Fidelity of meiotic gene conversion in yeast

Summary Gene conversion was studied in a sample of 3869 unselected meiotic tetrads obtained from three diploids, respectively; heterozygous for a single ochre mutant, heteroallelic for a pair of ochre alleles, and heterozygous for an ochre specific suppressor. Although the genetic system were sufficiently sensitive to detect single base changes at the mutant codon level, none were found among 36 conversions (1+:3m) of the ochre mutants and 153 conversions (3S:1+) of the suppressor locus. These findings lead to the conclusion that the informational transfer in gene conversion occurred with complete fidelity. Gene conversion conserved and did not generate new genetic information. The error level of conversion was estimated as less than 10^-2/N.P.     

Expansions and contractions of the genetic map relative to the physical map of yeast chromosome III.

To examine the relationship between genetic and physical chromosome maps, we constructed a diploid strain of the yeast Saccharomyces cerevisiae heterozygous for 12 restriction site mutations within a 23-kilobase (5-centimorgan) interval of chromosome III. Crossovers were not uniformly distributed along the chromosome, one interval containing significantly more and one interval significantly fewer crossovers than expected. One-third of these crossovers occurred within 6 kilobases of the centromere. Approximately half of the exchanges were associated with gene conversion events. The minimum length of gene conversion tracts varied from 4 base pairs to more than 12 kilobases, and these tracts were nonuniformly distributed along the chromosome. We conclude that the chromosomal sequence or structure has a dramatic effect on meiotic recombination.     

Induction and repair of gene conversion in UV-sensitive mutants of yeast

Summary Photoreactivation effect on UV-induced allelic recombination has been examined using various combinations of leu 1 alleles in UV-sensitive and wild type diploid yeast, Saccharomyces cerevisiae. The frequencies of UV-induced heteroallelic reversion in UV-sensitive strains, presumably lacking dark-repair, are strikingly enhanced compared to those in wild type at the same doses under dark condition. However, these enhanced frequencies of reversion are diminished by photoreactivation almost to the level of those in wild type. The induced frequencies of homoallelic reversion (mutation) of relevant alleles are apparently lower than those of heteroallelic reversion. Phenotypic analysis for linked gene leu 1 on UV-induced heteroallelic revertants has shown that most of the revertants are of the nonreciprocal type recombination (mitotic gene conversion). These results would indicate that most of the dark-repairable damage leading to mitotic gene conversion after UV-light is due to pyrimidine dimers.On leave of absence from Radiation Center of Osaka Prefecture, Shinke-cho Sakai, Osaka, Japan.     

Involvement of the essential yeast DNA polymerases in induced gene conversion

In the yeast Saccharomyces cerevisiae three different DNA polymerases alpha, delta and epsilon are involved in DNA replication. DNA polymerase alpha is responsible for initiation of DNA synthesis and polymerases delta and epsilon are required for elongation of DNA strand during replication. DNA polymerases delta and epsilon are also involved in DNA repair. In this work we studied the role of these three DNA polymerases in the process of recombinational synthesis. Using thermo-sensitive heteroallelic mutants in genes encoding DNA polymerases we studied their role in the process of induced gene conversion. Mutant strains were treated with mutagens, incubated under permissive or restrictive conditions and the numbers of convertants obtained were compared. A very high difference in the number of convertants between restrictive and permissive conditions was observed for polymerases alpha and delta, which suggests that these two polymerases play an important role in DNA synthesis during mitotic gene conversion. Marginal dependence of gene conversion on the activity of polymerase epsilon indicates that this DNA polymerase may be involved in this process but rather as an auxiliary enzyme.     

Alkylation induced gene conversion in yeast: Use in fine structure mapping

Summary Methyl-methanesulfonate (MMS) causes gene conversions in heteroallelic diploids of Saccharomyces cerevisiae. The frequency of production of prototrophic convertants is linearly proportional to the square of the time of MMS treatment, and the regression of prototrophs on dose varies depending upon the particular pair of alleles present in the diploid. The regressions show an additivity relationship, in that when a triad of heteroallelic diploids of the type m₁/m₂, m₂/m₃, and m₁/m₃ is considered, two of the regressions add up approximately to the third. MMS can, therefore, be used in fine structure mitotic mapping of genes. Good agreement was found both in relative order and spacing of alleles at the histidine 1 locus of yeast when the fine structure map based on the X-ray mapping method was compared with that based on MMS.     

Population bottlenecks in Polynesia revealed by minisatellites

Summary Tandem-repetitive highly variable loci in the human genome (minisatellites) have been used in gene mapping and as DNA fingerprints, but they have not yet found much application in population genetics. We have investigated the capacity of six minisatellites to discriminate between four populations in Oceania. We find that in comparison to Melanesians, Polynesians have a significant loss of heterozygosity (or gene diversity), not noted using more traditional markers. We show also that the number of alleles, the allele distribution and the mutation rates at the Polynesian minisatellite loci do not deviate from those predicted by the neutral mutation/infinite allele model. The low gene diversity is therefore likely to be a result of the maintenance of small population sizes and bottleneck effects during the colonization of the Pacific.     

Minisatellite variants generated in yeast meiosis involve DNA removal during gene conversion

Two yeast minisatellite alleles were cloned and inserted into a genetically defined interval in Saccharomyces cerevisiae. Analysis of flanking markers in combination with sequencing allowed the determination of the meiotic events that produced minisatellites with altered lengths. Tetrad analysis revealed that gene conversions, deletions, or complex combinations of both were involved in producing minisatellite variants. Similar changes were obtained following selection for nearby gene conversions or crossovers among random spores. The largest class of events involving the minisatellite was a 3:1 segregation of parental-size alleles, a class that would have been missed in all previous studies of minisatellites. Comparison of the sequences of the parental and novel alleles revealed that DNA must have been removed from the recipient array while a newly synthesized copy of donor array sequences was inserted. The length of inserted sequences did not appear to be constrained by the length of DNA that was removed. In cases where one or both sides of the insertion could be determined, the insertion endpoints were consistent with the suggestion that the event was mediated by alignment of homologous stretches of donor/recipient DNA.     

Direct selection of mutants influencing gene conversion in the yeast Schizosaccharomyces pombe

Summary In Schizosaccharomyces pombe, a suppressor-active mutation at the anticodon site of the tRNA _Ser ^UCA gene sup3 leads to opal (UGA)-specific suppression. Second-site mutations (rX) in sup3 inactivate the suppressor. The sup3-UGA, rX double mutants are genetically unstable in meiotic selfings, due to the intergenic transfer of information between sup3 and the unlinked genes sup9 and sup12 (Hofer et al. 1979; Munz and Leupold 1981; Munz et al. 1982). These three genes have considerable sequence homology over about 200 base pairs (Hottinger et al. 1982).Mutants showing a decrease or an increase of the meiotic instability at sup3 have been selected. One mutation (rec3-8) increases both the genetic instability and the frequency of intragenic recombination in sup3 by one order of magnitude. It has no effect on the stability of the nonsense alleles arg1-230 (UAA), ade6-704 and ura1-61 (UGA) or on the frequency of crossing-over between sup3 and the closely linked gene cdc8.The existence of a common genetic control over intragenic recombination and genetic instability at sup3 provides a direct way of selecting for rec mutants in homothallic haploid strains of S. pombe carrying a suppressor-inactive allele of sup3. It also supports the hypothesis that the instability of mutant alleles of this gene is due to chromosome mispairing at meiosis allowing sup3 to pair with sup9 or sup12 and then to undergo recombination by gene conversion restoring the suppressor-active allele sup3-UGA from the suppressor-inactive allele sup3-UGA,rX. Two mutations (rec2-5 and rec5-11) have no effect on intragenic recombination, but considerably reduce the meiotic instability of the sup3-UGA,rX alleles. They may suppress illegitimate pairing between sup3 and sup9 or sup12.     

Possible involvement of the yeast POLIII DNA polymerase in induced gene conversion

Summary In Saccharomyces cerevisiae, three different DNA polymerase complexes, POLI, POLII and POLIII, are known to be involved in DNA replication. The catalytic subunit of POLIII is encoded by the essential CDC2 gene. The existence of different thermosensitive non-complementing mutants of CDC2 offers the possibility of using a genetic approach to investigate the involvement of POLIII in induced gene conversion. When cdc2 heteroallelic cells were irradiated and incubated under restrictive conditions, almost no induction of thermoresistant cells could be detected, suggesting an essential role for POLIII in mitotic gene conversion events.     

Failed gene conversion leads to extensive end processing and chromosomal rearrangements in fission yeast

Loss of heterozygosity (LOH), a causal event in cancer and human genetic diseases, frequently encompasses multiple genetic loci and whole chromosome arms. However, the mechanisms by which such extensive LOH arises, and how it is suppressed in normal cells is poorly understood. We have developed a genetic system to investigate the mechanisms of DNA double‐strand break (DSB)‐induced extensive LOH, and its suppression, using a non‐essential minichromosome, Ch¹⁶, in fission yeast. We find extensive LOH to arise from a new break‐induced mechanism of isochromosome formation. Our data support a model in which Rqh1 and Exo1‐dependent end processing from an unrepaired DSB leads to removal of the broken chromosome arm and to break‐induced replication of the intact arm from the centromere, a considerable distance from the initial lesion. This process also promotes genome‐wide copy number variation. A genetic screen revealed Rhp51, Rhp55, Rhp57 and the MRN complex to suppress both isochromosome formation and chromosome loss, in accordance with these events resulting from extensive end processing associated with failed homologous recombination repair.     

Induction of gene conversion in yeast cells continuously cultured at high radiation background

The induction of genetic damage was investigated by culturing diploid yeastSaccharomyces cerevisiae D7 cells continuously at radiation levels ranging from 0.383 µSv/h to 1.275 mSv/h by selecting appropriate concentrations of tritiated water in the growth medium. These radiation levels correspond to 3–10000 times the natural background. Parameters such as growth kinetics, gene conversion frequency at background radiation and after a challenging dose of acute gamma-radiation or alkylating agentN-methyl-N-nitro-N-nitrosoguanidine (MNNG) were assessed. The gene conversion frequency in most of the assays was in the range of 5–10 convertants per 10⁶ cells, as in the case of controls. However, a number of the cultures showed conversion frequencies above 20 per 106 viable cells. This stochastic phenomenon occurred more frequently in cells which were incubated at higher radiation levels and for longer durations. This suggests that radiation is responsible for the phenomenon. When subculturing continued beyond 900 h, gene conversion frequencies reverted back to normal values in all cultures in spite of elevated background radiation levels, thus suggesting an adaptive response. The generation time of the cells was 78 min in all cultures irrespective of the radiation level. The response of the cells cultured at elevated background radiation levels to subsequent challenging treatment with gamma-radiation or MNNG was identical to that of the control cultures. Our results suggest that in eukaryotic yeast, low-level radiation may induce an adaptive response to chronic radiation, whereas no such response could be detected when the cells were challenged with acute high-dose exposure or with MNNG.     

Unequal crossing-over and gene conversion at the amplified CUP1 locus of yeast

Summary Meiotic recombination was analyzed between two twelve-copy arrays of a gene amplification at theCUP1 locus ofSaccharomyces cerevisiae. Utilizing Southern analysis to identify spores with non-parental repeat arrays, we find that approximately 11% of a sample with 202 unselected tetrads possess at least one nonparental spore array. Both reciprocal and non-reciprocal changes are observed. The data suggest a model in which frequent mispairing among identical copies of the 2.0 kb repeat unit leads to the formation of unpaired loops containing integral numbers of repeat units. In this model, conversions involving the loops lead to non-reciprocal changes in arrays: about half are associated with reciprocal exchange, and net increases in repeat unit numbers occur about as frequently as net decreases. Thus, the known properties of gene conversion can account for all the segregations we observe.     

A yeast centromere acts in cis to inhibit meiotic gene conversion of adjacent sequences

The centromere of chromosome III (CEN3) of yeast has been examined for its ability to inhibit meiotic recombination in adjacent sequences. The effect of the centromere was investigated when it was adjacent to both of the recombining sequences (homozygous) or adjacent to only one of the two recombining DNA segments (hemizygous). When homozygous, CEN3 exerts a bidirectional repression of crossing over and a strong inhibition of gene conversion. This suggests that CEN3 reduces the frequency of crossing over by interfering with the initiation of proximal recombination events. When hemizygous, CEN3 impairs the ability of adjacent sequences to act as the recipient of genetic information during gene conversion. These results support the idea that the initiating event in yeast meiotic recombination involves the recipient molecule.     

Multiple heterologies increase mitotic double-strand break-induced allelic gene conversion tract lengths in yeast.

Spontaneous and double-strand break (DSB)-induced allelic recombination in yeast was investigated in crosses between ura3 heteroalleles inactivated by an HO site and a +1 frameshift mutation, with flanking markers defining a 3.4-kbp interval. In some crosses, nine additional phenotypically silent RFLP mutations were present at approximately 100-bp intervals. Increasing heterology from 0.2 to 1% in this interval reduced spontaneous, but not DSB-induced, recombination. For DSB-induced events, 75% were continuous tract gene conversions without a crossover in this interval; discontinuous tracts and conversions associated with a crossover each comprised approximately 7% of events, and 10% also converted markers in unbroken alleles. Loss of heterozygosity was seen for all markers centromere distal to the HO site in 50% of products; such loss could reflect gene conversion, break-induced replication, chromosome loss, or G2 crossovers. Using telomere-marked strains we determined that nearly all allelic DSB repair occurs by gene conversion. We further show that most allelic conversion results from mismatch repair of heteroduplex DNA. Interestingly, markers shared between the sparsely and densely marked interval converted at higher rates in the densely marked interval. Thus, the extra markers increased gene conversion tract lengths, which may reflect mismatch repair-induced recombination, or a shift from restoration- to conversion-type repair.     

One-step gene replacement in yeast by cotransformation

A general method to replace chromosomal DNA sequences of Saccharomyces cerevisiae by any in vitro modified DNA sequence has been developed and was applied to the PHO5 locus on chromosome II. A recipient strain was constructed in which part of the chromosomal PHO5 sequence was substituted by the URA3 gene. Replacement of this pho5-URA3 substitution by pho5 mutant alleles was achieved in one step by cotransformation with a pho5 DNA fragment and the self-replicating plasmid YEp13, which contains the LEU2 gene as a selectable marker. Leu+ transformants were selected, and the replacement events at the PHO5 locus were detected by their Ura- phenotype (1-4% of the Leu+ were Ura-). In a similar way the PHO5 coding sequence was replaced by the sequence coding for human tissue-type plasminogen activator (t-PA).