Received 10 October 2001, Accepted 13 November 2001Eukaryotic replication protein A (RPA) is a single-stranded(ss) DNA binding protein with multiple functionsin DNA replication, repair, and genetic recombination. The70-kDa subunit of eukaryotic RPA contains a conservedfour cysteine-type zinc-finger motif that has beenimplicated in the regulation of DNA replication and repair.Recently, we described a novel function for the zinc-fingermotif in the regulation of human RPA’s ssDNA bindingactivity through reduction-oxidation (redox). Here, weshow that yeast RPA’s ssDNA binding activity is regulatedby redox potential through its RPA32 and/or RPA14subunits. Yeast RPA requires a reducing agent, such asdithiothreitol (DTT), for its ssDNA binding activity. Also,under non-reducing conditions, its DNA binding activitydecreases 20 fold. In contrast, the RPA70 subunit does notrequire DTT for its DNA binding activity and is notaffected by the redox condition. These results suggest thatall three subunits are required for the regulation of RPA’sDNA binding activity through redox potential.Keywords: Replication protein A (RPA), RPA70, Single-stranded DNA binding protein, Redox