Molecular and genetic analysis of the gene encoding the Saccharomyces cerevisiae strand exchange protein Sep1.
Vegetatively grown Saccharomyces cerevisiae cells contain an activity that promotes a number of homologous pairing reactions. A major portion of this activity is due to strand exchange protein 1 (Sep1), which was originally purified as a 132,000-Mr species (R. Kolodner, D. H. Evans, and P. T. Morrison, Proc. Natl. Acad. Sci. USA 84:5560-5564, 1987). The gene encoding Sep1 was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-Mr protein. The protein encoded by this open reading frame was overproduced and purified and had a relative molecular weight of approximately 160,000. The 160,000-Mr protein was at least as active in promoting homologous pairing as the original 132,000-Mr species, which has been shown to be a fragment of the intact 160,000-Mr Sep1 protein. The SEP1 gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn10LUK insertion mutations in the SEP1 gene were characterized. sep1 mutants grew more slowly than wild-type cells, showed a two- to fivefold decrease in the rate of spontaneous mitotic recombination between his4 heteroalleles, and were delayed in their ability to return to growth after UV or gamma irradiation. Sporulation of sep1/sep1 diploids was defective, as indicated by both a 10- to 40-fold reduction in spore formation and reduced spore viability of approximately 50%. The majority of sep1/sep1 diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sep1/sep1 his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His+ recombinants than did isogenic SEP1/SEP1 strains. sep1/sep1 mutants also showed an increased frequency of exchange between HIS4, LEU2, and MAT and a lack of positive interference between these markers compared with wild-type controls. The interaction between sep1, rad50, and spo13 mutations suggested that SEP1 acts in meiosis in a pathway that is parallel to the RAD50 pathway.
Tishkoff
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Division of Cellular and Molecular Biology, Dana-Farber Cancer Institute, Boston, Massachusetts.
Johnson
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Kolodner
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D000595:Amino Acid Sequence; D001483:Base Sequence; D015825:Chromosomes, Fungal; D003001:Cloning, Molecular; D003434:Crossing Over, Genetic; D005095:Exoribonucleases; D005656:Fungal Proteins / Q000235:genetics* / Q000302:isolation & purification; D005800:Genes, Fungal; D005838:Genotype; D008540:Meiosis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D029701:Saccharomyces cerevisiae Proteins; D013172:Spores, Fungal / Q000502:physiology
D005656:Fungal Proteins; D015345:Oligonucleotide Probes; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D005095:Exoribonucleases; C068398:XRN1 protein, S cerevisiae