Functional characterization of zinc-finger motif in redox regulation of RPA-ssDNA interaction

J. S. You, M. Wang, S. H. Lee

Research output: Contribution to journalArticlepeer-review

24 Scopus citations

Abstract

The 70-kDa subunit of eukaryotic replication protein A (RPA) contains a conserved four cysteine-type zinc-finger motif that has been implicated in regulation of DNA replication and repair. Unlike other zinc-finger proteins, RPA zinc-finger motif is not a DNA-binding component, and deletion of the zinc-finger had very little effect on its ssDNA binding activity. Recently, we described a novel function for the zinc-finger motif in regulation of RPA's ssDNA binding activity through reduction oxidation (redox). In this study, we carried out a detailed analysis of wild-type RPA and zinc-finger mutants in redox regulation of their ssDNA binding activity. Any mutation at a zinc-finger cysteine abolished its redox role in regulation of RPA-ssDNA interaction, Suggesting that all four zinc-finger cysteines are required for redox regulation. Reactivity of cysteine residues to 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) indicated that wild-type RPA contained 8.2 reactive thiols/molecule including all four cysteines in the zinc-finger motif. Zinc-finger cysteines slowly reacted to DTNB as compared to others. Zn(II) was not only essential but also uniquely qualified for redox regulation of RPA-ssDNA interaction, suggesting that Zn(II)-cysteine coordination is crucial for the zinc-finger function. Redox status significantly affected initial interaction of RPA with ssDNA but had no effect after RPA formed a stable complex with DNA. Together, our results suggest that the zinc-finger motif mediates the transition of RPA-ssDNA interaction to a stable RPA-ssDNA complex in a redox-dependent manner.

Original languageEnglish (US)
Pages (from-to)12953-12958
Number of pages6
JournalBiochemistry
Volume39
Issue number42
DOIs
StatePublished - Oct 24 2000

ASJC Scopus subject areas

  • Biochemistry

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