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首页> 外文期刊>Molecular and Cellular Biology >Single-stranded-DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase.
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Single-stranded-DNA binding alters human replication protein A structure and facilitates interaction with DNA-dependent protein kinase.

机译：单链DNA结合改变了人类复制蛋白A的结构，并促进了与DNA依赖性蛋白激酶的相互作用。

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Human replication protein A (hRPA) is an essential single-stranded-DNA-binding protein that stimulates the activities of multiple DNA replication and repair proteins through physical interaction. To understand DNA binding and its role in hRPA heterologous interaction, we examined the physical structure of hRPA complexes with single-stranded DNA (ssDNA) by scanning transmission electron microscopy. Recent biochemical studies have shown that hRPA combines with ssDNA in at least two binding modes: by interacting with 8 to 10 nucleotides (hRPA8nt) and with 30 nucleotides (hRPA30nt). We find the relatively unstable hRPA8nt complex to be notably compact with many contacts between hRPA molecules. In contrast, on similar lengths of ssDNA, hRPA30nt complexes align along the DNA and make few intermolecular contacts. Surprisingly, the elongated hRPA30nt complex exists in either a contracted or an extended form that depends on ssDNA length. Therefore, homologous-protein interaction and available ssDNA length both contribute to the physical changes that occur in hRPA when it binds ssDNA. We used activated DNA-dependent protein kinase as a biochemical probe to detect alterations in conformation and demonstrated that formation of the extended hRPA30nt complex correlates with increased phosphorylation of the hRPA 29-kDa subunit. Our results indicate that hRPA binds ssDNA in a multistep pathway, inducing new hRPA alignments and conformations that can modulate the functional interaction of other factors with hRPA.
机译：人复制蛋白A（hRPA）是一种必不可少的单链DNA结合蛋白，可刺激多种DNA复制的活性并通过物理相互作用来修复蛋白。为了了解DNA结合及其在hRPA异源相互作用中的作用，我们通过扫描透射电子显微镜检查了hRPA复合物与单链DNA（ssDNA）的物理结构。最近的生化研究表明，hRPA与ssDNA至少以两种结合方式结合：通过与8至10个核苷酸（hRPA8nt）和30个核苷酸（hRPA30nt）相互作用。我们发现相对不稳定的hRPA8nt复合物在hRPA分子之间有许多接触时非常紧凑。相反，在类似长度的ssDNA上，hRPA30nt复合物沿DNA排列，几乎没有分子间接触。令人惊讶地，伸长的hRPA30nt复合物以取决于ssDNA长度的收缩或延伸形式存在。因此，同源蛋白相互作用和可用的ssDNA长度都有助于hRPA结合ssDNA时发生的物理变化。我们使用活化的DNA依赖性蛋白激酶作为生化探针来检测构象变化，并证明扩展的hRPA30nt复合物的形成与hRPA 29-kDa亚基的磷酸化增加有关。我们的结果表明，hRPA在多步途径中与ssDNA结合，诱导了新的hRPA比对和构象，可调节其他因素与hRPA的功能相互作用。

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