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PubMed1988

Genetic and physical analysis of the M26 recombination hotspot of Schizosaccharomyces pombe.

The ade6-M26 mutation of Schizosaccharomyces pombe has previously been reported to stimulate ade6 intragenic meiotic recombination. We report here that the ade6-M26 mutation is a single G----T nucleotide change, that M26 stimulated recombination within ade6 but not at other distinct loci, and that M26 stimulated meiotic but not mitotic recombination. In addition, M26 stimulated recombination within ade6 when M26 is homozygous; this result demonstrates that a base-pair mismatch at the M26 site was not required for the stimulation. These results are consistent with the ade6-M26 mutation creating a meiotic recombination initiation site.

Crosses, Genetic↗
PubMed1996

Double-strand break-induced mitotic intrachromosomal recombination in the fission yeast Schizosaccharomyces pombe.

The Saccharomyces cerevisiae HO gene and MATa cutting site were used to introduce site-specific double-strand breaks (DSBs) within intrachromosomal recombination substrates in Schizosaccharomyces pombe. The recombination substrates consisted of nontandem direct repeats of ade6 heteroalleles. DSB induction stimulated the frequency of recombinants 2000-fold. The spectrum of DSB-induced recombinants depended on whether the DSB was introduced within one of the ade6 repeats or in intervening unique DNA. When the DSB was introduced within unique DNA, over 99.8% of the recombinants lacked the intervening DNA but retained one copy of ade6 that was wild type or either one of the heteroalleles. When the DSB was located in duplicated DNA, 77% of the recombinants were similar to the deletion types described above, but the single ade6 copy was either wild type or exclusively that of the uncut repeat. The remaining 23% of the induced recombinants were gene convertants with two copies of ade6 and the intervening sequences; the ade6 heteroallele in which the DSB was induced was the recipient of genetic information. Half-sectored colonies were isolated, analyzed and interpreted as evidence of heteroduplex DNA formation. The results are discussed in terms of current models for recombination.

Chromosomes, Fungal↗
PubMed1995

Active and inactive transplacement of the M26 recombination hotspot in Schizosaccharomyces pombe.

The ade6-M26 mutation of the fission yeast Schizosaccharomyces pombe creates a meiotic recombination hotspot that elevates ade6 intragenic recombination approximately 10-15-fold. A heptanucleotide sequence including the M26 point mutation is required but not sufficient for hotspot activity. We studied the effects of plasmid and chromosomal context on M26 hotspot activity. The M26 hotspot was inactive on a multicopy plasmid containing M26 embedded within 3.0 or 5.9 kb of ade6 DNA. Random S. pombe genomic fragments totaling approximately 7 Mb did not activate the M26 hotspot on a plasmid. M26 hotspot activity was maintained when 3.0-, 4.4-, and 5.9-kb ade6-M26 DNA fragments, with various amounts of non-S. pombe plasmid DNA, were integrated at the ura4 chromosomal locus, but only in certain configurations relative to the ura4 gene and the cointegrated plasmid DNA. Several integrations created new M26-independent recombination hotspots. In all cases the non-ade6 DNA was located > 1 kb from the M26 site, and in some cases > 2 kb. Because the chromosomal context effect was transmitted over large distances, and did not appear to be mediated by a single discrete DNA sequence element, we infer that the local chromatin structure has a pronounced effect on M26 hotspot activity.

Chromosomes, Fungal↗
PubMed1994

Region-specific activators of meiotic recombination in Schizosaccharomyces pombe.

Schizosaccharomyces pombe rec mutants were previously isolated on the basis of their deficiency in meiotic recombination at the ade6 locus. We surveyed their meiotic recombination deficiencies at and between other loci. In rec10 mutants recombinant frequencies in the approximately 2-Mb region surrounding the ade6 locus were reduced 10- to 100-fold, but recombinant frequencies at or between nine other unlinked loci were reduced < 3-fold. The rec10 mutations are recessive and are on chromosome I; the ade6 region is on chromosome III. These results indicate that the rec10 gene product is required for activation of meiotic recombination in the approximately 2-Mb region surrounding ade6 but not in the other regions surveyed. Similar ade6 regional specificities were observed for rec8 and rec11. We infer that there are multiple activators of meiotic recombination, each specific for a limited set of loci, and we discuss how these regional activators may work.

Chromosomes, Fungal↗
PubMed1982

Cyclic AMP may not be involved in catabolite repression in Saccharomyes cerevisiae: evidence from mutants capable of utilizing it as an adenine source.

Mutants able to utilize 5'-AMP or cyclic AMP as the adenine source were isolated from an ade6 ade10 double mutant by ethyl methane sulfonate mutagenesis. A single amp1 mutation, primarily selected on 5'-AMP medium, confers the phenotype for utilization of exogenous 5'-AMP as the adenine source. From the ade6 ade10 amp1 triple mutant, a mutant able to utilize cyclic AMP was isolated, and the mutant phenotype was proven to be due to the simultaneous occurrence of triple mutations designated as cam1, cam2, and cam3. The cam3 mutation, but not cam1 or cam2, also confers the phenotype for utilizing 5'-AMP, the same phenotype as the amp1 mutation. All of these mutations are recessive to the respective wild-type counterparts. Cells having the ade6 ade10 amp1 cam1 cam2 cam3 genotype showed significant ability to take up exogenous cyclic AMP, whereas no differences were observed in cyclic AMP phosphodiesterase activity in comparison with that of the original strains used in the mutant isolation. Since glucose severely repressed galactokinase synthesis in the constitutive GAL81 mutant having the ade6 ade10 amp1 cam1 cam2 cam3 genotype, irrespective of the presence or absence of cyclic AMP in the medium, it was suggested that cyclic AMP is not involved in the mechanism of catabolite repression in Saccharomyces cerevisiae. It does, however, have a stimulative effect on the galactokinase synthesis in the GAL81 mutant in the absence of glucose.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1989

Meiotic recombination-deficient mutants of Schizosaccharomyces pombe.

A mutant screen employing the ade6-M26 recombination hotspot was developed and used to isolate Schizosaccharomyces pombe mutants deficient in meiotic recombination. Nine rec mutations were recessive, defining six complementation groups, and reduced ade6 meiotic recombination 3-fold to greater than or equal to 300-fold when homozygous. Three recessive rec mutations analyzed further also reduced meiotic intragenic recombination at ura4 on chromosome III and intergenic recombination between pro2 and arg3 on chromosome I. The observed non-co-ordinate reductions of the recombinant frequencies in the three test intervals suggest a degree of locus (or intragenic vs. intergenic) specificity of the corresponding rec+ gene products. None of the mutations specifically inactivated the ade6-M26 hotspot. Additional rec genes may be identified with these methods.

Crosses, Genetic↗
PubMed1992

Seventeen complementation groups of mutations decreasing meiotic recombination in Schizosaccharomyces pombe.

We have analyzed 43 recessive mutations reducing meiotic intragenic recombination in Schizosaccharomyces pombe. These mutations were isolated by a screen for reduced plasmid-by-chromosome recombination at the ade6 locus. Sixteen of the mutations define 10 new complementation groups, bringing to 17 the number of genes identified to be involved in meiotic recombination. The mutations were grouped into three discrete classes depending on the severity of the recombination deficiency in crosses involving the ade6-M26 recombination hotspot. Class I mutations caused at least a 1000-fold reduction in M26-stimulated intragenic recombination at the ade6 locus. Class II mutations reduced M26-stimulated recombination approximately 100-fold. Class III mutations caused a 3-10-fold reduction in either M26-stimulated or non-hotspot recombination. We obtained multiple alleles of class I and class II mutations, suggesting that we may be nearing saturation for mutations of this type. As a first step toward mapping, we used mitotic segregation to assign fourteen of the rec genes to chromosomes. Mutations in the six rec genes tested also caused a decrease in intragenic recombination at the ura4 locus; five of these mutations also reduced intergenic recombination between the pro2 and arg3 genes. These results indicate that these multiple rec gene products are required for high level meiotic recombination throughout the S. pombe genome.

Alleles↗
PubMed2001

Crossing over is rarely associated with mitotic intragenic recombination in Schizosaccharomyces pombe.

Chromosomal rearrangements can result from crossing over during ectopic homologous recombination between dispersed repetitive DNA. We have previously shown that meiotic ectopic recombination between artificially dispersed ade6 heteroalleles in the fission yeast Schizosaccharomyces pombe frequently results in chromosomal rearrangements. The same recombination substrates have been studied in mitotic recombination. Ectopic recombination rates in haploids were approximately 1-4 x 10(-6) recombinants per cell generation, similar to allelic recombination rates in diploids. In contrast, ectopic recombination rates in heterozygous diploids were 2.5-70 times lower than allelic recombination or ectopic recombination in haploids. These results suggest that diploid-specific factors inhibit ectopic recombination. Very few crossovers occurred in ade6 mitotic recombination, either allelic or ectopic. Allelic intragenic recombination was associated with 2% crossing over, and ectopic recombination between multiple different pairing partners showed 1-7% crossing over. These results contrast sharply with the 35-65% crossovers associated with meiotic ade6 recombination and suggest either differential control of resolution of recombination intermediates or alternative pathways of recombination in mitosis and meiosis.

Alleles↗
PubMed2005

Optimizing the nucleotide sequence of a meiotic recombination hotspot in Schizosaccharomyces pombe.

The ade6-M26 mutation of Schizosaccharomyces pombe created a meiotic recombination hotspot. Previous analyses indicated that the heptamer 5'-ATGACGT-3' was necessary and sufficient for hotspot activity; the Atf1-Pcr1 transcription factor binds to this sequence and activates M26. After finding cases in which the M26 heptamer in ade6 was, surprisingly, not active as a hotspot, we used an in vitro selection method (SELEX) that revealed an 18-bp consensus sequence for Atf1-Pcr1 binding, 5'-GNVTATGACGTCATNBNC-3', containing the M26 heptamer at its core. Using this consensus sequence as a guide, we made mutations on each side of the heptamer at two separate sites in ade6. These mutations increased the intracellular hotspot activity of the heptamer, in some cases by >15-fold. These results show that M26, the eukaryotic recombination hotspot with the most precisely defined nucleotide sequence, is larger than previously thought, and they provide valuable information for clarifying the role of M26, and perhaps other hotspots, in meiotic recombination.

Alleles↗
PubMed1988

Induction of mitotic chromosome loss in the diploid yeast Saccharomyces cerevisiae D61.M by genotoxic carcinogens and tumor promoters.

Three genotoxic carcinogens and eight tumor promoters were tested for induction of aneuploidy, specifically chromosome loss, in Saccharomyces cerevisiae D61.M. This is a heterozygous diploid yeast strain that permits the scoring of segregants expressing three linked recessive markers (cyhR2, ade6, and leu1), two of which (ade6 and leu1) are located close to the centromere on opposite arms of chromosome VII. The centromere marker leu was routinely checked, and a positive control (bavistan) was run with every experiment. The three genotoxic carcinogens aflatoxin B1, benzo(a)pyrene, and 7,12-dimethylbenz(a)anthracene did not induce aneuploidy, independent of the presence or absence of an exogenous metabolic activation system (rat liver homogenate; S9). Four of the eight tumor promoters tested induced chromosome loss but not mitotic recombination or mutation: cholic acid, lithocholic acid, phenobarbital, and saccharin. Diethylstilbestrol (DES) led to positive as well as to negative results in several independent experiments. In the case of the positive experiment, DES also induced putative recombinants. Three tumor promoters induced neither chromosome loss nor mitotic recombination: anthralin, 4,4'-dichloro-diphenyl-ethane (DDT) and gamma-hexachlorcyclohexane (lindane). From our experiments it can be concluded that the hypothesis put forward by Parry et al. [Nature; 294:263-265], according to which tumor promoters induce chromosome loss in yeast, is not correct in a general sense. In our set of eight tumor promoters, only one half distinctly induced chromosome loss.

Aneuploidy↗
PubMed1994

Telomere-associated chromosome breakage in fission yeast results in variegated expression of adjacent genes.

The sequence requirements for in vivo telomere function in the fission yeast, Schizosaccharomyces pombe, have been investigated. A 258 bp tract of previously characterized cloned fission yeast terminal repeats adjacent to 800 bp of telomere-associated sequences is sufficient to seed new telomeres onto linearized ars-containing plasmids when introduced into cells. The resulting transformants contain unrearranged, acentric, linear episomes. Cloned telomeres, with and without telomere-associated sequences adjacent to the 258 bp terminal repeats, were utilized to introduce chromosome breaks at specific sites in a non-essential minichromosome. Truncated minichromosome derivatives were recovered containing the ura4 or ade6 gene adjacent to a newly formed telomere. These telomeres exert reversible position effects on the expression of the adjacent ura4 or ade6 genes.

Adenine↗
PubMed1989

An abnormal cell division cycle in an AIR carboxylase-deficient mutant of the fission yeast Schizosaccharomyces pombe.

Adenine-requiring mutant strains of S. pombe enter the stationary phase after depleting a culture medium of adenine or its analogues. Stationary phase cells of six mutants defective at different stages of the purine nucleotide synthetic pathway were examined for cell volume and DNA content, and then compared in these respects with those of a prototrophic wild-type strain. The cell cycle of the wild-type strain was arrested in the G2 phase (2C state) in the nitrogen rich medium, as is evident from DNA content per cell (0.0425 pg) and cell volume (47.7 microns 3). An AIR carboxylase-deficient (ade6) mutant strain was found to have an unusual cell volume (307.4 microns 3) and DNA content (0.1187 pg). By DAPI fluorescence microscopy, each mutant cell was seen to contain only one enlarged nucleus, which indicates the absence of cell populations containing cells in the 4C state of the S phase following nuclear division. It then follows that in ade6 mutant cells, DNA synthesis occurs in the absence of a completed nuclear division. Thus in S. pombe cells, the completion of nuclear division is not necessarily required for the next cycle initiation of DNA synthesis under certain physiological conditions.

Adenine↗
PubMed2002

Meiotic DNA breaks at the S. pombe recombination hot spot M26.

The ade6-M26 allele of Schizosaccharomyces pombe creates a well-defined meiotic recombination hot spot that requires a specific sequence, 5'-ATGACGT-3', and the Atf1*Pcr1 transcription factor for activity. We find that M26 stimulates the formation of meiosis-specific double-strand DNA breaks at multiple sites surrounding M26. Like hot spot activity, breakage requires the M26 heptamer, Pcr1, and the general recombination factor Rec12. When the M26 heptamer is moved to new positions within ade6, new break sites are observed spanning approximately 0.5-2 kb around the moved heptamer. Break frequency is strongly correlated with recombination frequency for these alleles. The occurrence of breaks at M26 suggests mechanistic similarities to hot spots in the distantly related yeast Saccharomyces cerevisiae.

Activating Transcription Factor 1↗
PubMed1976

Recombination in Saccharomyces cerevisiae: a DNA repair mutation associated with elevated mitotic gene conversion.

A mutant haploid strain of Saccharomyces cerevisiae has been isolated that is sensitive to the alkylating agent methyl methanesulfonate at a concentration of 0.01% (vol/vol). The strain also shows sensitivities to x-rays and ultra-violet light, which cosegregate with sensitivity to methyl methanesulfonate as a single gene defect. An analysis of the sensitivity to ultraviolet light indicates that the mutation interferes with the excision of pyrimidine dimers. Diploids homozygous for the mutant gene exhibit elevated frequencies of spontaneous mitotic recombination at the ade6 locus. The results indicate that all the events are due to gene conversion. Mitotic recombination was also found to be elevated for three loci other than ade6. Thus, the recombinational effect seems not to be locus specific. Linkage and allelism tests indicate that the mutation is an allele of the known radiation-sensitive gene rad18. The various effects of this new rad18 allele (rad18-3) are discussed in terms of a defect in DNA repair mechanisms.

Alleles↗
PubMed1997

Position- and orientation-independent activity of the Schizosaccharomyces pombe meiotic recombination hot spot M26.

The activity of the M26 meiotic recombination hot spot of Schizosaccharomyces pombe depends on the presence of the heptamer 5'-ATGACGT-3'. Transplacement of DNA fragments containing the ade6-M26 gene to other chromosomal loci has previously demonstrated that the heptamer functions in some, but not all, transplacements, suggesting that hot spot activity depends on chromosomal context. In this study, hot spot activity was tested in the absence of gross DNA changes by using site-directed mutagenesis to create the heptamer sequence at novel locations in the genome. When created by mutagenesis of 1-4 bp in the ade6 and ura4 genes, the heptamer was active as a recombination hot spot, in an orientation-independent manner, at all locations tested. Thus, the heptamer sequence can create an active hot spot in other chromosomal contexts, provided that the gross chromosomal structure is not altered; this result is consistent with the hypothesis that a specific higher-order chromatin structure is required for M26 hot spot activity.

DNA, Fungal↗
PubMed1997

Transcription factor Mts1/Mts2 (Atf1/Pcr1, Gad7/Pcr1) activates the M26 meiotic recombination hotspot in Schizosaccharomyces pombe.

Homologous recombination hotspots increase the frequency of recombination in nearby DNA. The M26 hotspot in the ade6 gene of Schizosaccharomyces pombe is a meiotic hotspot with a discrete, cis-acting nucleotide sequence (5'-ATGACGT-3') defined by extensive mutagenesis. A heterodimeric M26 DNA binding protein, composed of subunits Mts1 and Mts2, has been identified and purified 40,000-fold. Cloning, disruption, and genetic analyses of the mts genes demonstrate that the Mts1/Mts2 heterodimer is essential for hotspot activity. This provides direct evidence that a specific trans-acting factor, binding to a cis-acting site with a unique nucleotide sequence, is required to activate this meiotic hotspot. Intriguingly, the Mts1/Mts2 protein subunits are identical to the recently described transcription factors Atf1 (Gad7) and Pcr1, which are required for a variety of stress responses. However, we report differential dependence on the Mts proteins for hotspot activation and stress response, suggesting that these proteins are multifunctional and have distinct activities. Furthermore, ade6 mRNA levels are equivalent in hotspot and nonhotspot meioses and do not change in mts mutants, indicating that hotspot activation is not a consequence of elevated transcription levels. These findings suggest an intimate but separable link between the regulation of transcription and meiotic recombination. Other studies have recently shown that the Mts1/Mts2 protein and M26 sites are involved in meiotic recombination elsewhere in the S. pombe genome, suggesting that these factors help regulate the timing and distribution of homologous recombination.

Activating Transcription Factor 1↗
PubMed1991

Isolation and characterization of Schizosaccharomyces pombe mutants affected in mitotic recombination.

A haploid Schizosaccharomyces pombe strain carrying a heteroallelic duplication of the ade6 gene was used to isolate mitotic recombination-deficient mutants. Recombination between the different copies of the ade6 gene can lead to Ade+ segregants. These are observed as growing papillae when colonies of a suitable size are replicated onto selective medium. We isolated mutants which show an altered papillation phenotype. With two exceptions, they exhibit a decrease in the frequency of mitotic recombination between the heteroalleles of the duplication. The two other mutants display a hyper-recombination phenotype. The 12 mutations were allocated to at least nine distinct loci by recombination tests. Of the eight rec mutants analyzed further, six were also affected in mitotic intergenic recombination in the intervals cen2-mat or cen3-arg 1. No effect on mitotic intragenic recombination was observed. These data suggest that mitotic gene conversion and crossing over can be separated mutationally. Meiotic recombination occurs at the wild-type frequency in all mutants investigated.

Adenine↗
PubMed1993

Marker effects of G to C transversions on intragenic recombination and mismatch repair in Schizosaccharomyces pombe.

G to C transversion mutations show very strong allele-specific marker effects on the frequency of wild-type recombinants in intragenic two-factor crosses. Here we present a detailed study of the marker effect of one representative, the ade6-M387 mutation of Schizosaccharomyces pombe. Crosses of M387 with other mutations at varying distance reveal highly increased prototroph frequencies in comparison with the C to T transition mutation ade6-51 (control without any known marker effect) located four nucleotides from M387. The marker effect of M387 is strongest (> 40-fold) for crosses with mutations less than 15 nucleotides from M387. It decreases to an intermediate level (5-10-fold) in crosses with mutations located 25-150 base pairs from M387/51 and is very low in crosses with mutations beyond 200 base pairs. On the basis of these results and the quantitation of the low efficiency of C/C mismatch repair presented in the accompanying publication we propose the existence of at least two different types of mechanisms for base mismatch repair in fission yeast. The major system is suggested to recognize all base mismatches except C/C with high efficiency and to generate long excision tracts (approximately 100 nucleotides unidirectionally). The minor system is proposed to recognize all base mismatches including C/C with low and variable efficiency and to have short excision tracts (approximately 10 nucleotides unidirectionally). We estimate from the M387 marker effect that the minor system accounts for approximately 1-8% repair of non-C/C mismatches (depending on the nature of the mutation) in fission yeast meiosis.

Cytosine↗