene silencing [81,82,155], telomere function [96] or (for BARD1) mRNA processing [156,157]. Both HR and non-HR functions of BRCA genes might account for the tissue specificity of cancer risk in BRCA gene mutation carriers [158] and the latter may be important modulators of the clinical phenotypes associated with BRCA gene inactivation.…”

DSS1 is required for the stability of BRCA2

“…Extensive studies have indicated that BRCA2 has an important function in DNA repair (Connor et al, 1997;Mizuta et al, 1997;Sharan et al, 1997;Wong et al, 1997;Chen et al, 1998;Patel et al, 1998). Recent reports indicate that BRCA2 acts in the homology-directed recombinational repair of double-strand DNA breaks (Davies et al, 2001;Moynahan et al, 2001;Xia et al, 2001;Kraakman-van der Zwet et al, 2002). The finding that BRCA2 is mutated in Faconi anemia patients further indicates that BRCA2 functions in a DNA damage-repair pathway (Howlett et al, 2002).…”

Deficiency in BRCA2 leads to increase in non-conservative homologous recombination

“…This cell line shows extreme sensitivity towards DNA crosslinking agents such as mitomycin C (MMC), increased sensitivity to X-rays (Overkamp et al, 1993) and display high chromosomal instability (Overkamp et al, 1993;Kraakman-van der Zwet et al, 2002). V-C8 cells have an impaired capacity of repair of DNA DSBs following g-irradiation (Verhaegh et al, 1995) and are defective in Rad51-foci formation after DNA damage (Kraakman-van der Zwet et al, 2002), suggesting that they are defective in Rad51-dependent recombination pathways.…”

“…The radiosensitive Chinese hamster cell mutant V-C8 has been recently shown to be defective in the BRCA2 gene (Kraakman-van der Zwet et al, 2002). This cell line shows extreme sensitivity towards DNA crosslinking agents such as mitomycin C (MMC), increased sensitivity to X-rays (Overkamp et al, 1993) and display high chromosomal instability (Overkamp et al, 1993;Kraakman-van der Zwet et al, 2002).…”

“…The V-C8 cell line with nonfunctional BRCA2 status is derived from Chinese hamster V79 fibroblasts (Zdzienicka et al, 1990;Overkamp et al, 1993;Kraakman-van der Zwet et al, 2002). To investigate whether the frequency and proportions of HR events are modulated in V-C8 cells, we examined the ability of these cells to perform recombination between two intrachromosomal sequences.…”