Role of zinc-finger motif in redox regulation of human replication protein A.
Replication protein A (RPA) is a heterotrimeric zinc-finger protein complex involved in DNA replication, repair, and genetic recombination. Unlike other zinc-finger proteins, RPA's zinc-finger motif is not essential for its single-stranded DNA (ssDNA) binding activity, but is involved in redox regulation of its single-stranded DNA (ssDNA) binding activity. To get an insight into the regulation of RPA-ssDNA interaction, wild-type RPA (wt-RPA) and zinc-finger mutant were examined for ssDNA binding activity using surface plasmon resonance technique. Interaction of wt-RPA with ssDNA under nonreducing conditions was very weak (KD x 2.33 x 10(-8) M) compared with that under reducing conditions (KD = 7.35 x 10(-11) M), whereas ssDNA binding affinity of the zinc-finger mutant was not affected by redox. The divalent ion chelator, o-phenanthroline, significantly reduced wt-RPA-ssDNA interaction, but had no effect on the zinc-finger mutant. The inhibitory effect of o-phenanthroline on RPA-ssDNA interaction was reversed by Zn(II), but not by other divalent cations, suggesting that Zn(II) is the unique metal coordinating the zinc-finger cysteines in redox regulation of RPA-ssDNA interaction. In DNA repair, redox affected RPA's interaction with damaged DNA, but not its role in stabilizing the xeroderma pigmentosum group A (XPA)-damaged DNA complex, suggesting that the zinc-finger motif may mediate the transition of RPA-XPA interaction to a stable RPA-XPA-damaged DNA complex in a redox-dependent manner.
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Department of Biochemistry and Molecular Biology, Indiana University Cancer Center, Indiana University School of Medicine, Indianapolis 46202, USA.
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Lee
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D000818:Animals; D015374:Biosensing Techniques; D002413:Cations, Divalent; D002614:Chelating Agents / Q000494:pharmacology; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D003199:Computer Systems; D004247:DNA / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair / Q000502:physiology; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D010084:Oxidation-Reduction; D010618:Phenanthrolines / Q000494:pharmacology; D010777:Photochemistry; D011485:Protein Binding / Q000187:drug effects; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D051716:Replication Protein A; D013329:Structure-Activity Relationship; D020349:Surface Plasmon Resonance; D014466:Ultraviolet Rays / Q000009:adverse effects; D014983:Xeroderma Pigmentosum / Q000235:genetics / Q000378:metabolism; D015032:Zinc / Q000737:chemistry / Q000502:physiology; D016335:Zinc Fingers / Q000502:physiology*
D002413:Cations, Divalent; D002614:Chelating Agents; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D010618:Phenanthrolines; C497167:RPA1 protein, human; D011993:Recombinant Fusion Proteins; D051716:Replication Protein A; D004247:DNA; D015032:Zinc; C025205:1,10-phenanthroline