Leading-strand template aberrations cause helicase-polymerase uncoupling and impede replication fork progression, but the details of how uncoupled forks are restarted remain uncertain. Using purified proteins from Saccharomyces cerevisiae, we have reconstituted translesion synthesis (TLS)-mediated restart of a eukaryotic replisome following collision with a cyclobutane pyrimidine dimer. We find that TLS functions 'on the fly' to promote resumption of rapid replication fork rates, despite lesion bypass occurring uncoupled from the Cdc45-MCM-GINS (CMG) helicase. Surprisingly, the main lagging-strand polymerase, Pol δ, binds the leading strand upon uncoupling and inhibits TLS. Pol δ is also crucial for efficient recoupling of leading-strand synthesis to CMG following lesion bypass. Proliferating cell nuclear antigen monoubiquitination positively regulates TLS to overcome Pol δ inhibition. We reveal that these mechanisms of negative and positive regulation also operate on the lagging strand. Our observations have implications for both fork restart and the division of labor during leading-strand synthesis generally.

中文翻译：

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跨损伤合成的重建揭示了真核 DNA 复制重启的机制。

前导链模板畸变导致解旋酶聚合酶解偶联并阻碍复制叉的进展，但如何重新启动解偶联叉的细节仍不确定。使用来自酿酒酵母的纯化蛋白，我们在与环丁烷嘧啶二聚体碰撞后重组了跨损伤合成 (TLS) 介导的真核复制体重新启动。我们发现 TLS 功能“动态”以促进快速复制叉率的恢复，尽管病变旁路发生与 Cdc45-MCM-GINS (CMG) 解旋酶无关。令人惊讶的是，主要的滞后链聚合酶 Pol δ 在解偶联时与前导链结合并抑制 TLS。Pol δ 对于病变旁路后前导链合成与 CMG 的有效再偶联也至关重要。增殖细胞核抗原单泛素化正向调节 TLS 以克服 Pol δ 抑制。我们揭示了这些负调节和正调节机制也在滞后链上起作用。我们的观察结果通常对前导链合成过程中的分叉重启和分工都有影响。