Detection of heteroduplex DNA molecules among the products of Saccharomyces cerevisiae meiosis.

Abstract

We have used denaturant-gel electrophoresis to provide a physical demonstration of heteroduplex DNA in the products of yeast meiosis. We examined heteroduplex formation at arg4-nsp, a G-C -COG transversion that displays a moderately high level of postmeiotic segregation. Of the two possible arg4-nsp/ARG4 mismatches (GG and CC), only CC was detected in spores from mismatch repair-competent (Pmsl+) diploids. In contrast, C-C and G G were present at nearly equal levels in spores from Pmsl diploids. These results confirm previous suggestions that postmeiotic segregation spores contain heteroduplex DNA at the site ofthe marker in question, that C C is repaired less frequently than is G-G, and that the PMS1 gene product plays a role in mismatch correction. Combined with the observation that Pms1+ ARG4/arg4-nsp diploids produce 3 times more 3+:5" (wildtype:mutant) tetrads (+, +, +/m, m) than 5+:3m tetrads (+, +/m, m, m), these results indicate that, during meiosis, formation of heteroduplex DNA at ARG4 involves preferential transfer of the sense (nontranscribed) strand of the DNA duplex.Central to current models of generalized recombination is an early step in which a single strand of DNA is transferred from one parental duplex to the other, forming a region of heteroduplex DNA (1). Such a structure ensures that the breakage and joining reactions of recombination occur with fidelity and provides an attractive way to search for homology when initiating meiotic chromosome pairing (2).In the yeast Saccharomyces cerevisiae, evidence for formation of heteroduplex DNA during meiosis has largely been provided by genetic examination of tetrads. Although diploids heterozygous for a mutation (m/+) usually yield two mutant (m) and two wild-type (+) spores, occasionally gene conversion tetrads are produced. These tetrads contain parental alleles in a non-Mendelian ratio, most commonly 6+ :2m or 2+ :6m (numbers refer to the eight single DNA strands in the four spores). Occasionally, postmeiotic segregation tetrads are observed. These contain a haploid spore that, upon outgrowth, segregates both parental alleles at a locus and most often take 3+:5m and 5+:3m (+, +/m, m, m and +, +,

DNA Loop Repair by Escherichia coli Cell Extracts

“…DISCUSSION The major finding of this study is that repair of loops in E. coli extracts can be very efficient. In vitro assays with loops of 8,12,16,22,27,45, and 429 nucleotides show a high level of correction for all substrates. Under these assay conditions, the efficiency of loop repair was comparable with that of methyldirected mismatch repair.…”

“…Such pairing errors may take the form of base-base mismatches or loops, in which one strand contains one or more unpaired nucleotides. Strand-specific correction of base-base and loop mismatches produced during DNA biosynthesis plays an important role in mutation avoidance (2, 5, 6), and mismatch repair within the recombination heteroduplex has been implicated in gene conversion (3,4,7,8).Base-base mispairs are subject to strand-specific correction by the mismatch repair system of both prokaryotes and eukaryotes (5, 6, 9, 10), but action of this system on loop mispairs is limited to fairly small heterologies. The Escherichia coli mismatch repair pathway will correct loops up to about 7 unpaired nucleotides, but larger heterologies are poorly processed by this system (11)(12)(13)(14).…”

“…Such pairing errors may take the form of base-base mismatches or loops, in which one strand contains one or more unpaired nucleotides. Strand-specific correction of base-base and loop mismatches produced during DNA biosynthesis plays an important role in mutation avoidance (2, 5, 6), and mismatch repair within the recombination heteroduplex has been implicated in gene conversion (3,4,7,8).…”

Mismatch Repair during Homologous and Homeologous Recombination

“…The occurrence of mismatches during meiotic recombination has been documented in genetic experiments via the manifestation of sectored PMS colonies, as well as through physical methods (White et al 1985;Lichten et al 1990). Correction of mismatched meiotic recombination intermediates by the MMR machinery results in gene conversion (Alani et al 1994).…”

Physical detection of heteroduplexes during meiotic recombination in the yeast Saccharomyces cerevisiae.

“…First, since the assay involves sectoring of spore colonies, the kinetics of heteroduplex formation during meiosis cannot be examined. Thus (16) developed a denaturing gel procedure to detect heteroduplexes formed at theARG4 locus during meiotic recombination. In this report, we describe a simple general method of detecting heteroduplexes.…”