Identification of the Structural Change of Human Replication Protein A (hRPA) in the ssDNA Binding and Redox Potential

Eukaryotic replication protein A (RPA) is comprised of a three subunits (70, 32, 14 kDa; p70, p32, p14 respectively). RPA has been identified as a human protein necessary for SV40 DNA replication in vitro and acting as a singlestrand DNA (ssDNA) binding protein with multiple functions in DNA replication, repair, and genetic recombination. Recent studies suggest that RPA functions are regulated in response DNA damage and that its ssDNA binding activity may be involved in this regulatory event. In our previous study, we found that RPA’s ssDNA binding activity is regulated by redox through cysteins in the putative zinc finger domain of the p70 subunit. A number of DNAbinding proteins have been identified in which their DNA binding activity is regulated by redox, although the regulatory role of the zinc finger domain is not clear. RPA70 has an evolutionarily conserved 4 cystein in the zinc-finger domain at 473 through 503 amino acids. Even though previous studies showed that the zinc-finger domain does not participate in DNA binding directly, it is still possible that the zinc-finger domain regulates DNA binding activity. To verify this possibility, we built mutant RPA (ZFD4) by substitution four cysteins for alanine and checked the DNA binding activity. The ZFD4 formed a stable complex with the ssDNA, even under oxidized conditions, and the amounts of DTT had no effect on its binding property (Figure 1a). The ZFD4 also showed strong binding activity in the non-reducing condition without DTT. The addition of H2O2 did not have much of effect on RPA-DNA complex formation (Figure 1a), whereas the wild-type RPA had H2O2 dependent activity. 7-9 These results suggest that the zinc-finger domain could regulate the ssDNA binding activity of RPA and that the cystein residues of the p70 subunit are essential in this process. We examined the effect of phosphorylation on ZFD4’s in regulation of ssDNA binding activity of RPA in the various redox conditions. The results showed phosphorylation of ZFD4 does not affect its DNA binding activity and suggests that phosphorylation could occur at other sites of the RPA complex (Figure 1a). In an effort to understand RPA’s regulatory function, we examined the structural changes of hRPA by the ssDNA binding using protease sensitive digestions. We examined