Modulation of replication protein A function by its hyperphosphorylation-induced conformational change involving DNA binding domain B.

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.