Redox-dependent Formation of Disulfide Bonds in Human Replication Protein A

Human replication protein A (RPA) is a single-stranded DNA (ssDNA)-binding protein with three subunits. The largest subunit, p70, contains a conserved (cysteine)(4)-type zinc-finger motif that has been implicated in the regulation of DNA replication and repair. Previous studies indicated that the ssDNA-binding activity of RPA could be redox-regulated via reversible oxidation of cysteines in the zinc-finger motif. We exposed recombinant human RPA to hydrogen peroxide and characterized the oxidized protein by liquid chromatography/tandem mass spectrometric (LC/MS/MS) analyses. Our results demonstrated that, upon H(2)O(2) treatment, four cysteines, which reside at the zinc-finger motif of the p70 subunit, could result in the formation of two pairs of intramolecular disulfides, Cys481-Cys486 and Cys500-Cys503; no cysteine sulfinic acid or cysteine sulfonic acid could be found. Moreover, the other 11 cysteines in this protein remained intact. The results demonstrated that the formation of disulfide bonds at the zinc-finger site was responsible for the redox regulation of the DNA-binding activity of RPA.