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M Lichten

Publications and source records attributed to M Lichten.

28 records · Page 2Linked to original sources
PubMed1993

Timing of molecular events in meiosis in Saccharomyces cerevisiae: stable heteroduplex DNA is formed late in meiotic prophase.

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 MAT. Remarkably, sequences in the ARG4 promoter that suffered double-strand cleavage at the normal ARG4 locus were not cut at significant levels when present at MAT::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.

DNA Repair↗
PubMed1991

The frequency of meiotic recombination in yeast is independent of the number and position of homologous donor sequences: implications for chromosome pairing.

We constructed diploids of Saccharomyces cerevisiae homozygous for LEU2 and carrying one, two, or four copies of leu2 at ectopic locations and determined the frequency of 3+:1- (LEU2:leu2) meiotic tetrads. Gene conversion between a LEU2 recipient and a leu2 ectopic donor occurred at the same frequency as did gene conversion between allelic copies of LEU2 and leu2. An increase in the number of possible ectopic donor loci did not lead to a proportional increase in the level of ectopic gene conversion. We suggest that the limiting step in meiotic recombination is the activation of a locus to become a recipient in recombination and that once activated, a locus can search the entire genome for a homologous partner with which to recombine. In this respect, this search for a homologous partner resembles the efficient premeiotic methylation/inactivation of duplicated sequences in Ascobolus and Neurospora. These observations support models in which strand exchange serves to align homologous chromosomes prior to their becoming much more fully synapsed by the elaboration of the synaptonemal complex.

Crosses, Genetic↗
PubMed1990

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

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----C.G transversion that displays a moderately high level of postmeiotic segregation. Of the two possible arg4-nsp/ARG4 mismatches (G.G and C.C), only C.C was detected in spores from mismatch repair-competent (Pms1+) diploids. In contrast, C.C and G.G were present at nearly equal levels in spores from Pms1- diploids. These results confirm previous suggestions that postmeiotic segregation spores contain heteroduplex DNA at the site of the 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+:5m (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.

Base Sequence↗
PubMed1989

Position effects in ectopic and allelic mitotic recombination in Saccharomyces cerevisiae.

We have examined the role that genomic location plays in mitotic intragenic recombination. Mutant alleles of the LEU2 gene were inserted at five locations in the yeast genome. Diploid and haploid strains containing various combinations of these inserts were used to examine both allelic recombination (between sequences at the same position on parental homologs) and ectopic recombination (between sequences at nonallelic locations). Chromosomal location had little effect on mitotic allelic recombination. The rate of recombination to LEU2 at five different loci varied less than threefold. This finding contrasts with previous observations of strong position effects in meiosis; frequencies of meiotic recombination at the same five loci differ by about a factor of forty. Mitotic recombination between dispersed copies of leu2 displayed strong position effects. Copies of leu2 located approximately 20 kb apart on the same chromosome recombined at rates 6-13-fold higher than those observed for allelic copies of leu2. leu2 sequences located on nonhomologous chromosomes or at distant loci on the same chromosome recombined at rates similar to those observed for allelic copies. We suggest that, during mitosis, parental homologs interact with each other no more frequently than do nonhomologous chromosomes.

Alleles↗
PubMed1987

Meiotic gene conversion and crossing over between dispersed homologous sequences occurs frequently in Saccharomyces cerevisiae.

We have examined meiotic recombination between two defined leu2 heteroalleles present at the normal LEU2 locus and in leu2-containing plasmids inserted at four other genomic locations. In diploids where the two leu2 markers were present at allelic locations on parental homologs, the frequency of Leu2+ spores varied 38-fold, in a location-dependent manner. These results indicate that recombination in a genetic interval can be modulated by sequences at least 2.7 kb outside that interval. Leu2+ meiotic segregants were also recovered from diploids where LEU2 was marked with one heteroallele, and the other leu2 heteroallele was inserted at another genomic location. These products of ectopic interactions, between dispersed copies of leu2 sharing only 2.2 kb of homology, were recovered at a frequency comparable to that observed in corresponding allelic crosses. This high frequency of ectopic meiotic recombination was observed in crosses where both recombining partners could potentially pair with sequences at an allelic position. In addition, a significant fraction (22-50%) of these ectopic recombinants were associated with crossing over of flanking sequences.

Alleles↗
PubMed1986

Analysis of meiosis-defective mutations in yeast by physical monitoring of recombination.

We have developed a method by which the extent of physical exchange of DNA molecules can be determined throughout meiosis in the yeast Saccharomyces cerevisiae. We have used this technique to analyze the effect of five meiosis-defective mutations (rad6, rad50, rad52, rad57 and spo11) on the physical exchange of DNA molecules. In the same experiments, we have also measured other meiotic parameters, such as premeiotic DNA synthesis, commitment to intragenic recombination, haploidization, ascus formation, and viability. rad50 and spo11 diploids make an undetectable amount of physically recombined DNA and less than 1% of wild-type levels of viable intragenic recombinants. In contrast, diploids homozygous for rad52, rad6 or rad57 all yield significant amounts of novel restriction fragments which arise by recombination. rad57 diploids make nearly wild-type levels of the recombined restriction fragments, although they produce less than 10% of the wild-type levels of viable intragenic recombinants. rad52 strains are also capable of a significant (33%) amount of exchange of DNA molecules, but make less than 1% of wild-type levels of viable intragenic recombinants. rad6 diploids are also capable of undergoing a high level of exchange, as measured by the appearance of the recombined restriction fragment. In addition, rad6 diploids show an unusual allele- or locus-specific variability in the level of viable intragenic recombinants produced. Although rad6 diploids produce no viable spores, they are able to complete a significant amount of haploidization upon return to vegetative growth conditions.

DNA Restriction Enzymes↗
PubMed1984

Evidence for inclusion of regions of nonhomology in heteroduplex products of bacteriophage lambda recombination.

Total intracellular DNA was isolated from replication-restricted bacteriophage lambda crosses in which the infecting parents were heteroallelic for wild-type and deletion mutant alleles. This DNA was examined for the presence of heteroduplex DNA molecules that contained wild-type sequences in one strand and deletion-mutant sequences in the other. Molecules hybrid for a 689-nucleotide deletion in the immunity region of lambda were detected at significant levels only in crosses in which both the red recombination system of lambda and the rec recombination system of Escherichia coli were active. Molecules hybrid for a 1300-nucleotide deletion in the central portion of the lambda genome were detected at significant levels in DNA isolated from both red+ and red- crosses in which recA function was present.

Bacteriophage lambda↗
PubMed1983

Effects of nonhomology on bacteriophage lambda recombination.

When crosses are performed under conditions severely restricting DNA synthesis, the presence of DNA sequence nonhomologies in the lac region of lambda plac5 limits the parental material contribution to and the yield of phage recombinant in a region bracketed by the nonhomologies. These observations are consistent with the expectation of a role for branch migration in the formation of heteroduplex structures under these conditions. Under conditions permissive for DNA replication, bracketing a region with nonhomologies has an only modest effect on the yield of recombinants within that interval. In addition, recombinants within such a bracketed interval manifest an excess of coincident exchange events in an adjacent region. These observations suggest the possibility that, under conditions permissive for DNA replication, regions of nonhomology can be included in heteroduplex structures.

Bacteriophage lambda↗