Abstract
The highly conserved RAD51 protein has a central role in homologous recombination. Five novel RAD51-like genes have been identified in mammalian cells, but little is known about their functions. A DNA damage-sensitive hamster cell line, irs3, was found to have a mutation in the RAD51L2 gene and an undetectable level of RAD51L2 protein. Resistance of irs3 to DNA-damaging agents was significantly increased by expression of the human RAD51L2 gene, but not by other RAD51-like genes or RAD51 itself. Consistent with a role for RAD51L2 in homologous recombination, irs3 cells show a reduction in sister chromatid exchange, an increase in isochromatid breaks, and a decrease in damage-dependent RAD51 focus formation compared with wild type cells. As recently demonstrated for human cells, we show that RAD51L2 forms part of two separate complexes of hamster RAD51-like proteins. Strikingly, neither complex of RAD51-like proteins is formed in irs3 cells. Our results demonstrate that RAD51L2 has a key role in mammalian RAD51-dependent processes, contingent on the formation of protein complexes involved in homologous recombination repair.
Highlights
The use of cultured mammalian cell lines selected for sensitivity to DNA-damaging agents to identify genes and gene functions has had a major impact on our understanding of DNA repair pathways
To check that the altered profile of cDNA products in irs3 was related to the RAD51L2 gene defect, we assayed the RAD51L2 cDNA profiles in two other radiation-sensitive mutants isolated in the same screen from mutagenized V79 cells
Our data show a direct correlation between the presence of the RAD51L2 cDNA and resistance to MMC in the irs3 cells, while the RAD51L1 and RAD51L3 cDNAs showed no ability to complement
Summary
Gene Cloning and Vector Construction—The cloning of RAD51L1 and RAD51L3 was described previously [11]. The human RAD51 gene was cloned from the same cDNA library into pIRESneo. Filters were probed with full-length human RAD51L2 cDNA, and DNA from positive clones was isolated using a Qiagen large-construct kit. 10 g of the RAD51L1, RAD51L2, RAD51L3, or RAD51 constructs were electroporated into 106 irs cells (BioRad Gene Pulser set at 500 F, 400 V) and transfected clones were selected in the appropriate drugs. 15 g of PAC DNA was co-transfected with 2 g of the vector pEGFP-N2 (CLONTECH) into irs cells as described above, followed by selection in 500 g/ml G418. The presence of the appropriate RAD51L gene (cDNA or PAC) was tested in drug-resistant irs clones by PCR using cellular DNA, and gene expression was checked by reverse transcriptase (RT-) PCR (Superscript II, Invitrogen) of cellular RNA with oligo(dT) as the first-strand primer. Complexes were washed four times in lysis buffer and visualized by Western blotting using monoclonal antibodies
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