CHARACTERIZATION OF DNA-BINDING AND STRAND-EXCHANGE STIMULATION PROPERTIES OF Y-RPA, A YEAST SINGLE-STRAND-DNA-BINDING PROTEIN

Single-stranded DNA binding proteins (SSBs) have been isolated from many organisms including Escherichia coli, Saccharomyces cerevisiae and humans. Characterization of these proteins suggests they are required for DNA replication and are active in homologous recombination. As an initial step towards understanding the role of the eukaryotic SSBs in DNA replication and recombination, we examined the DNA binding and strand exchange stimulation properties of the S. cerevisiae single-strand binding protein y-RPA (yeast replication protein A). y-RPA was found to bind to single-stranded DNA (ssDNA) as a 115,000 Mr heterotrimer containing 70,000, 36,000 and 14,000. Mr subunits. It saturated ssDNA at a stoichiometry of one heterotrimer per 90 to 100 nucleotides and binding occurred with high affinity (Kω > 109m-1) and co-operativity (ω = 10,000 to 100,000) Electron microscopic analysis revealed that y-RPA binding was highly co-operative and that the ssDNA present in y-RPA-ssDNA complexes was compacted fourfold, arranged into nucleosome-like structures, and was free of secondary structure. y-RPA was also tested for its ability to stimulate the yeast Sep1 and E. coli RecA strand-exchange proteins. In an assay that measures the pairing of circular ssDNA with homologous linear duplex DNA y-RPA stimulated the strand-exchange activity of Sep1 approximately threefold and the activity of RecA protein to the same extent as did E. coli SSB. Maximal stimulation of Sep1 occurred at a stoichiometry of one y-RPA heterotrimer per 95 nucleotides of ssDNA. y-RPA stimulated RecA and Sep1 mediated strand exchange reactions in a manner similar to that observed for the stimulation of RecA by E. coli SSB; in both of these reactions, y-RPA inhibited the aggregation of ssDNA and promoted the co-aggregation of single-stranded and double-stranded linear DNA. These results demonstrate that the E. coli and yeast SSBs display similar DNA-binding properties and support a model in which y-RPA functions as an E. coli SSB-like protein in yeast. © 1992.