Center for Personalized Cancer Therapy, University of Massachusetts, Boston, MA.
Department of Biology, University of Massachusetts, Boston, MA.
J Cell Biol. 2020 Jun 1;219(6). doi: 10.1083/jcb.201908192.
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.
BRCA1/2 通过稳定停滞的叉来帮助维持基因组完整性。在这里，我们确定 E3 连接酶 RFWD3 是 BRCA2 缺陷细胞中稳定停滞叉的重要调节剂，并表明 RFWD3 的共缺失可挽救复制应激时叉的降解、崩溃和细胞敏感性。BRCA2 缺陷细胞中的停滞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。这种中间产物的产生需要 SMARCAL1，表明它依赖于停滞叉的反转。我们表明，在 BRCA2 缺陷细胞中，挽救叉的降解可能不足以确保叉的修复。在 BRCA2 缺陷细胞中耗尽 MRE11 确实可以阻止叉的降解，但在存在复制应激时，它并不能阻止叉的崩溃和细胞敏感性。BRCA1 缺陷细胞中不会形成这种 ubq-pRPA 中间产物，我们的结果表明 BRCA1 可能在停滞叉修复途径中在上游作用于 BRCA2。总之，我们的数据揭示了 RFWD3 在 BRCA2 缺陷细胞中破坏叉的新机制。
