Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester MA 01605.
Graduate School of Biomedical Sciences, University of Massachusetts Medical School, Worcester MA 01605.
Proc Natl Acad Sci U S A. 2020 Sep 22;117(38):23571-23580. doi: 10.1073/pnas.2007437117. Epub 2020 Sep 9.
DNA replication requires the sliding clamp, a ring-shaped protein complex that encircles DNA, where it acts as an essential cofactor for DNA polymerases and other proteins. The sliding clamp needs to be opened and installed onto DNA by a clamp loader ATPase of the AAA+ family. The human clamp loader replication factor C (RFC) and sliding clamp proliferating cell nuclear antigen (PCNA) are both essential and play critical roles in several diseases. Despite decades of study, no structure of human RFC has been resolved. Here, we report the structure of human RFC bound to PCNA by cryogenic electron microscopy to an overall resolution of ∼3.4 Å. The active sites of RFC are fully bound to adenosine 5'-triphosphate (ATP) analogs, which is expected to induce opening of the sliding clamp. However, we observe the complex in a conformation before PCNA opening, with the clamp loader ATPase modules forming an overtwisted spiral that is incapable of binding DNA or hydrolyzing ATP. The autoinhibited conformation observed here has many similarities to a previous yeast RFC:PCNA crystal structure, suggesting that eukaryotic clamp loaders adopt a similar autoinhibited state early on in clamp loading. Our results point to a "limited change/induced fit" mechanism in which the clamp first opens, followed by DNA binding, inducing opening of the loader to release autoinhibition. The proposed change from an overtwisted to an active conformation reveals an additional regulatory mechanism for AAA+ ATPases. Finally, our structural analysis of disease mutations leads to a mechanistic explanation for the role of RFC in human health.
DNA 复制需要滑动夹，这是一种环绕 DNA 的环形蛋白复合物，作为 DNA 聚合酶和其他蛋白质的必需辅助因子发挥作用。滑动夹需要由 AAA+家族的夹载 ATP 酶打开并安装到 DNA 上。人类夹载器复制因子 C（RFC）和滑动夹增殖细胞核抗原（PCNA）都是必需的，在多种疾病中发挥着关键作用。尽管经过了几十年的研究，但仍未解析出人类 RFC 的结构。在这里，我们通过低温电子显微镜将与人 RFC 结合的 PCNA 结构报告至约 3.4Å 的整体分辨率。RFC 的活性位点完全与腺苷 5'-三磷酸（ATP）类似物结合，预计这将诱导滑动夹的打开。然而，我们观察到复合物处于 PCNA 打开之前的构象，夹载器 ATP 酶模块形成了无法结合 DNA 或水解 ATP 的过度扭曲螺旋。这里观察到的自动抑制构象与先前的酵母 RFC:PCNA 晶体结构有许多相似之处，表明真核夹载器在夹载过程中早期采用类似的自动抑制状态。我们的结果表明存在“有限变化/诱导契合”机制，其中夹首先打开，然后结合 DNA，诱导加载器打开以释放自动抑制。从过度扭曲到活跃构象的变化揭示了 AAA+ATP 酶的另一种调节机制。最后，我们对疾病突变的结构分析导致了 RFC 在人类健康中的作用的机制解释。
