Purification of a strand exchange stimulatory factor from Saccharomyces cerevisiae.
The SEP1 strand exchange protein of Saccharomyces cerevisiae catalyzes the formation of heteroduplex DNA joints between single-strand circles and homologous linear duplexes in vitro. Previous work [Kolodner, R., Evans, D. H., & Morrison, P. T. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 5560-5564] showed that the optimal stoichiometry of SEP1 in this reaction was 1 SEP1 monomer per 12-14 nucleotides of single-stranded DNA. The work presented here describes the purification and characterization of a 33,000-dalton yeast protein that permits SEP1 to catalyze joint molecule formation at much lower stoichiometries. In the presence of this second factor, which has been designated SF1 for stimulatory factor 1, the optimal amount of SEP1 dropped to 1 SEP1 monomer per 725 nucleotides of single-stranded DNA. At this concentration of SEP1, the rate of joint molecule formation increased approximately 3-fold over that seen in the unstimulated reaction (no SF1). Titration experiments indicated that when the concentration of SEP1 was reduced over 300-fold to 1 SEP1 molecule per 5800 nucleotides of single-stranded DNA, the stimulated reaction had the same rate and extent of joint molecule formation as the unstimulated reaction. The optimal amount of SF1 was 1 molecule of SF1 per 20 nucleotides of single-stranded DNA. Electron microscopic analysis showed that a bona fide strand exchange reaction produced the joint molecules in the stimulated reaction. The stimulated reaction had requirements that were essentially identical with those seen in the unstimulated reaction, including a lack of dependence on ATP. SF1 aggregated single-stranded and double-stranded DNA.(ABSTRACT TRUNCATED AT 250 WORDS)
Norris
D
D
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Kolodner
R
R
D004271:DNA, Fungal / Q000032:analysis*; D004277:DNA, Single-Stranded / Q000032:analysis*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D005656:Fungal Proteins / Q000302:isolation & purification*; D008938:Mitosis; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000648:ultrastructure
D004271:DNA, Fungal; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; D005656:Fungal Proteins