No abstract

Eukaryotic cells repair DNA double-strand breaks (DSBs) by at least two pathways, homologous recombination (HR) and non-homologous end-joining (NHEJ).Rad54 participates in the first recombinational repair pathway while Ku proteins are involved in NHEJ. To investigate the distinctive as well as redundant roles of these two repair pathways, we analyzed the mutants RAD54 -/-, KU70 -/-and RAD54 -/-/KU70 -/-, generated from the chicken B-cell line DT40. We found that the NHEJ pathway plays a dominant role in repairing γ-radiation-induced DSBs during G 1 -early S phase while recombinational repair is preferentially used in late S-G 2 phase. RAD54 -/-/KU70 -/-cells were profoundly more sensitive to γ-rays than either single mutant, indicating that the two repair pathways are complementary. Spontaneous chromosomal aberrations and cell death were observed in both RAD54 -/-and RAD54 -/-/KU70 -/-cells, with RAD54 -/-/KU70 -/-cells exhibiting significantly higher levels of chromosomal aberrations than RAD54 -/-cells. These observations provide the first genetic evidence that both repair pathways play a role in maintaining chromosomal DNA during the cell cycle.

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.

The RAD51 protein functions in the processes of DNA repair and in mitotic and meiotic genetic recombination in the yeast Saccharomyces cerevisiae. The protein has adenosine triphosphate-dependent DNA binding activities similar to those of the Escherichia coli RecA protein, and the two proteins have 30 percent sequence homology. RAD51 polymerized on double-stranded DNA to form a helical filament nearly identical in low-resolution, three-dimensional structure to that formed by RecA. Like RecA, RAD51 also appears to force DNA into a conformation of approximately a 5.1-angstrom rise per base pair and 18.6 base pairs per turn. As in other protein families, its structural conservation appears to be stronger than its sequence conservation. Both the structure of the protein polymer formed by RecA and the DNA conformation induced by RecA appear to be general properties of a class of recombination proteins found in prokaryotes as well as eukaryotes.

In Saccharomyces cerevisiae, the RAD51 and RAD52 genes are involved in recombination and in repair of damaged DNA. The RAD51 gene is a structural and functional homologue of the recA gene and the gene product participates in strand exchange and single-stranded-DNA-dependent ATP hydrolysis by means of nucleoprotein filament formation. The RAD52 gene is important in RAD51-mediated recombination. Binding of this protein to Rad51 suggests that they cooperate in recombination. Homologues of both Rad51 and Rad52 are conserved from yeast to humans, suggesting that the mechanisms used for pairing homologous DNA molecules during recombination may be universal in eukaryotes. Here we show that Rad52 protein stimulates Rad51 reactions and that binding to Rad51 is necessary for this stimulatory effect. We conclude that this binding is crucial in recombination and that it facilitates the formation of Rad51 nucleoprotein filaments.