Department of Radiation Genetics, Kyoto University, Graduate School of Medicine, Yoshidakonoe, Sakyo-ku, Kyoto, Japan.
Cancer Res. 2010 Nov 1;70(21):8467-74. doi: 10.1158/0008-5472.CAN-10-0267. Epub 2010 Oct 19.
The tumor suppressor BRCA1 functions in DNA homologous recombination, and mutations in BRCA1 increase the risk of breast and ovarian cancers. RAP80 is a component of BRCA1-containing complexes that is required for recruitment of BRCA1 to sites of DNA damage. To evaluate the role of RAP80 in DNA damage repair, we genetically disrupted both RAP80 alleles in the recombinogenic avian DT40 cell line. The resulting RAP80(-/-) cells were proficient at homologous recombination and nonhomologous end-joining (NHEJ), but were specifically sensitized to the topoisomerase II inhibitor etoposide. Notably, doubly mutant RAP80(-/-)BRCA1(-/-) cells were more sensitive to etoposide than were BRCA1(-/-) cells, revealing that RAP80 performs a BRCA1-independent repair function. Moreover, jointly impairing the function of CtIP, a distinct BRCA1 effector protein, rendered RAP80(-/-) cells more sensitive to etoposide compared with singly mutant cells, again illustrating a BRCA1-independent role of RAP80. Based on our findings, we propose that RAP80 exerts a specific function in repair of the topoisomerase-cleavage complex, such as the removal of covalently bound polypeptides from double-strand break ends independently of BRCA1.
肿瘤抑制因子 BRCA1 参与 DNA 同源重组，BRCA1 突变会增加乳腺癌和卵巢癌的风险。RAP80 是 BRCA1 包含复合物的一个组成部分，对于 BRCA1 招募到 DNA 损伤部位是必需的。为了评估 RAP80 在 DNA 损伤修复中的作用，我们在具有重组能力的禽类 DT40 细胞系中遗传敲除了 RAP80 的两个等位基因。由此产生的 RAP80(-/-)细胞在同源重组和非同源末端连接（NHEJ）方面非常有效，但对拓扑异构酶 II 抑制剂依托泊苷（etoposide）具有特异性敏感性。值得注意的是，双突变 RAP80(-/-)BRCA1(-/-)细胞比 BRCA1(-/-)细胞对依托泊苷更为敏感，表明 RAP80 发挥了 BRCA1 非依赖性修复功能。此外，共同破坏 CtIP 的功能，一种独特的 BRCA1 效应蛋白，使 RAP80(-/-)细胞对依托泊苷的敏感性比单突变细胞更高，再次表明 RAP80 具有 BRCA1 非依赖性作用。基于我们的发现，我们提出 RAP80 在修复拓扑异构酶切割复合物方面发挥特定功能，例如独立于 BRCA1 从双链断裂末端去除共价结合的多肽。
