Replication fork reversal is an important mechanism to protect the stability of stalled forks and thereby preserve genomic integrity. While multiple enzymes have been identified that can remodel forks, their regulation remains poorly understood. Here, we demonstrate that the ubiquitin ligase RFWD3, whose mutation causes Fanconi Anemia, promotes recruitment of the DNA translocase ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. Using electron microscopy, we show that RFWD3 stimulates fork remodeling in a ZRANB3-epistatic manner. Fork reversal is known to promote nascent DNA degradation in BRCA2-deficient cells. Consistent with a role for RFWD3 in fork reversal, inactivation of RFWD3 in these cells rescues fork degradation and collapse, analogous to ZRANB3 inactivation. RFWD3 loss impairs ZRANB3 localization to spontaneous nuclear foci induced by inhibition of the PCNA deubiquitinase USP1. We demonstrate that RFWD3 promotes PCNA ubiquitination and interaction with ZRANB3, providing a mechanism for RFWD3-dependent recruitment of ZRANB3. Together, these results uncover a new role for RFWD3 in regulating ZRANB3-dependent fork remodeling.

中文翻译：

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RFWD3 促进 ZRANB3 募集以调节停滞复制叉的重塑

复制叉逆转是保护停滞叉稳定性并从而保持基因组完整性的重要机制。虽然已经鉴定出多种可以重塑分叉的酶，但它们的调节仍然知之甚少。在这里，我们证明泛素连接酶 RFWD3（其突变会导致范可尼贫血）促进 DNA 易位酶 ZRANB3 募集到停滞的复制叉和 DNA 损伤的泛素化位点。使用电子显微镜，我们发现 RFWD3 以 ZRANB3 上位方式刺激叉重塑。众所周知，叉逆转会促进 BRCA2 缺陷细胞中新生 DNA 的降解。与 RFWD3 在叉逆转中的作用一致，这些细胞中 RFWD3 的失活可以挽救叉的降解和崩溃，类似于 ZRANB3 失活。 RFWD3 缺失会损害 ZRANB3 对 PCNA 去泛素酶 USP1 抑制诱导的自发核灶的定位。我们证明 RFWD3 促进 PCNA 泛素化以及与 ZRANB3 的相互作用，为 RFWD3 依赖性 ZRANB3 募集提供机制。总之，这些结果揭示了 RFWD3 在调节 ZRANB3 依赖性分叉重塑中的新作用。