Molecular and Genetic Analysis of the Gene Encoding the Saccharomyces cerevisiae Strand Exchange Protein Sepl

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 (Sepl), 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 Sepl was cloned, and analysis of the cloned gene revealed a 4,587-bp open reading frame capable of encoding a 175,000-M, 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 Sepl protein. The SEP] gene mapped to chromosome VII within 20 kbp of RAD54. Three Tn1OLUK insertion mutations in the SEPI gene were characterized. sepi 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 y irradiation. Sporulation of sepilsepl 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 sepilsepl diploid cells arrested in meiosis after commitment to recombination but prior to the meiosis I cell division. Return-to-growth experiments showed that sepilsepi his4X/his4B diploids exhibited a five- to sixfold greater meiotic induction of His' recombinants than did isogenic SEPI/SEPI strains. sepilsepi 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 sepi, radSO, and spol3 mutations suggested that SEP] acts in meiosis in a pathway that is parallel to the RADSO pathway. cleotide # 970 was chosen for the initial screen. Oligonucleotide 970 was 5' 32P labeled (63) and used to screen the YCp50 library of cloned S. cerevisiae chromosomal DNA fragments (66) by using the method of Wood et al. (75). The positive clones from the initial screening were rescreened by using oligonucleotides 970, 928, 934, and 937; this resulted in the identification of two plasmids (pRDK251 and pRDK252) containing the SEP] gene; pRDK252 contains a 7.5-kbp Sau3A fragment of S. cerevisiae chromosomal DNA in the BamHI site of YCpSO, and pRDK251 is a similar plasmid containing an overlapping 12-kbp Sau3A fragment. DNA sequence analysis. DNA sequencing was performed on CsCl purified plasmid DNA by using a Sequenase kit from U.S. Biochemicals. The SEP] region of pRDK252 was sequenced on both strands by using appropriate oligonucle- otide primers. Correspondence between cloned DNA sequences and genomic sequences was checked by using pairs of sequencing primers to amplify both cloned and genomic DNA by the polymerase chain reaction to show that the two DNAs yielded identical fragments. TnJOLUK insertion points were determined by using the oligonucleotide 5'- CAAGATGTGTATCCACC-3' that hybridizes to the lacZ end of TnJOLUK and directs DNA synthesis outward. The DNA and translated protein sequences were analyzed by using the Pearson-Lipman algorithm (58) available on the Eugene program package (LARK Sequencing Technologies, Ltd., Tex.) for homology searches against the PIR protein sequence data base (release no. 23.0) and against the GenBank DNA sequence data base (release no. 63.0). The translated protein sequence was searched against a library of 152 protein signature sequences by using the Prosite search program (Molecular