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

被引:32
作者
Dong, JW
Park, JS
Lee, SH [1 ]
机构
[1] Indiana Univ, Sch Med, Dept Biochem & Mol Biol, Indianapolis, IN 46202 USA
[2] St Jude Childrens Res Hosp, Dept Virol & Mol Biol, Memphis, TN 38105 USA
[3] Indiana Univ, Sch Med, Ctr Canc, Indianapolis, IN 46202 USA
[4] Indiana Univ, Sch Med, Walther Oncol Ctr, Indianapolis, IN 46202 USA
关键词
DNA replication; DNA repair; Xeroderma pigmentosum group A complementing protein; single-stranded DNA binding;
D O I
10.1042/0264-6021:3370311
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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.
引用
收藏
页码:311 / 317
页数:7
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