Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.
Cryo-EM structures reveal high-resolution mechanism of a DNA polymerase sliding clamp loader.
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DOI：
10.7554/elife.74175
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发表时间：
2022-02-18
期刊：
影响因子：
7.7
通讯作者：
Kelch BA
中科院分区：
文献类型：
--
作者：
Gaubitz C;Liu X;Pajak J;Stone NP;Hayes JA;Demo G;Kelch BA
滑动夹是环形蛋白质复合物，是所有生命DNA复制机制的组成部分。打开滑动夹并通过夹加载器AAA+ ATP酶复合物安装到DNA上。然而，夹具加载器如何打开和关闭DNA周围的滑动夹具仍然是未知的。在这里，我们描述了结构的酿酒酵母钳加载复制因子C（RFC）绑定到其同源滑动钳增殖细胞核抗原（PCNA）途中成功加载。RFC首先以动态的封闭构象结合PCNA，阻断ATP酶活性和DNA结合。然后RFC通过RFC和PCNA的大规模“蟹爪”扩展打开PCNA环，这解释了RFC如何更喜欢PCNA而不是DNA的初始结合。接下来，开放RFC：PCNA复合物结合DNA，并使用令人惊讶的碱基翻转机制询问引物-模板连接。我们的结构表明，最初的PCNA开放和随后的关闭周围的DNA不需要ATP水解，但由结合能驱动。RFC释放所必需的ATP水解是由与PCNA和DNA的相互作用触发的，这解释了RFC的开关样ATP酶活性。我们的工作揭示了AAA+机器如何经历戏剧性的构象变化，以实现结合偏好和底物重塑。
Sliding clamps are ring-shaped protein complexes that are integral to the DNA replication machinery of all life. Sliding clamps are opened and installed onto DNA by clamp loader AAA+ ATPase complexes. However, how a clamp loader opens and closes the sliding clamp around DNA is still unknown. Here, we describe structures of the Saccharomyces cerevisiae clamp loader Replication Factor C (RFC) bound to its cognate sliding clamp Proliferating Cell Nuclear Antigen (PCNA) en route to successful loading. RFC first binds to PCNA in a dynamic, closed conformation that blocks both ATPase activity and DNA binding. RFC then opens the PCNA ring through a large-scale ‘crab-claw’ expansion of both RFC and PCNA that explains how RFC prefers initial binding of PCNA over DNA. Next, the open RFC:PCNA complex binds DNA and interrogates the primer-template junction using a surprising base-flipping mechanism. Our structures indicate that initial PCNA opening and subsequent closure around DNA do not require ATP hydrolysis, but are driven by binding energy. ATP hydrolysis, which is necessary for RFC release, is triggered by interactions with both PCNA and DNA, explaining RFC’s switch-like ATPase activity. Our work reveals how a AAA+ machine undergoes dramatic conformational changes for achieving binding preference and substrate remodeling.