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. 2001 Apr;21(8):2858-66.
doi: 10.1128/MCB.21.8.2858-2866.2001.

Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs

M Takata  1 , M S Sasaki, S Tachiiri, T Fukushima, E Sonoda, D Schild, L H Thompson, S Takeda
Affiliations

VSports注册入口 - Chromosome instability and defective recombinational repair in knockout mutants of the five Rad51 paralogs

"V体育官网" M Takata et al. Mol Cell Biol. 2001 Apr.

Abstract

The Rad51 protein, a eukaryotic homologue of Escherichia coli RecA, plays a central role in both mitotic and meiotic homologous DNA recombination (HR) in Saccharomyces cerevisiae and is essential for the proliferation of vertebrate cells. Five vertebrate genes, RAD51B, -C, and -D and XRCC2 and -3, are implicated in HR on the basis of their sequence similarity to Rad51 (Rad51 paralogs). We generated mutants deficient in each of these proteins in the chicken B-lymphocyte DT40 cell line and report here the comparison of four new mutants and their complemented derivatives with our previously reported rad51b mutant. The Rad51 paralog mutations all impair HR, as measured by targeted integration and sister chromatid exchange. Remarkably, the mutant cell lines all exhibit very similar phenotypes: spontaneous chromosomal aberrations, high sensitivity to killing by cross-linking agents (mitomycin C and cisplatin), mild sensitivity to gamma rays, and significantly attenuated Rad51 focus formation during recombinational repair after exposure to gamma rays VSports手机版. Moreover, all mutants show partial correction of resistance to DNA damage by overexpression of human Rad51. We conclude that the Rad51 paralogs participate in repair as a functional unit that facilitates the action of Rad51 in HR. .

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Figures

FIG. 1
FIG. 1
Level of spontaneous death in cells defective in Rad51 paralogs. (A) Cell viability was assessed by flow cytometric analysis using PI uptake (y axis) and forward scatter, representing cell size (x axis). Numbers show the percentage of dead cells (PI bright and PI dim or small), and the solid line separates live from dead cells. (B) Bars represent the level of spontaneous cell death in indicated genotypes. Complemented mutant clones were transfected with the corresponding human (rad51c, xrcc2, and xrcc3) or mouse (rad51d) cDNA. The means and standard deviations for three independent experiments are shown. WT, wild type.
FIG. 2
FIG. 2
Levels of SCE per cell before and after MMC treatment. For each preparation, 150 cells were analyzed. The mean number of SCEs per cell is indicated in each panel. The comparison of each Rad51 paralog mutant with a wild-type (WT) control cell is statistically significant (P < 0.001, Bonferroni/Dunn test).
FIG. 3
FIG. 3
Sensitivity of knockout cell lines to DNA-damaging agents. (A) Survival curves after treatments with gamma radiation and MMC. Data shown are representative of at least three independent experiments. Sensitivity data of rad51b cells was taken from our previous study (59). (B) Partial correction of cisplatin sensitivity in knockout mutants by overexpression of human Rad51. Data shown are representative of at least three independent experiments. (C) Western blot analysis of human Rad51 transformants derived from knockout mutants. Transformants have much higher steady-state levels of cDNA-derived human Rad51 than endogenous Rad51. Although highly conserved (95.6% identity), human Rad51 migrates slightly faster than the chicken counterpart in SDS-PAGE analysis (55). Given this high degree of conservation, it is likely that the antibody used, which was made against human Rad51, efficiently recognizes the chicken homolog. WT, wild type.
FIG. 4
FIG. 4
Immunofluorescence visualization of Rad51 subnuclear foci after irradiation (8 Gy). A, wild-type; B and F, rad51c; C and G, rad51d; D and H, xrcc2; E and I, xrcc3. F to I, mutant cells complemented with the corresponding human (rad51c, xrcc2, and xrcc3) or mouse (rad51d) cDNA.
FIG. 5
FIG. 5
Induction of Rad51 foci by IR treatments. Cells were analyzed at the indicated time points after gamma irradiation (8 Gy). (A) A cell containing more than four distinct foci was scored as positive. Each bar represents the results of scoring at least 100 cells. (B) Average number of Rad51 foci per cell in cells scored as positive at 8 h after 8 Gy of IR.
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