Abstract
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.