DNA polymerases, deoxyribonucleases, and recombination during meiosis in Saccharomyces cerevisiae.

Abstract

We utilized strains of Saccharomyces cerevisiae that exhibit high efficiency of synchrony of meiosis to examine several aspects of meiosis including sporulation, recombination, DNA synthesis, DNA polymerase I and II, and Mg2+-dependent alkaline DNases. The kinetics of commitment to intragenic recombination and sporulation are similar. The synthesis of DNA, as measured directly with diphenylamine, appears to precede the commitment to recombination. Both DNA polymerase I and H activities and total DNA-synthesizing activity in crude extracts increase two-to threefold before the beginning of meiotic DNA synthesis. Increases of 10-to 20-fold over mitotic levels are found for Mg2+-dependent alkaline DNase activity in crude extracts before and during the commitment to meiotic intragenic recombination. Of particular interest is the comparable increase in a nuclease under the control of the RAD52 gene; this enzyme has been identified by the use of antibody raised against a similar enzyme from Neurospora crassa. Since the RAD52 gene is essential for meiotic recombination, the nuclease is implicated in the high levels of recombination observed during meiosis. The effects observed in this report are meiosis specific since they are not observed in an aa strain.

Functional and physical interaction between Sgs1 and Top3 and Sgs1-independent function of Top3 in DNA recombination repair.

“…The yeast strains used in this study were as follows: MR101 112 trp1-289 his1-7 ) (Resnick et al, 1984) 112 gal4D gal80D cyhr2 LYS2::GAL1-HIS3-HIS3 MEL1 URA3::GAL1-GAL1-lacZ ). The complete null top3 ∆ mutant from 1 to 2000nt (open reading frame;1-1971nt) was constructed by one-step gene replacement using a PCR-based gene deletion method as described (Longtine et al, 1998), with PCR primers (5'-GCGCTCGCTTCAGCTACGTAAAGGTG-TATTATAGACAATACGGATCCCCGGGTTAATTAA-3' and 5'-ATATGCAGTCAGCTGAATCATGCAATTAAG-CGGAGAGCTTGAATTCGAGCTCGTTTAAAC-3') and template (pFA6a-kanMX6).…”

ATP-independent DNA strand transfer catalyzed by protein(s) from meiotic cells of the yeast Saccharomyces cerevisiae.

“…To date, it has been possible to identify DNA changes associated with recombination (3,4) and to charactenze some enzymes that may be involved (5)(6)(7). Based on studies with several organisms and human cells (see ref.…”

“…Conditions for growth and sporulation of yeast strains have been described (5). Cells were collected by centrifugation, resuspended in 10% sucrose/50 mM Tris-HCI, pH 7.5/10 mM EDTA, and frozen in liquid nitrogen (5 (5% NaDodSO4/25% glycerol/0.5% bromophenol blue) was added, and the total sample was loaded onto 1% agarose gels in TBE buffer (12) and electrophoresed at room temperature at 4 V/cm for 5-16 hr.…”

“…Conditions for growth and sporulation of yeast strains have been described (5). Cells were collected by centrifugation, resuspended in 10% sucrose/50 mM Tris-HCI, pH 7.5/10 mM EDTA, and frozen in liquid nitrogen (5 (5% NaDodSO4/25% glycerol/0.5% bromophenol blue) was added, and the total sample was loaded onto 1% agarose gels in TBE buffer (12) and electrophoresed at room temperature at 4 V/cm for 5-16 hr. Occasionally, the reactions were terminated by addition of 10 mM EDTA/1% NaDodSO4 and the products were treated with 0.1 mg of proteinase K per ml at 25C for 30 min; this was followed by phenol extraction and ethanol precipitation (12).…”

Yeast and human genes that affect the Escherichia coli SOS response

“…To increase our chances of isolating yeast cDNAs affecting genome stability, we purified poly(A) ϩ mRNA from the diploid yeast strain MR48 (14) at various times in meiosis (0.5, 1, 2, 4 and 6 h; after 6 h, spore formation was visually confirmed). During meiosis, there is a significant induction of various DNA metabolic activities including replication and recombination, as well as increases in specific activities of enzymes associated with these processes (14). Cells were harvested and guanidium isothiocyanateprepared meiotic RNA was isolated as described (15).…”