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 metabolic processes: replication, repair and recombination. Recent deletion analysis demonstrated that the large subunit of RPA, p70, has multiple functional domains, including a DNA polymerase alpha-stimulation domain and a single-stranded DNA-binding domain. It also contains a putative pe (Cys-Xaa(4)-Cys-Xaa(13)-Cys-Xaa(2)-Cys)that is highly conserved among eukaryotes. To study the role of this domain in DNA metabolism, Re created various p70 mutants that lack the zinc-finger motif (by Cys --> Ala substitutions). Mutation at the zinc-finger domain (ZFM) abolished RPA's function in nucleotide excision repair (NER), but had very little impact on DNA replication. The failure of zinc-finger mutant RPA in NER may be explained by the observation that wild-type RPA significantly stimulated DNA polymerase delta activity, whereas 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 abolished phosphorylation of the p34 subunit by DNA-dependent protein kinase, but not that by cyclin-dependent kinase, Taker together, our results strongly suggest a positive role for RPA's zinc finger domain in its function.