Insights into hRPA32 C-terminal domain-mediated assembly of the simian virus 40 replisome

被引:68
作者
Arunkumar, AI
Klimovich, V
Jiang, XH
Ott, RD
Mizoue, L
Fanning, E
Chazin, WJ
机构
[1] Vanderbilt Univ, Dept Biol Sci, Nashville, TN 37232 USA
[2] Vanderbilt Univ, Dept Biochem, Nashville, TN 37232 USA
[3] Vanderbilt Univ, Dept Phys, Nashville, TN 37232 USA
[4] Vanderbilt Univ, Ctr Struct Biol, Nashville, TN 37232 USA
关键词
D O I
10.1038/nsmb916
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:332 / 339
页数:8
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