Insights into hRPA32 C-terminal domain-mediated assembly of the simian virus 40 replisome
Insights into hRPA32 C-terminal domain-mediated assembly of the simian virus 40 replisome
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DOI：
10.1038/nsmb916
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发表时间：
2005-04-01
影响因子：
16.8
通讯作者：
Chazin, WJ
中科院分区：
文献类型：
--
作者：
Arunkumar, AI;Klimovich, V;Chazin, WJ
猿猴病毒40（Simian virus 40，SV 40）是研究真核生物DNA复制的一个模型系统，其病毒蛋白大T抗原（Large T antigen，Tag）可引导人类蛋白质复制病毒微染色体。SV 40复制需要Tag与宿主单链DNA结合蛋白，复制蛋白A（hRPA）相互作用。hRPA 32亚基的C-末端结构域（RPA 32 C）促进复制的起始，但它是否与Tag相互作用尚不清楚。亲和层析和NMR揭示了hRPA 32 C和标签来源DNA结合结构域之间的物理相互作用，并确定了复合物的结构模型。然后设计点突变以逆转结合位点中的电荷，导致结合亲和力大幅降低。相应的突变引入到完整的hRPA损伤的复制和启动子活性，这意味着这种相互作用具有组装和SV 40复制体的进展中的关键作用。
Simian virus 40 (SV40) provides a model system for the study of eukaryotic DNA replication, in which the viral protein, large T antigen ( Tag), marshals human proteins to replicate the viral minichromosome. SV40 replication requires interaction of Tag with the host single-stranded DNA-binding protein, replication protein A (hRPA). The C-terminal domain of the hRPA32 subunit (RPA32C) facilitates initiation of replication, but whether it interacts with Tag is not known. Affinity chromatography and NMR revealed physical interaction between hRPA32C and the Tag origin DNA - binding domain, and a structural model of the complex was determined. Point mutations were then designed to reverse charges in the binding sites, resulting in substantially reduced binding affinity. Corresponding mutations introduced into intact hRPA impaired initiation of replication and primosome activity, implying that this interaction has a critical role in assembly and progression of the SV40 replisome.