Purification and characterization of a protein from Saccharomyces cerevisiae that binds tightly to single-stranded DNA and stimulates a cognate strand exchange protein.
A single-stranded DNA binding protein (yeast SSB protein) was purified to near-homogeneity from mitotic Saccharomyces cerevisiae cells. The Mr 34,000 protein specifically eluted at high salt (approximately 1200 mM NaCl) during chromatography on a single-stranded DNA-cellulose column. The protein formed stable complexes with single-stranded DNA in an apparent cooperative fashion. As judged from titration and competition experiments, the affinity of the protein was much higher for single-stranded DNA than for double-stranded DNA or single-stranded RNA. The SSB protein also was found to stimulate the strand exchange reaction between linear M13mp19 RF DNA and circular M13mp19 viral DNA as catalyzed by a yeast strand exchange protein previously purified in this laboratory [Kolodner, R., Evans, D. H., & Morrison, P. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5660-5664]. Titration experiments showed maximum stimulation of joint molecule formation at a stoichiometry of about 1 Mr 34,000 monomer yeast SSB per 18 nucleotides of single-stranded DNA. Kinetic experiments demonstrated at least an 18-fold increase in the rate of strand exchange due to the presence of the SSB in reactions where the amount of strand exchange protein was limiting. The yeast SSB protein stimulated the Escherichia coli RecA protein in the strand exchange reaction involving linear M13mp19 RF DNA and circular M13mp19 viral DNA as efficiently as E. coli SSB. However, the E. coli SSB protein did not substitute for the yeast SSB protein in reactions with the yeast strand exchange protein. This suggests that the stimulation of the yeast strand exchange protein by the yeast SSB may involve specific protein/protein interactions.
Heyer
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WD
Division of Cell and Molecular Biology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Kolodner
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RD
D002846:Chromatography, Affinity; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011938:Rec A Recombinases / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000254:growth & development / Q000378:metabolism*
D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; D011938:Rec A Recombinases