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.

FIG. 1.

(a) Immunofluorescent visualization of Rad51 nuclear foci: wild-type V79 (A to C), V-C8 (D to F), V-C8 cells with human chromosome 13 providing the BRCA2 gene (V-C8+#13) (G to I), and V-C8 containing a BAC with the murine Brca2 gene (V-C8+Brca2) (J to L). Cells were analyzed for 8 h after treatment with either 12 Gy of X rays (B, E, H, and K) or 2.4 μg of MMC/ml for 1 h (C, F, I, and L). (b) Quantification of Rad51 focus-positive cells. A cell containing more than five distinct foci was considered positive. Each bar represents the result of scoring at least 100 cells. The error bars represent the standard error of the mean (SEM).

FIG. 2.

Cell survival after exposure to MMC (a), X rays (b), or MMS (c) of wild-type V79 (⧫), V-C8 (•), V-C8 cells with human chromosome 13 providing the BRCA2 gene (V-C8+#13) (shaded area, four independent clones), and V-C8 containing a BAC with the murine Brca2 gene (V-C8+Brca2, two independent clones; ▵ and ▿). The error bars represent the SEM.

FIG. 3.

Dose-response curve of the rate of DNA synthesis after gamma-irradiation in V-C8 cells with human chromosome 13 providing the BRCA2 gene. The curves shown are for wild-type V79 cells (⧫), V-C8 cells (•), V-C8+#13 (□, two independent clones), and the AT-like hamster cell line V-E5 (⧫). The error bars represent the SEM.

FIG. 4.

Immunoblot analysis of the hamster Brca2 protein in V-C8 and V79 cells with antibody pep-4 raised against the 13 most carboxy-terminal amino acids of the mouse Brca2 protein (a) and the human BRCA2 protein in V-C8+#13 microcell hybrids with antibody Ab-1, raised against amino acids 1651 to 1821 of human BRCA2 (b). The wild-type simian virus 40-transformed human fibroblast cell line MRC5V1 was used as a positive control.

FIG. 5.

(a) Centrosome analysis in interphase cells of wild-type V79, Brca2-deficient V-C8, V-C8 with human chromosome 13 providing the BRCA2 gene (V-C8+#13, three independent clones), and V-C8 containing a BAC with the murine Brca2 gene (V-C8+Brca2, two independent clones). (b) Immunofluorescent visualization of normal centrosomes in V79 cells (left panel) and abnormally structured centrosomes in V-C8 cells (right panel).

FIG. 6.

Subcellular localization of Rad51-GFP in V-C8 cells. Stable transfectants of wild-type V79 or V-C8 cells expressing Rad51-GFP were analyzed by immunofluorescence (a) or immunoblotting with anti-Rad51 antibodies (b) at 6 h after mock treatment or irradiation with 12 Gy of gamma-irradiation.