Functional characterization of zinc-finger motif in redox regulation of RPA-ssDNA interaction.
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.
You
J S
JS
Department of Biochemistry and Molecular Biology, Indiana University Cancer Center and Walther Oncology Center, Indiana University School of Medicine, 635 Barnhill Drive, Indianapolis, Indiana 46202, USA.
Wang
M
M
Lee
S H
SH
D015374:Biosensing Techniques; D002614:Chelating Agents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D004261:DNA Replication; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D004228:Dithionitrobenzoic Acid / Q000737:chemistry; D046911:Macromolecular Substances; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000737:chemistry; D051716:Replication Protein A; D015032:Zinc / Q000737:chemistry; D016335:Zinc Fingers
D002614:Chelating Agents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D010618:Phenanthrolines; D051716:Replication Protein A; D004228:Dithionitrobenzoic Acid; D015032:Zinc; D003545:Cysteine; C025205:1,10-phenanthroline