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

同源重组介导的DNA修复是促进基因组稳定和预防肿瘤发生的重要机制。在这个项目中，我们破坏了鸡细胞系DT40中可能存在的HR调控因子，包括5个Rad51类似物和Fanconi贫血基因。(1)缺乏每一种Rad51类似物的细胞都表现出非常相似的HR缺陷，如顺铂敏感性、染色体畸变增加、Rad51病灶形成或基因靶向效率的丧失。由于Rad51平行体相互作用，这些结果表明，Rad51平行体作为一个功能单元共同作用，通过Rad51介导HR反应。(2)我们发现Rad52 （HR因子）和Xrcc3 （Rad51的一个类似物）在DT40细胞中表现出合成致死性，这表明这两个分子之间存在一些重叠的功能。(3)缺乏FA基因的DT40突变体表现出HR缺陷，如基因靶向效率降低，免疫球蛋白位点基因转换频率降低，限制性内切酶诱导的染色体双链断裂的HR修复受损。这些结果首次正式证明了FA基因在HR中的作用。(4)我们发现，在SCE频率和对MMC或顺铂的敏感性方面，BLM解旋酶和FA途径之间存在优势。BLM解旋酶似乎是FA通路的效应分子之一。

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.