A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA.
A second DNA binding site on RFC facilitates clamp loading at gapped or nicked DNA.
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DOI：
10.7554/elife.77483
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发表时间：
2022-06-22
期刊：
影响因子：
7.7
通讯作者：
Kelch, Brian A.
中科院分区：
文献类型：
--
作者：
Liu, Xingchen;Gaubitz, Christl;Pajak, Joshua;Kelch, Brian A.
夹子加载器将环形滑动夹子蛋白放置在DNA上，以便与夹子结合的配对蛋白可以合成、扫描和修复基因组。带有缺口或小的单链间隙的DNA是DNA修复中常见的钳制加载目标，但由于已知的引物-模板DNA结合机制，这些底物会被空间阻塞。在这里，我们报告了在酵母夹加载器复制因子C(RFC)中发现了第二个DNA结合位点，它有助于与有缺口的DNA结合。这个DNA结合部位在外表面，只有在开放构象的RFC中才能接触到。因此，在这个部位的初始DNA结合提供了进入中央小室中的主要DNA结合部位的途径。此外，我们发现该位点可以部分解开DNA，为RFC中央小室的DNA结合和随后的ATPase激活创造一个延长的单链间隙。最后，我们证明了BRCT结构域的缺失，这是外部DNA结合位点的主要组成部分，在DNA损伤存在的情况下导致酵母生长缺陷，其中DNA中间体出现缺口或缺口。我们认为RFC的外部DNA结合位点可以增强DNA结合和钳制负载，特别是在DNA修复中常见的DNA结构。
Clamp loaders place circular sliding clamp proteins onto DNA so that clamp-binding partner proteins can synthesize, scan, and repair the genome. DNA with nicks or small single-stranded gaps are common clamp-loading targets in DNA repair, yet these substrates would be sterically blocked given the known mechanism for binding of primer-template DNA. Here, we report the discovery of a second DNA binding site in the yeast clamp loader replication factor C (RFC) that aids in binding to nicked or gapped DNA. This DNA binding site is on the external surface and is only accessible in the open conformation of RFC. Initial DNA binding at this site thus provides access to the primary DNA binding site in the central chamber. Furthermore, we identify that this site can partially unwind DNA to create an extended single-stranded gap for DNA binding in RFC’s central chamber and subsequent ATPase activation. Finally, we show that deletion of the BRCT domain, a major component of the external DNA binding site, results in defective yeast growth in the presence of DNA damage where nicked or gapped DNA intermediates occur. We propose that RFC’s external DNA binding site acts to enhance DNA binding and clamp loading, particularly at DNA architectures typically found in DNA repair.