Purification and characterization of a DNA-pairing and strand transfer activity from mitotic Saccharomyces cerevisiae.
An enzyme catalyzing homologous pairing of DNA chains has been extensively purified from mitotic yeast. The most highly purified fractions are enriched for a polypeptide with a molecular mass of approximately 120 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Protein-dependent pairing of single-stranded DNAs requires a divalent cation (Mg2+ or Ca2+) but proceeds rapidly in the absence of any nucleoside triphosphates. The kinetics of reassociation are extremely rapid, with more than 60% of the single-stranded DNA becoming resistant to S1 nuclease within 1 min at a ratio of 1 protein monomer/50 nucleotides. The results of enzyme titration and DNA challenge experiments suggest that this protein does not act catalytically during renaturation but is required stoichiometrically. The protein promotes formation of joint molecules between linear M13 replicative form DNA (form III) containing short single-stranded tails and homologous single-stranded M13 viral DNA. Removal of approximately 50 nucleotides from the ends of the linear duplex using either exonuclease III (5' ends) or T7 gene 6 exonuclease (3' ends) activates the duplex for extensive strand exchange. Electron microscopic analysis of product molecules suggests that the homologous circular DNA initially associates with the single-stranded tails of the duplexes, and the heteroduplex region is extended with displacement of the noncomplementary strand. The ability of this protein to pair and to promote strand transfer using either exonuclease III or T7 gene 6 exonuclease-treated duplex substrates suggests that this activity promotes heteroduplex extension in a nonpolar fashion. The biochemical properties of the transferase are consistent with a role for this protein in heteroduplex joint formation during mitotic recombination in Saccharomyces cerevisiae.
Halbrook
J
J
Department of Biological Chemistry, UCLA School of Medicine.
McEntee
K
K
D002413:Cations, Divalent; D002845:Chromatography; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004277:DNA, Single-Stranded / Q000378:metabolism*; D004279:DNA, Viral / Q000378:metabolism / Q000648:ultrastructure; D017886:Durapatite; D006882:Hydroxyapatites; D007700:Kinetics; D008854:Microscopy, Electron; D008938:Mitosis; D009695:Nucleic Acid Renaturation; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*
D002413:Cations, Divalent; D004277:DNA, Single-Stranded; D004279:DNA, Viral; D006882:Hydroxyapatites; D017886:Durapatite