The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain
The RPA32 subunit of human replication protein A contains a single-stranded DNA-binding domain
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DOI：
10.1074/jbc.273.7.3932
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发表时间：
1998-02-13
影响因子：
4.8
通讯作者：
Bochkarev, A
中科院分区：
文献类型：
--
作者：
Bochkareva, E;Frappier, L;Bochkarev, A
复制蛋白A(RPA)是一种保守的核单链DNA(SsDNA)结合蛋白，人RPA(HrpA)由约70、32和14 kDa的三个亚基(hRPA70、hRPA32和hRPA14)组成，RPA通过RPA70亚基中的两个单链DNA结合域与ssDNA结合。在这里，我们证明了hRPA32和hRPA14的复合体具有一个单链DNA结合域，hRPA14.32复合体的有限蛋白降解定义了由hRPA32(氨基酸43-171)和RPA14的中心区域组成的CARS双链。核心二聚体与单链DNA的亲和力约为10-50微米，比完整二聚体的DNA结合亲和力至少高100倍。对hRPA32二级结构的分析表明，hRPA32的63-150位氨基酸形成了一个与hRPA70的结构相似的单链DNA结合区，hRPA14与hRPA32-(43-171)的复合体与hRPA70的C-端区(氨基酸436-616)形成了三聚体复合体，该三聚体与单链DNA的亲和力比单独的hRPA14.32-(43-171)高3~5倍，表明hRPA70在单链DNA结合中的作用距离hRPA70的C端较远。
Replication protein A (RPA) is a conserved nuclear single-stranded DMA (ssDNA)-binding protein, Human RPA (hRPA) comprises three subunits of approximately 70, 32, and 14 kDa (hRPA70, hRPA32 and hRPA14), RPA is known to bind ssDNA through two ssDNA-binding domains in the RPA70 subunit. Here, we demonstrate that the complex of hRPA32 and hRPA14 has an ssDNA-binding domain, Limited proteolysis of the hRPA14.32 complex defined a cars dimes composed of the central region of hRPA32 (amino acids 43-171) and RPA14. The core dimes bound ssDNA with an affinity of approximately 10-50 mu M, which is at least 100-fold more avid than the DNA-binding affinity of the intact dimer. Analysis of the predicted secondary structure of hRPA32 suggests that amino acids 63-150 of hRPA32 form an ssDNA-binding domain similar in structure to each of those in hRPA70, The complex of hRPA14 and hRPA32-(43-171) in turn formed a trimeric complex with the C-terminal region of hRPA70 (amino acids 436-616), The ssDNA-binding affinity of this trimeric complex was 3 to 5-fold higher than hRPA14.32-(43-171) alone, suggesting a role far the C terminus of hRPA70 in ssDNA binding.