Reconstitution of translesion synthesis reveals a mechanism of eukaryotic DNA replication restart
Reconstitution of translesion synthesis reveals a mechanism of eukaryotic DNA replication restart
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DOI：
10.1038/s41594-020-0418-4
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发表时间：
2020-04-27
影响因子：
16.8
通讯作者：
Yeeles, Joseph T. P.
中科院分区：
文献类型：
--
作者：
Guilliam, Thomas A.;Yeeles, Joseph T. P.
前导链模板异常会导致解旋酶-聚合酶解偶联，并阻碍复制分叉的进展，但解偶联分叉如何重新启动的细节仍不确定。使用从酿酒酵母中纯化的蛋白，我们已经重组了跨损伤合成(TLS)介导的真核复制体在与环丁烷嘧啶二聚体碰撞后的重新启动。我们发现，尽管病变旁路发生在与CDc45-MCM-gins(CMG)解旋酶分离的情况下，但TLS仍起到促进快速复制分叉速率恢复的作用。令人惊讶的是，主要的滞后链聚合酶，Pol Delta，在解偶联时结合领先链并抑制TLS。POL增量对于病变搭桥后引导链合成到CMG的有效重新偶联也是至关重要的。增殖细胞核抗原单素化正向调节TLS以克服Pol Delta抑制。我们发现，这些负调控和正调控机制也作用于滞后链。我们的观察对分叉重新启动和前导链合成过程中的分工都有意义。在体外重建跨病变合成介导的复制分叉重新启动显示了DNA Pol ETA是如何在酵母复制体的背景下被招募来绕过前导链上的CPD损伤的。
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 delta, binds the leading strand upon uncoupling and inhibits TLS. Pol delta 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 delta 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.In vitro reconstitution of translesion synthesis-mediated replication fork restart shows how DNA Pol eta is recruited to bypass a CPD lesion on the leading strand in the context of the yeast replisome.