BRCA1/2 help maintain genomic integrity by stabilizing stalled forks. Here, we identify the E3 ligase RFWD3 as an essential modulator of stalled fork stability in BRCA2-deficient cells and show that codepletion of RFWD3 rescues fork degradation, collapse, and cell sensitivity upon replication stress. Stalled forks in BRCA2-deficient cells accumulate phosphorylated and ubiquitinated replication protein A (ubq-pRPA), the latter of which is mediated by RFWD3. Generation of this intermediate requires SMARCAL1, suggesting that it depends on stalled fork reversal. We show that in BRCA2-deficient cells, rescuing fork degradation might not be sufficient to ensure fork repair. Depleting MRE11 in BRCA2-deficient cells does block fork degradation, but it does not prevent fork collapse and cell sensitivity in the presence of replication stress. No such ubq-pRPA intermediate is formed in BRCA1-deficient cells, and our results suggest that BRCA1 may function upstream of BRCA2 in the stalled fork repair pathway. Collectively, our data uncover a novel mechanism by which RFWD3 destabilizes forks in BRCA2-deficient cells.

中文翻译：

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E3 连接酶 RFWD3 是 BRCA2 缺陷细胞中停滞叉稳定性的新型调节剂

BRCA1/2 通过稳定停滞的分叉来帮助维持基因组完整性。在这里，我们将 E3 连接酶 RFWD3 确定为 BRCA2 缺陷细胞中停滞叉稳定性的重要调节剂，并表明 RFWD3 的共缺失可以挽救叉降解、崩溃和复制应激时的细胞敏感性。BRCA2 缺陷细胞中停滞的分叉会积累磷酸化和泛素化的复制蛋白 A (ubq-pRPA)，后者由 RFWD3 介导。该中间体的生成需要 SMARCAL1，这表明它取决于停滞的分叉反转。我们发现，在 BRCA2 缺陷细胞中，挽救叉降解可能不足以确保叉修复。在 BRCA2 缺陷细胞中耗尽 MRE11 确实可以阻止叉降解，但它不能防止叉崩溃和存在复制应激时的细胞敏感性。在 BRCA1 缺陷细胞中不会形成这样的 ubq-pRPA 中间体，我们的结果表明，BRCA1 可能在停滞叉修复途径中的 BRCA2 上游发挥作用。总的来说，我们的数据揭示了 RFWD3 使 BRCA2 缺陷细胞中的叉不稳定的新机制。