Functional analysis of genes involved in maintenance of chromosome stability and homologous recombination

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DNA repair mediated by homologous recombination (HR) is an important mechanism in promoting genome stability and preventing carcinogenesis. In this project, we disrupted putative HR regulators including five Rad51 paralogs and Fanconi anemia genes in chicken cell line DT40.(1) Cells lacking each of Rad51 paralogs all displayed very similar HR defects such as cisplatin sensitivity, increased chromosomal aberrations, and loss of Rad51 focus formation or gene targeting efficiencies. Since Rad51 paralogs interact with each other, these results suggest that Rad51 paralogs work together as a functional unit in mediating HR reactions through Rad51.(2) We found that Rad52 (an HR factor) and Xrcc3 (one of Rad51 paralogs) exhibited synthetic lethality in DT40 cells, indicating that there are some overlapping functions between these two molecules.(3) DT40 mutants lacking FA genes displayed HR defects such as decreased gene targeting efficiencies, a decreased gene conversion frequency at immunoglobulin locus, and impaired HR repair of restriction enzyme-induced chromosonal double strand breaks. These results were the first formal demonstration of the role of FA genes in HR.(4) We showed epistasis between BLM helicase and the FA pathway in terms of SCE frequency and sensitivities to MMC or cisplatin. BLM helicase seems to be one of the effector molecules of the FA pathway.