Yeast rad51 mutants are viable, but extremely sensitive to γ-rays due to defective repair of double-strand breaks. In contrast, disruption of the murine RAD51 homologue is lethal, indicating an essential role of Rad51 in vertebrate cells. We generated clones of the chicken B lymphocyte line DT40 carrying a human RAD51 transgene under the control of a repressible promoter and subsequently disrupted the endogenous RAD51 loci. Upon inhibition of the RAD51 transgene, Rad51 -cells accumulated in the G 2 /M phase of the cell cycle before dying. Chromosome analysis revealed that most metaphase-arrested Rad51 -cells carried isochromatid-type breaks. In conclusion, Rad51 fulfils an essential role in the repair of spontaneously occurring chromosome breaks in proliferating cells of higher eukaryotes.
rad54 mutants of the yeast Saccharomyces cerevisiae are extremely X-ray sensitive and have decreased mitotic recombination frequencies because of a defect in double-strand break repair. A RAD54 homolog was disrupted in the chicken B cell line DT40, which undergoes immunoglobulin gene conversion and exhibits unusually high ratios of targeted to random integration after DNA transfection. Homozygous RAD54-/- mutant clones were highly X-ray sensitive compared to wildtype cells. The rate of immunoglobulin gene conversion was 6- to 8-fold reduced, and the frequency of targeted integration was at least two orders of magnitude decreased in the mutant clones. Reexpression of the RAD54 cDNA restored radiation resistance and targeted integration activity. The reported phenotype provides the first genetic evidence of a link between double-strand break repair and homologous recombination in vertebrate cells.
Homologous recombination is a versatile DNA damage repair pathway requiring Rad51 and Rad54. Here we show that a mammalian Rad54 paralog, Rad54B, displays physical and functional interactions with Rad51 and DNA that are similar to those of Rad54. While ablation of Rad54 in mouse embryonic stem (ES) cells leads to a mild reduction in homologous recombination efficiency, the absence of Rad54B has little effect. However, the absence of both Rad54 and Rad54B dramatically reduces homologous recombination efficiency. Furthermore, we show that Rad54B protects ES cells from ionizing radiation and the interstrand DNA cross-linking agent mitomycin C. Interestingly, at the ES cell level the paralogs do not display an additive or synergic interaction with respect to mitomycin C sensitivity, yet animals lacking both Rad54 and Rad54B are dramatically sensitized to mitomycin C compared to either single mutant. This suggests that the paralogs possibly function in a tissue-specific manner. Finally, we show that Rad54, but not Rad54B, is needed for a normal distribution of Rad51 on meiotic chromosomes. Thus, even though the paralogs have similar biochemical properties, genetic analysis in mice uncovered their nonoverlapping roles.DNA double-strand breaks (DSBs) are among a plethora of lesions that threaten the integrity of the genome. If not properly processed, DSBs can lead to cell cycle arrest or illegitimate DNA rearrangements such as translocations, inversions, or deletions. These rearrangements can contribute to cell dysfunction, cell death, or carcinogenesis (22). DSBs can arise through the action of exogenous DNA-damaging agents, but they also arise from endogenous sources, such as oxidative DNA damage and as a consequence of DNA replication (10,22). Homologous recombination is a major DNA repair pathway by which DSBs are repaired. Homologous recombination is generally a precise way of resolving DSBs, because it uses homologous sequence, usually provided on the sister chromatid, as a repair template (54).Homologous recombination is a complex process requiring a number of proteins of the RAD52 epistasis group, including Rad51 and Rad54. Rad51 is the key player in this process because it is critical for homology recognition and performs strand exchange between recombining DNA molecules. A pivotal intermediate in these reactions is the Rad51 nucleoprotein filament. This forms when Rad51 polymerizes on singlestranded DNA that results from DNA damage processing (54). Rad54 is an important accessory factor for Rad51 (56). A number of biochemical characteristics of Rad54 have been well defined for different species ranging from yeasts to humans (8,18,24,31,37,38,42,47,48,53,55,59). Rad54 is a doublestranded-DNA-dependent ATPase that can translocate on DNA, thereby affecting DNA topology. Biochemically, Rad54 has been implicated in participation in multiple steps of homologous recombination. It can stabilize the Rad51 nucleoprotein filament in an early stage of recombination (30). At a subsequent stage it can promote chromatin rem...
Rad52 plays a pivotal role in double-strand break (DSB) repair and genetic recombination in Saccharomyces cerevisiae, where mutation of this gene leads to extreme X-ray sensitivity and defective recombination. Yeast Rad51 and Rad52 interact, as do their human homologues, which stimulates Rad51-mediated DNA strand exchange in vitro, suggesting that Rad51 and Rad52 act cooperatively. To define the role of Rad52 in vertebrates, we generated RAD52 ؊/؊ mutants of the chicken B-cell line DT40. Surprisingly, RAD52 ؊/؊ cells were not hypersensitive to DNA damages induced by ␥-irradiation, methyl methanesulfonate, or cis-platinum(II)diammine dichloride (cisplatin). Intrachromosomal recombination, measured by immunoglobulin gene conversion, and radiation-induced Rad51 nuclear focus formation, which is a putative intermediate step during recombinational repair, occurred as frequently in RAD52؊/؊ cells as in wild-type cells. Targeted integration frequencies, however, were consistently reduced in RAD52 ؊/؊ cells, showing a clear role for Rad52 in genetic recombination. These findings reveal striking differences between S. cerevisiae and vertebrates in the functions of RAD51 and RAD52.The many strategies that have evolved to deal with DNA damage attest to the vital importance of chromosomal integrity to all organisms. Double-strand breaks (DSBs) can lead to immediate cell death if unrepaired or to chromosomal loss or translocation if not repaired correctly. Aside from environmentally induced damage, DSBs of genomic DNA are generated during several biological processes such as meiotic recombination or the development of the vertebrate immune system. Accordingly, the enzymes and systems of DSB repair are of great interest in biology.The RAD52 epistasis group of genes (RAD50, RAD51, RAD52, RAD54, RAD55, RAD57, RAD59, MRE11, and XRS2) has been defined by the respective Saccharomyces cerevisiae mutants, which are hypersensitive to ionizing radiation and exhibit mitotic and meiotic recombination defects. While phenotypic differences between mutants distinguish between these genes genetically, it is clear that they are constituents of a pathway for the repair of DSB damage by homologous recombination (reviewed in references 10, 22, and 27). The high degree of conservation of the RAD52 group of genes from yeast to vertebrates (4,8,14,23,25) suggests a similar role for these proteins. However, while vertebrate RAD54 Ϫ/Ϫ cells reflect the yeast phenotype, namely, extreme sensitivity to ␥-ray and defective recombination (5, 9), RAD51 deficiency results in the death of vertebrate cells, indicating that Rad51 is essential for cell proliferation (16,29,34).Rad52 mutants show the most pronounced phenotype among RAD52 epistasis group mutants in S. cerevisiae. Rad52 is essential for an intermediate stage after the formation of DSBs but before the appearance of stable recombinants during gene conversion (26). Rad52 is also involved in single-strand annealing (13) and other RAD51-independent forms of recombination (30), which may explain the ...
The tissue-specific expression of mHR54 is consistent with a role for the gene in recombination. The complementation experiments show that the DNA repair function of Rad54 is conserved from yeast to humans. Our findings underscore the fundamental importance of DNA repair pathways: even though they are complex and involve multiple proteins, they seem to be functionally conserved throughout the eukaryotic kingdom.
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