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J. Biol. Chem., Vol. 280, Issue 38, 32775-32783, September 23, 2005

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Modulation of Replication Protein A Function by Its Hyperphosphorylation-induced Conformational Change Involving DNA Binding Domain B^*

Yiyong Liu, Mamuka Kvaratskhelia, Sonja Hess¶, Youxing Qu||, and Yue Zou1

From the Department of Biochemistry and Molecular Biology, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, Tennessee 37614, The Ohio State University Health Sciences Center, College of Pharmacy, Center for Retrovirus Research and Comprehensive Cancer Center, Columbus, Ohio 43210, ^¶Proteomics and Mass Spectrometry Facility, NIDDK, National Institutes of Health, Bethesda, Maryland 20892, and ^||Department of Biochemistry and Molecular Biology, University of Georgia, Athens, Georgia 30602-7229

Human replication protein A (RPA), composed of RPA70, RPA32, and RPA14 subunits, undergoes hyperphosphorylation in cells in response to DNA damage. Hyperphosphorylation that occurs predominately in the N-terminal region of RPA32 is believed to play a role in modulating the cellular activities of RPA essential for almost all DNA metabolic pathways. To understand how the hyperphosphorylation modulates the functions of RPA, we compared the structural characteristics of full-length native and hyperphosphorylated RPAs using mass spectrometric protein footprinting, fluorescence spectroscopy, and limited proteolysis. Our mass spectrometric data showed that of 24 lysines and 18 arginines readily susceptible to small chemical reagent modification in native RPA, the three residues Lys-343, Arg-335, and Arg-382, located in DNA binding domain B (DBD-B) of RPA70, were significantly shielded in the hyperphosphorylated protein. Tryptophan fluorescence studies indicated significant quenching of Trp-361, located in the DBD-B domain, induced by hyperphosphorylation of RPA. Consistently, DBD-B became more resistant to the limited proteolysis by chymotrypsin after RPA hyperphosphorylation. Taken together, our results indicate that upon hyperphosphorylation of RPA32 N terminus (RPA32N), RPA undergoes a conformational change involving the single-stranded DNA binding cleft of DBD-B. Comparison of the interactions of native and hyperphosphorylated RPAs with short single-stranded oligonucleotides or partial DNA duplexes with a short 5' or 3' single-stranded DNA tails showed reduced affinity for the latter protein. We propose that the hyperphosphorylation may play a role in modulating the cellular pathways by altering the DBD-B-mediated RPA-DNA and RPA-protein interactions, hypothetically via the interaction of hyperphosphorylated RPA32N with DBD-B.

Received for publication, May 25, 2005 , and in revised form, July 8, 2005.

^* This study was supported by NCI, National Institutes of Health Grant CA86927 (to Y. Z.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 423-439-2124; Fax: 423-439-2030; E-mail: zouy{at}etsu.edu.

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