Mass Spectrometric Identification of Lysines Involved in the Interaction of Human Replication Protein A with Single-Stranded DNA
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- 作者：Steven M. Shell ; Sonja Hess ; Mamuka Kvaratskhelia ; and Yue Zou
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：January 25, 2005
- 年：2005
- 卷：44
- 期：3
- 页码：971 - 978
- 全文大小：511K
- 年卷期：v.44,no.3(January 25, 2005)
- ISSN：1520-4995
文摘
Human replication protein A (hRPA), a heterotrimeric single-stranded DNA (ssDNA) bindingprotein, is required for many cellular pathways including DNA damage repair, recombination, andreplication as well as the ATR-mediated DNA damage response. While extensive effort has been devotedto understanding the structural relationships between RPA and ssDNA, information is currently limitedto the RPA domains, the trimerization core, and a partial cocrystal structure. In this work, we employeda mass spectrometric protein footprinting method of single amino acid resolution to investigate theinteractions of the entire heterotrimeric hRPA with ssDNA. In particular, we monitored surface accessibilityof RPA lysines with NHS-biotin modification in the contexts of the free protein and the nucleoproteincomplex. Our results not only indicated excellent agreement with the available crystal structure data forRPA70 DBD-AB-ssDNA complex but also revealed new protein contacts in the nucleoprotein complex.In addition to two residues, K263 and K343 of p70, previously identified by cocrystallography as directDNA contacts, we observed protection of five additional lysines (K183, K259, K489, K577, and K588 ofp70) upon ssDNA binding to RPA. Three residues, K489, K577, and K588, are located in ssDNA bindingdomain C and are likely to establish the direct contacts with cognate DNA. In contrast, no ssDNA-contacting lysines were identified in DBD-D. In addition, two lysines, K183 and K259, are positionedoutside the putative ssDNA binding cleft. We propose that the protection of these lysines could resultfrom the RPA interdomain structural reorganization induced by ssDNA binding.