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首页> 外文期刊>Biochemistry >Structural Characterization of Human RPA Sequential Binding to Single-Stranded DNA Using ssDNA as a Molecular Ruler
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Structural Characterization of Human RPA Sequential Binding to Single-Stranded DNA Using ssDNA as a Molecular Ruler

机译：使用RssDNA作为分子标尺的人RPA顺序结合单链DNA的结构表征

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摘要

Human replication protein A (RPA), a heterotrimer composed of RPA70, RPA32, and RPA14 subunits, contains four single-stranded DNA (ssDNA) binding domains (DBD): DBD-A, DBD-B, and DBD-C in RPA70 and DBD-D in RPA32. Although crystallographic or NMR structures of these DBDs and a trimerization core have been determined, the structure of the full length of RPA or the RPA- ssDNA complex remains unknown. In this article, we have examined the structural features of RPA interaction with ssDNA by fluorescence spectroscopy. Using a set of oligonucleotides (dT) with varying lengths as a molecular ruler and also as the substrate, we have determined at single-nucleotide resolution the relative positions of the ssDNA with interacting intrinsic tryptophans of RPA. Our results revealed that Trp528 in DBD-C and Trp107 in DBD-D contact ssDNA at the 16th and 24th nucleotides (nt) from the 5'-end of the substrate, respectively. Evaluation of the relative spatial arrangement of RPA domains in the RPA-ssDNA complex suggested that DBD-B and DBD-C are spaced by about 4 nt (~19 A) apart, whereas DBD-C and DBD-D are spaced by about 7 nt (~34 A). On the basis of these geometric constraints, a global structure model for the binding of the major RPA DBDs to ssDNA was proposed.
机译：人复制蛋白A（RPA）是由RPA70，RPA32和RPA14亚基组成的异源三聚体，在RPA70和RPA70中包含四个单链DNA（ssDNA）结合域（DBD）：DBD-A，DBD-B和DBD-C RPA32中的DBD-D。尽管已经确定了这些DBD的晶体学或NMR结构以及三聚核，但RPA或RPA-ssDNA复合物全长的结构仍然未知。在本文中，我们通过荧光光谱检查了RPA与ssDNA相互作用的结构特征。使用一组长度可变的寡核苷酸（dT）作为分子尺和底物，我们已经确定了单核苷酸分辨率下ssDNA与RPA相互作用固有色氨酸的相对位置。我们的结果表明，DBD-C中的Trp528和DBD-D中的Trp107分别与底物5'端第16和第24个核苷酸（nt）接触ssDNA。对RPA-ssDNA复合体中RPA域的相对空间排列的评估表明，DBD-B和DBD-C的间隔约为4 nt（〜19 A），而DBD-C和DBD-D的间隔约为7 nt。 nt（〜34 A）。基于这些几何约束，提出了一种主要的RPA DBD与ssDNA结合的全局结构模型。

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