PCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication
PCNA-mediated stabilization of E3 ligase RFWD3 at the replication fork is essential for DNA replication
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DOI：
10.1073/pnas.1814521115
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发表时间：
2018-12-26
影响因子：
11.1
通讯作者：
Prasanth, Supriya G.
中科院分区：
文献类型：
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作者：
Lin, Yo-Chuen;Wang, Yating;Prasanth, Supriya G.
RING FING和WD重复结构域包含蛋白3(RFWD3)是一种E3连接酶，通过从DNA损伤部位去除复制蛋白A(RPA)和RAD51来促进同源重组。此外，RPA介导的RFWD3在停滞的复制叉上的招募对于链间交叉修复是必不可少的。在这里，我们报告了在未受干扰的人类细胞中，RFWD3定位于复制叉处，并通过其增殖细胞核抗原相互作用蛋白(PIP)基序与其结合。增殖细胞核抗原的结合对于RFWD3的稳定性和DNA复制是至关重要的。缺乏RFWD3的细胞表现出较慢的分叉进程，延长的S时相，以及几个复制分叉组件在染色质上的负载增加。这些发现都表明，在没有RFWD3的情况下，叉子失速的频率会增加。WT RFWD3可以挽救S相的缺陷，但PIP突变体不能，这表明RFWD3与增殖细胞核抗原的相互作用是DNA复制的关键。最后，我们观察到在缺乏RFWD3的细胞中RPA泛素化减少。我们的结论是，增殖细胞核抗原在复制分叉处稳定了RFWD3，使RPA的多泛素化及其随后的降解为适当的DNA复制提供了可能。
RING finger and WD repeat domain-containing protein 3 (RFWD3) is an E3 ligase known to facilitate homologous recombination by removing replication protein A (RPA) and RAD51 from DNA damage sites. Further, RPA-mediated recruitment of RFWD3 to stalled replication forks is essential for interstrand cross-link repair. Here, we report that in unperturbed human cells, RFWD3 localizes at replication forks and associates with proliferating cell nuclear antigen (PCNA) via its PCNA-interacting protein (PIP) motif. PCNA association is critical for the stability of RFWD3 and for DNA replication. Cells lacking RFWD3 show slower fork progression, a prolonged S phase, and an increase in the loading of several replication-fork components on the chromatin. These findings all point to increased frequency of stalled forks in the absence of RFWD3. The S-phase defect is rescued by WT RFWD3, but not by the PIP mutant, suggesting that the interaction of RFWD3 with PCNA is critical for DNA replication. Finally, we observe reduced ubiquitination of RPA in cells lacking RFWD3. We conclude that the stabilization of RFWD3 by PCNA at the replication fork enables the polyubiquitination of RPA and its subsequent degradation for proper DNA replication.