The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading
The replication factor C clamp loader requires arginine finger sensors to drive DNA binding and proliferating cell nuclear antigen loading
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DOI：
10.1074/jbc.m606090200
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发表时间：
2006-11-17
影响因子：
4.8
通讯作者：
O'Donnell, Mike
中科院分区：
文献类型：
--
作者：
Johnson, Aaron;Yao, Nina Y.;O'Donnell, Mike
复制因子C(RFC)是一种AAA+异五聚体，它将ATP结合和水解的能量与DNA聚合酶加工性钳制--增殖细胞核抗原(PCNA)--负载到DNA上。RFC由螺旋排列的五个亚基(RFC-A、-B、-C、-D和-E，分别对应于亚基Rfc1、RFC4、RFC3、Rfc2和RFC5)组成。RFC亚基是AAA+家族蛋白，该复合体包含位于亚基界面的四个ATP位点(A、B、C和D)。在每个ATP位点，一个亚基的精氨酸残基位于相邻亚基结合的ATP的伽马-磷酸附近。这些精氨酸起着“精氨酸手指”的作用，可以潜在地执行两个功能：感知ATP的结合和催化ATP的水解。在这项研究中，RFC中的精氨酸手指发生突变，以检测RFC与ATP结合后发生的增殖细胞核抗原加载机制中的步骤。这份报告发现，在将增殖细胞核抗原装载到DNA上的过程中，RFC的ATP位点以不同的步骤发挥作用。与RFC结合的ATP促进了增殖细胞核抗原的募集和开放，并激活了ATP C位点上的一个促进DNA结合的伽马-磷酸传感器。在D位的ATP水解是由增殖细胞核抗原唯一地刺激的，我们认为这一事件与DNA周围的增殖细胞核抗原的关闭有关，从而开始了环周围的有序水解。增殖细胞核抗原封闭切断了与RFC亚单位D和E(Rfc2和RFC5)的接触，关闭了ATP位点C的γ-磷酸感受器，导致RFC与DNA的低亲和力，并将RFC从增殖细胞核抗原装载部位排出。
Replication factor C (RFC) is an AAA+ heteropentamer that couples the energy of ATP binding and hydrolysis to the loading of the DNA polymerase processivity clamp, proliferating cell nuclear antigen (PCNA), onto DNA. RFC consists of five subunits in a spiral arrangement (RFC-A, -B, -C, -D, and -E, corresponding to subunits RFC1, RFC4, RFC3, RFC2, and RFC5, respectively). The RFC subunits are AAA+ family proteins and the complex contains four ATP sites (sites A, B, C, and D) located at subunit interfaces. In each ATP site, an arginine residue from one subunit is located near the gamma-phosphate of ATP bound in the adjacent subunit. These arginines act as "arginine fingers" that can potentially perform two functions: sensing that ATP is bound and catalyzing ATP hydrolysis. In this study, the arginine fingers in RFC were mutated to examine the steps in the PCNA loading mechanism that occur after RFC binds ATP. This report finds that the ATP sites of RFC function in distinct steps during loading of PCNA onto DNA. ATP binding to RFC powers recruitment and opening of PCNA and activates a gamma-phosphate sensor in ATP site C that promotes DNA association. ATP hydrolysis in site D is uniquely stimulated by PCNA, and we propose that this event is coupled to PCNA closure around DNA, which starts an ordered hydrolysis around the ring. PCNA closure severs contact to RFC subunits D and E (RFC2 and RFC5), and the gamma-phosphate sensor of ATP site C is switched off, resulting in low affinity of RFC for DNA and ejection of RFC from the site of PCNA loading.