Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp.
Human CTF18-RFC clamp-loader complexed with non-synthesising DNA polymerase ε efficiently loads the PCNA sliding clamp.
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DOI：
10.1093/nar/gkx096
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发表时间：
2017-05-05
影响因子：
14.9
通讯作者：
Tsurimoto T
中科院分区：
文献类型：
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作者：
Fujisawa R;Ohashi E;Hirota K;Tsurimoto T
选择性增殖细胞核抗原（PCNA）装载剂CTF 18-RFC与DNA聚合酶ε（Polε）形成稳定的复合物。我们观察到，在接近生理条件下，CTF 18-RFC单独无效率地负载PCNA，但当与Polε复合时有效地负载PCNA。在有效的PCNA负载过程中，CTF 18-RFC和Polε协同组装在3 μ m引物-模板连接处，并将PCNA引导至负载位点。在引物-模板连接处直接相互作用的蛋白质的位点特异性光交联显示出类似的合作结合，其中Polε的催化N-末端部分充当主要对接蛋白。在含有ATPγS的PCNA负载中间体中，CTF 18与DNA结构的结合增加，表明CTF 18-RFC瞬时接近引物末端。Polε在DNA合成模式中使用具有脱氧的3 ′引物末端的底物DNA，不刺激PCNA加载，这表明DNA合成和PCNA加载在3 ′引物-模板连接处是相互排斥的。此外，PCNA与CTF 18-RFC-Polε复合物在模板DNA上进行了稳定的三聚体组装，并有效地合成了DNA。因此，CTF 18-RFC似乎通过与Polε的相互作用参与了前导链DNA的合成，并且可以在Polε不处于DNA合成模式时将PCNA负载到DNA上以恢复DNA合成。
The alternative proliferating-cell nuclear antigen (PCNA)-loader CTF18-RFC forms a stable complex with DNA polymerase ε (Polε). We observed that, under near-physiological conditions, CTF18-RFC alone loaded PCNA inefficiently, but loaded it efficiently when complexed with Polε. During efficient PCNA loading, CTF18-RFC and Polε assembled at a 3΄ primer–template junction cooperatively, and directed PCNA to the loading site. Site-specific photo-crosslinking of directly interacting proteins at the primer–template junction showed similar cooperative binding, in which the catalytic N-terminal portion of Polε acted as the major docking protein. In the PCNA-loading intermediate with ATPγS, binding of CTF18 to the DNA structures increased, suggesting transient access of CTF18-RFC to the primer terminus. Polε placed in DNA synthesis mode using a substrate DNA with a deoxidised 3΄ primer end did not stimulate PCNA loading, suggesting that DNA synthesis and PCNA loading are mutually exclusive at the 3΄ primer–template junction. Furthermore, PCNA and CTF18-RFC–Polε complex engaged in stable trimeric assembly on the template DNA and synthesised DNA efficiently. Thus, CTF18-RFC appears to be involved in leading-strand DNA synthesis through its interaction with Polε, and can load PCNA onto DNA when Polε is not in DNA synthesis mode to restore DNA synthesis.