In vitro analysis of the zinc-finger motif in human replication protein A

Human replication protein A (RPA) is composed of 70, 34 and 11 kDa subunits (p70, p34 and pll respectively) and functions in all three major DNA metab olic processes: replication, repair and recombination. Recent deletion anal ysis demonstrated that the large subunit of RPA, p70, has multiple function al domains, including a DNA polymerase alpha-stimulation domain and a singl e-stranded DNA-binding domain. It also contains a putative pe (Cys-Xaa(4)-C ys-Xaa(13)-Cys-Xaa(2)-Cys)that is highly conserved among eukaryotes. To stu dy the role of this domain in DNA metabolism, Re created various p70 mutant s that lack the zinc-finger motif (by Cys --> Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide exci sion repair (NER), but had very little impact on DNA replication. The failu re of zinc-finger mutant RPA in NER may be explained by the observation tha t wild-type RPA significantly stimulated DNA polymerase delta activity, whe reas only marginal stimulation was observed with zinc-finger mutant RPA. We also observed that ZFM reduced RPA's single-stranded DNA-binding activity by 2-3-fold in the presence of low amounts of RPA. Interestingly, the ZFM a bolished phosphorylation of the p34 subunit by DNA-dependent protein kinase , but not that by cyclin-dependent kinase, Taker together, our results stro ngly suggest a positive role for RPA's zinc finger domain in its function.