Mass spectrometric identification of lysines involved in the interaction of human replication protein a with single-stranded DNA.
Human replication protein A (hRPA), a heterotrimeric single-stranded DNA (ssDNA) binding protein, is required for many cellular pathways including DNA damage repair, recombination, and replication as well as the ATR-mediated DNA damage response. While extensive effort has been devoted to understanding the structural relationships between RPA and ssDNA, information is currently limited to the RPA domains, the trimerization core, and a partial cocrystal structure. In this work, we employed a mass spectrometric protein footprinting method of single amino acid resolution to investigate the interactions of the entire heterotrimeric hRPA with ssDNA. In particular, we monitored surface accessibility of RPA lysines with NHS-biotin modification in the contexts of the free protein and the nucleoprotein complex. Our results not only indicated excellent agreement with the available crystal structure data for RPA70 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 direct DNA contacts, we observed protection of five additional lysines (K183, K259, K489, K577, and K588 of p70) upon ssDNA binding to RPA. Three residues, K489, K577, and K588, are located in ssDNA binding domain 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 positioned outside the putative ssDNA binding cleft. We propose that the protection of these lysines could result from the RPA interdomain structural reorganization induced by ssDNA binding.
Shell
Steven M
SM
Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, USA.
Hess
Sonja
S
Kvaratskhelia
Mamuka
M
Zou
Yue
Y
D000595:Amino Acid Sequence; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D008239:Lysine / Q000032:analysis* / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D011485:Protein Binding; D051716:Replication Protein A; D017386:Sequence Homology, Amino Acid
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D008239:Lysine