Plasmid recombination intermediates generated in a Saccharomyces cerevisiae cell-free recombination system.

Abstract

We have developed an assay utilizing Saccharomyces cerevisiae cell extracts to catalyze recombination in vitro between homologous plasmids containing different mutant alleles of the tet gene. Electrophoretic analysis of product DNA indicated that a number of novel DNA species were formed during the reaction. These species migrated through agarose gels as distinct bands with decreased electrophoretic mobility compared with the substrate DNA. The DNA from each individual band was purified and shown to be enriched 5-to 100-fold for tetracycline-resistant recombinants by using a transformation assay. The structure of the DNA molecules present in these bands was determined by electron microscopy. Recombination between circular substrates appeared to involve the formation and processing of figure-eight molecules, while recombination between circular and linear substrates involved the formation of molecules in which a circular monomer had a monomer-length linear tail attached at a region of homology.Genetic recombination is the process by which DNA molecules undergo rearrangement to generate new combinations of genes or parts of genes. Because meiotic recombination in Saccharomyces cerevisiae generally yields 6:2, 2:6, 5:3, and 3:5 segregants and rarely yields aberrant 4:4 segregants, recombination models involving the formation of asymmetric heteroduplex DNA have been proposed (18). This is in contrast to the original model proposed by Holliday, which hypothesizes the formation of symmetric regions of heteroduplex DNA (13). Asymmetric strand exchange could be initiated at the site of single-strand breaks in DNA. In support of this model, it has been shown that single-strand breaks in DNA accumulate during meiosis at the time of commitment to recombination (24). Double-strand breaks in DNA have been shown to be efficiently repaired by a recombination reaction in S. cerev'isiae (20,21,25). These data have led to the proposal of a model in which recombination initiates at the site of double-strand breaks and results in the formation of a joint molecule containing two Holliday junctions (28). This model can explain certain features of yeast meiotic recombination, such as the predominance of 5:3, 3:5, 6:2, and 2:6 segregants over aberrant 4:4 segregants and the observation that initiation of recombination frequently occurs on the chromatid that is the recipient of genetic information during gene conversion events (11,12

Mismatch correction catalyzed by cell-free extracts of Saccharomyces cerevisiae.

“…The data presented in Table 3 and Fig. 5 have been used to calculate that the approximate length of the A-C mismatch correction associated excision/resynthesis tract is on the order of 10-20 nucleotides.t DISCUSSION Our results indicate that the S. cerevisiae cell-free mitotic recombination system developed in this laboratory (32,34) will catalyze mismatch correction. The repair reactions were efficient, with the specific activity for repair of the A-C mispair (2 pmol/mg of protein, Table 2) representing approximately 300 repair events per cellular equivalent of protein.…”

Partial purification of an enzyme from Saccharomyces cerevisiae that cleaves Holliday junctions.

“…S. cerevisiae strain AP-1 was grown to 5 x 107 cells per ml in yeast extract/peptone/dextrose broth, harvested by centrifugation, resuspended in 50 mM Tris HCl (pH 7.5)/10% (wt/vol) sucrose/i mM EDTA at 2.5 x 109 cells per ml and stored at -70°C as described (21,33). Cells (160 ml) were thawed at room temperature and placed on ice, and the following additions were made: 4 M KCl to a final concentration of 400 mM, 0.1 M spermidine (pH 8.0) to a final concentration of 5 mM, 0.5 M EDTA (pH 8.0) to a final concentration of 1 mM, 2-mercaptoethanol to a final concentration of 14.3 mM, and 10 mg of Zymolyase 100T per ml to a final concentration of 0.4 mg/ml.…”

“…The T4 and T7 enzymes differ from the Int protein in that they have endonuclease activity on single-stranded substrates and lack sequence specificity whereas the Int protein has no single-stranded DNA-specific endonuclease activity and only cleaves Holliday junctions constructed from X att sites (14,18,19). We have recently demonstrated that DNA molecules having a figure-8 configuration are generated in a Saccharomyces cerevisiae cell-free recombination system and that these molecules appear to be processed during the reaction (20,21). Here we describe an enzymatic activity from yeast that cleaves Holliday structures.…”

Double-strand-break repair and recombination catalyzed by a nuclear extract of Saccharomyces cerevisiae.

“…Although a direct analysis of the reaction products by Southern blot hybridization revealed the presence of XhoI resistant molecules that could be due to either heteroduplex DNA, or corrected DNA, at one of the mutant sites, reciprocal exchange products were not detected. Branched DNA intermediates were observed by electron microscopy (Symington et al, 1985) and a slight stimulatory effect on 987 recombination was noted when a double-strand break was introduced into one of the substrates (Symington et al, 1983).…”