Structural characterization of human RPA sequential binding to single-stranded DNA using ssDNA as a molecular ruler
Structural characterization of human RPA sequential binding to single-stranded DNA using ssDNA as a molecular ruler
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DOI：
10.1021/bi7004976
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发表时间：
2007-07-17
期刊：
影响因子：
2.9
通讯作者：
Zou, Yue
中科院分区：
文献类型：
--
作者：
Cai, Lifeng;Roginskaya, Marina;Zou, Yue
人复制蛋白A（RPA）是由RPA 70、RPA 32和RPA 14亚基组成的异源三聚体，含有四个单链DNA（ssDNA）结合结构域（DBD）：RPA 70中的DBD-A、DBD-B和DBD-C以及RPA 32中的DBD-D。尽管已经确定了这些DBD和三聚核心的晶体学或NMR结构，但RPA或RPA-ssDNA复合物的全长结构仍然未知。在这篇文章中，我们已经研究了RPA与ssDNA相互作用的荧光光谱的结构特征。使用一组不同长度的寡核苷酸（dT）作为分子标尺，也作为基板，我们已经确定了在单核苷酸分辨率的相对位置的ssDNA与相互作用的内在的RPA的双链。我们的研究结果表明，DBD-C中的Trp 528和DBD-D中的Trp 107分别在底物5 '端的第16和第24个核苷酸（nt）处与ssDNA接触。RPA-ssDNA复合物中RPA结构域的相对空间排列的评估表明，DBD-B和DBD-C间隔约4 nt（类似于），而DBD-C和DBD-D间隔约7 nt（类似于）。基于这些几何约束，提出了主要RPA DBD与ssDNA结合的全局结构模型。
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 (similar to 19 A) apart, whereas DBD-C and DBD-D are spaced by about 7 nt (similar to 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.