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...

Chromosome 6 may harbor a repressor of hTERT transcription, the loss of which may be involved in HPV-mediated immortalization.

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DNA-dependent protein kinase (DNA-PK) plays an important role in DNA double-strand break (DSB) repair and V(D)J recombination. We have isolated a new X-ray-sensitive CHO cell line, XR-C1, which is impaired in DSB repair and which was assigned to complementation group 7, the group that is defective in the XRCC7 / SCID ( Prkdc ) gene encoding the catalytic subunit of DNA-PK (DNA-PKcs). Consistent with this complementation analysis, XR-C1 cells lackeddetectable DNA-PKcs protein, did not display DNA-PK catalytic activity and were complemented by the introduction of a single human chromosome 8 (providing the Prkdc gene). The impact of the XR-C1 mutation on V(D)J recombination was quite different from that found in most rodent cells defective in DNA-PKcs, which are preferentially blocked in coding joint formation, whereas XR-C1 cells were defective in forming both coding and signal joints. These results suggest that DNA-PKcs is required for both coding and signal joint formation during V(D)J recombination and that the XR-C1 mutant cell line may prove to be a useful tool in understanding this pathway.

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