Molecular mechanisms determining methylation patterns in eukaryotic genomes still remain unresolved. We have characterized, in Ascobolus, a gene for de novo methylation. This novel eukaryotic gene, masc1, encodes a protein that has all motifs of the catalytic domain of eukaryotic C5-DNA-methyltransferases but is unique in that it lacks a regulatory N-terminal domain. The disruption of masc1 has no effect on viability or methylation maintenance but prevents the de novo methylation of DNA repeats, which takes place after fertilization, through the methylation induced premeiotically (MIP) process. Crosses between parents harboring the masc1 disruption are arrested at an early stage of sexual reproduction, indicating that the activity of Masc1, the product of the gene, is crucial in this developmental process.

To better understand the means by which chromosomes pair and recombine during meiosis, we have determined the time of appearance of heteroduplex DNA relative to the times of appearance of double-strand DNA breaks and of mature recombined molecules. Site-specific double-strand breaks appeared early in meiosis and were formed and repaired with a timing consistent with a role for breaks as initiators of recombination. Heteroduplex-containing molecules appeared about 1 h after double-strand breaks and were followed shortly by crossover products and the first meiotic nuclear division. We conclude that parental chromosomes are stably joined in heteroduplex-containing structures late in meiotic prophase and that these structures are rapidly resolved to yield mature crossover products. If the chromosome pairing and synapsis observed earlier in meiotic prophase is mediated by formation of biparental DNA structures, these structures most likely either contain regions of non-Watson-Crick base pairs or contain regions of heteroduplex DNA that either are very short or dissociate during DNA purification. Two loci were examined in this study: the normal ARG4 locus, and an artificial locus consisting of an arg4-containing plasmid inserted at AUT. Remarkably, sequences in the ARG4 promoter that suffered double-strand cleavage at the normalARG4 locus were not cut at significant levels when present at AL4T:arg4. These results indicate that the formation of double-strand breaks during meiosis does not simply involve the specific recognition and cleavage of a short nucleotide sequence.

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 +, +,