Lee Min-Soo, Yu Mi, Kim Kyoung-Yeon, Park Geun-Hee, Kwack KyuBum, Kim Keun P
Department of Life Science, Chung-Ang University, Seoul, Korea.
Department of Biomedical Science, CHA University, Seongnam, Korea.
PLoS One. 2015 May 4;10(5):e0124152. doi: 10.1371/journal.pone.0124152. eCollection 2015.
Systems for the repair of DNA double-strand breaks (DSBs) are necessary to maintain genome integrity and normal functionality of cells in all organisms. Homologous recombination (HR) plays an important role in repairing accidental and programmed DSBs in mitotic and meiotic cells, respectively. Failure to repair these DSBs causes genome instability and can induce tumorigenesis. Rad51 and Rad52 are two key proteins in homologous pairing and strand exchange during DSB-induced HR; both are highly conserved in eukaryotes. In this study, we analyzed pathogenic single nucleotide polymorphisms (SNPs) in human RAD51 and RAD52 using the Polymorphism Phenotyping (PolyPhen) and Sorting Intolerant from Tolerant (SIFT) algorithms and observed the effect of mutations in highly conserved domains of RAD51 and RAD52 on DNA damage repair in a Saccharomyces cerevisiae-based system. We identified a number of rad51 and rad52 alleles that exhibited severe DNA repair defects. The functionally inactive SNPs were located near ATPase active site of Rad51 and the DNA binding domain of Rad52. The rad51-F317I, rad52-R52W, and rad52-G107C mutations conferred hypersensitivity to methyl methane sulfonate (MMS)-induced DNA damage and were defective in HR-mediated DSB repair. Our study provides a new approach for detecting functional and loss-of-function genetic polymorphisms and for identifying causal variants in human DNA repair genes that contribute to the initiation or progression of cancer.
DNA双链断裂（DSB）修复系统对于维持所有生物体中细胞的基因组完整性和正常功能至关重要。同源重组（HR）分别在有丝分裂和减数分裂细胞中修复偶然和程序性DSB方面发挥着重要作用。无法修复这些DSB会导致基因组不稳定，并可能诱发肿瘤发生。Rad51和Rad52是DSB诱导的HR过程中同源配对和链交换的两个关键蛋白；二者在真核生物中高度保守。在本研究中，我们使用多态性表型分析（PolyPhen）和容忍度不耐受排序（SIFT）算法分析了人类RAD51和RAD52中的致病性单核苷酸多态性（SNP），并在基于酿酒酵母的系统中观察了RAD51和RAD52高度保守结构域中的突变对DNA损伤修复的影响。我们鉴定出了一些表现出严重DNA修复缺陷的rad51和rad52等位基因。功能失活的SNP位于Rad51的ATP酶活性位点附近以及Rad52的DNA结合结构域。rad51-F317I、rad52-R52W和rad52-G107C突变赋予了对甲磺酸甲酯（MMS）诱导的DNA损伤的超敏性，并且在HR介导的DSB修复中存在缺陷。我们的研究为检测功能性和功能丧失性基因多态性以及鉴定导致癌症发生或进展的人类DNA修复基因中的因果变异提供了一种新方法。
