Department of Pathology, Yale University School of Medicine, New Haven, CT, USA.
Blood. 2011 May 12;117(19):5078-87. doi: 10.1182/blood-2010-10-311761. Epub 2011 Feb 25.
Fanconi anemia (FA) is a rare genetic disorder characterized by bone marrow failure, congenital abnormalities, and an increased risk for cancer and leukemia. Components of the FA-BRCA pathway are thought to function in the repair of DNA interstrand cross-links. Central to this pathway is the monoubiquitylation and chromatin localization of 2 FA proteins, FA complementation group D2 (FANCD2) and FANCI. In the present study, we show that RAD18 binds FANCD2 and is required for efficient monoubiquitylation and chromatin localization of both FANCD2 and FANCI. Human RAD18-knockout cells display increased sensitivity to mitomycin C and a delay in FANCD2 foci formation compared with their wild-type counterparts. In addition, RAD18-knockout cells display a unique lack of FANCD2 and FANCI localization to chromatin in exponentially growing cells. FANCD2 ubiquitylation is normal in cells containing a ubiquitylation-resistant form of proliferating cell nuclear antigen, and chromatin loading of FA core complex proteins appears normal in RAD18-knockout cells. Mutation of the RING domain of RAD18 ablates the interaction with and chromatin loading of FANCD2. These data suggest a key role for the E3 ligase activity of RAD18 in the recruitment of FANCD2 and FANCI to chromatin and the events leading to their ubiquitylation during S phase.
范可尼贫血症（FA）是一种罕见的遗传疾病，其特征为骨髓衰竭、先天异常以及癌症和白血病风险增加。FA-BRCA 途径的成分被认为在 DNA 链间交联的修复中发挥作用。该途径的核心是 2 种 FA 蛋白，即 FA 互补群 D2（FANCD2）和 FANCI 的单泛素化和染色质定位。在本研究中，我们表明 RAD18 与 FANCD2 结合，并且是 FANCD2 和 FANCI 的有效单泛素化和染色质定位所必需的。与野生型细胞相比，人类 RAD18 敲除细胞对丝裂霉素 C 更敏感，并且 FANCD2 焦点形成延迟。此外，RAD18 敲除细胞在指数生长期细胞中表现出独特的缺乏 FANCD2 和 FANCI 向染色质的定位。在含有增殖细胞核抗原的泛素化抗性形式的细胞中，FANCD2 的泛素化是正常的，并且 RAD18 敲除细胞中的 FA 核心复合物蛋白的染色质加载似乎正常。RAD18 的 RING 结构域突变会破坏与 FANCD2 的相互作用以及 FANCD2 的染色质加载。这些数据表明 RAD18 的 E3 连接酶活性在招募 FANCD2 和 FANCI 到染色质以及在 S 期导致它们泛素化的事件中起着关键作用。
