RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination
RFWD3-Mediated Ubiquitination Promotes Timely Removal of Both RPA and RAD51 from DNA Damage Sites to Facilitate Homologous Recombination
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DOI：
10.1016/j.molcel.2017.04.022
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发表时间：
2017-06-01
期刊：
影响因子：
16
通讯作者：
Takata, Minoru
中科院分区：
文献类型：
--
作者：
Inano, Shojiro;Sato, Koichi;Takata, Minoru
RFWD 3是最近鉴定的Fanconi贫血蛋白FANCW，其针对RPA的E3连接酶活性在同源重组（HR）修复中是必需的。然而，RPA泛素化如何促进HR仍不清楚。在这里，我们确定了中心HR蛋白RAD 51作为RFWD 3的另一个靶点。我们表明，RFWD 3 polyubiquitinates RPA和RAD 51在体外和体内。ATR和ATM激酶的磷酸化是体内这种活性所必需的。RFWD 3通过促进VCP/p97介导的蛋白动力学和随后的降解来抑制持续的丝裂霉素C（MMC）诱导的RAD 51和RPA灶。此外，MMC诱导的MCM 8和RAD 54的染色质加载在具有失活RFWD 3或表达泛素化缺陷突变体RAD 51的细胞中是有缺陷的。总的来说，我们的数据揭示了一种机制，有助于从DNA损伤位点及时去除RPA和RAD 51，这对于进展到晚期HR和抑制FA表型至关重要。
RFWD3 is a recently identified Fanconi anemia protein FANCW whose E3 ligase activity toward RPA is essential in homologous recombination (HR) repair. However, how RPA ubiquitination promotes HR remained unknown. Here, we identified RAD51, the central HR protein, as another target of RFWD3. We show that RFWD3 polyubiquitinates both RPA and RAD51 in vitro and in vivo. Phosphorylation by ATR and ATM kinases is required for this activity in vivo. RFWD3 inhibits persistent mitomycin C (MMC)-induced RAD51 and RPA foci by promoting VCP/p97-mediated protein dynamics and subsequent degradation. Furthermore, MMC-induced chromatin loading of MCM8 and RAD54 is defective in cells with inactivated RFWD3 or expressing a ubiquitination-deficient mutant RAD51. Collectively, our data reveal a mechanism that facilitates timely removal of RPA and RAD51 from DNA damage sites, which is crucial for progression to the late-phase HR and suppression of the FA phenotype.