Mitotic Recombination In Yeast Research Articles

DNA strand-exchange patterns associated with double-strand break-induced and spontaneous mitotic crossovers in Saccharomyces cerevisiae.

Mitotic recombination can result in loss of heterozygosity and chromosomal rearrangements that shape genome structure and initiate human disease. Engineered double-strand breaks (DSBs) are a potent initiator of recombination, but whether spontaneous events initiate with the breakage of one or both DNA strands remains unclear. In the current study, a crossover (CO)-specific assay was used to compare heteroduplex DNA (hetDNA) profiles, which reflect strand exchange intermediates, associated with DSB-induced versus spontaneous events in yeast. Most DSB-induced CO products had the two-sided hetDNA predicted by the canonical DSB repair model, with a switch in hetDNA position from one product to the other at the position of the break. Approximately 40% of COs, however, had hetDNA on only one side of the initiating break. This anomaly can be explained by a modified model in which there is frequent processing of an early invasion (D-loop) intermediate prior to extension of the invading end. Finally, hetDNA tracts exhibited complexities consistent with frequent expansion of the DSB into a gap, migration of strand-exchange junctions, and template switching during gap-filling reactions. hetDNA patterns in spontaneous COs isolated in either a wild-type background or in a background with elevated levels of reactive oxygen species (tsa1Δ mutant) were similar to those associated with the DSB-induced events, suggesting that DSBs are the major instigator of spontaneous mitotic recombination in yeast.

Mitotic recombination in yeast: elements controlling its incidence.

Mitotic recombination is an important mechanism of DNA repair in eukaryotic cells. Given the redundancy of the eukaryotic genomes and the presence of repeated DNA sequences, recombination may also be an important source of genomic instability. Here we review the data, mainly from the budding yeast S. cerevisiae, that may help to understand the spontaneous origin of mitotic recombination and the different elements that may control its occurrence. We cover those observations suggesting a putative role of replication defects and DNA damage, including double-strand breaks, as sources of mitotic homologous recombination. An important part of the review is devoted to the experimental evidence suggesting that transcription and chromatin structure are important factors modulating the incidence of mitotic recombination. This is of great relevance in order to identify the causes and risk factors of genomic instability in eukaryotes.

Mismatch repair proteins regulate heteroduplex formation during mitotic recombination in yeast.

Mismatch repair (MMR) proteins actively inhibit recombination between diverged sequences in both prokaryotes and eukaryotes. Although the molecular basis of the antirecombination activity exerted by MMR proteins is unclear, it presumably involves the recognition of mismatches present in heteroduplex recombination intermediates. This recognition could be exerted during the initial stage of strand exchange, during the extension of heteroduplex DNA, or during the resolution of recombination intermediates. We previously used an assay system based on 350-bp inverted-repeat substrates to demonstrate that MMR proteins strongly inhibit mitotic recombination between diverged sequences in Saccharomyces cerevisiae. The assay system detects only those events that reverse the orientation of the region between the recombination substrates, which can occur as a result of either intrachromatid crossover or sister chromatid conversion. In the present study we sequenced the products of mitotic recombination between 94%-identical substrates in order to map gene conversion tracts in wild-type versus MMR-defective yeast strains. The sequence data indicate that (i) most recombination occurs via sister chromatid conversion and (ii) gene conversion tracts in an MMR-defective strain are significantly longer than those in an isogenic wild-type strain. The shortening of conversion tracts observed in a wild-type strain relative to an MMR-defective strain suggests that at least part of the antirecombination activity of MMR proteins derives from the blockage of heteroduplex extension in the presence of mismatches.

Modulation of bleomycin-induced mitotic recombination in yeast by the aminothiols cysteamine and WR-1065.

The cancer chemotherapy drug bleomycin (BLM) is a potent inducer of genetic damage in a wide variety of assays. The radioprotectors cysteamine (CSM) and WR-1065 have been shown in previous studies to potentiate the induction of micronuclei and chromosome aberrations by BLM in Go human lymphocytes. By contrast, WR-1065 is reported to reduce the induction of hprt mutations by BLM in Chinese hamster cells. To elucidate the basis for these interactions, we examined the effects of CSM and WR-1065 on the induction of mitotic gene conversion by BLM in the yeast Saccharomyces cerevisiae. Treatment with BLM causes a dose-dependent increase in the frequency of mitotic gene conversion and gene mutations. Unlike its potentiation of BLM in Go lymphocytes, WR-1065 protected against the recombinagenicity of BLM in yeast. CSM was also strongly-antirecombinagenic under, some conditions, but the nature of the interaction depended strongly on the treatment conditions. Under hypoxic conditions, cysteamine protected against BLM, but under oxygen-rich conditions CSM potentiated the genetic activity of BLM. The protective effect of aminothiols against BLM may be ascribed to the depletion of oxygen required for the activation of BLM and the processing of BLM-induced damage. Aminothiols may potentiate the effect of BLM by acting as an electron source for the activation of BLM and/or by causing conformational alterations that make DNA more accessible to BLM. The results indicate that aminothiols have a strong modulating influence on the genotoxicity of BLM in yeast as they do in other genetic assays. Moreover, the modulation differs markedly depending on physiological conditions. Thus, yeast assays help to explain why aminothiols have been observed to potentiate BLM in some genetic systems and to protect against it in others.

Homologous recombination in plant cells is enhanced by in vivo induction of double strand breaks into DNA by a site-specific endonuclease.

Induction of double strand breaks (DSBs) is coupled to meiotic and mitotic recombination in yeast. We show that also in a higher eukaryote induction of DSBs is directly correlated with a strong enhancement of recombination frequencies. We cotransfected Nicotiana plumbaginifolia protoplasts with a plasmid carrying a synthetic I-SceI gene, coding for a highly sequence specific endonuclease, together with recombination substrates carrying an I-SceI-site adjacent to their homologous sequences. We measured efficiencies of extrachromosomal recombination, using a well established transient beta-glucuronidase (GUS) assay. GUS enzyme activities were strongly increased when a plasmid carrying the I-SceI gene in sense but not in antisense orientation with respect to the promoter was included in the transfections. The in vivo induced DSBs were detected in the recombination substrates by Southern blotting, demonstrating that the yeast enzyme is functional in plant cells. At high ratios of transfected I-SceI-genes to I-SceI-sites the majority of the I-SceI-sites in the recombination substrates are cleaved, indicating that the induction of the DSBs is the rate limiting step in the described recombination reaction. These results imply that in vivo induction of transient breaks at specific sites in the plant genome could allow foreign DNA to be targeted to these sites via homologous recombination.

Somatic and germinal recombination of a direct repeat in Arabidopsis.

Homologous recombination between a pair of directly repeated transgenes was studied in Arabidopsis. The test construct included two different internal, non-overlapping deletion alleles of npt (neomycin phosphotransferase) flanking an active HPT (hygromycin phosphotransferase) gene. This construct was introduced into Arabidopsis by agrobacterium-mediated transformation with selection for resistance to hygromycin, and two independent single-insert lines were analyzed. Selection for active NPT by resistance to kanamycin gave both fully and partly (chimeric) recombinant seedlings. Rates for one transgenic line were estimated at less than 2 x 10(-5) events per division for germinal and greater than 10(-6) events per division for somatic recombination, a much smaller difference than between meiotic and mitotic recombination in yeast. Southern analysis showed that recombinants could be formed by either crossing over or gene conversion. A surprisingly high fraction (at least 2/17) of the recombinants, however, appeared to result from the concerted action of two or more independent simple events. Some evolutionary implications are discussed.

A novel recombinator in yeast based on gene II protein from bacteriophage f1.

Interchromosomal mitotic recombination in yeast can be stimulated by the protein encoded by gene II of bacteriophage f1. The normal role of the gene II enzyme is to make a site-specific cleavage of a particular strand of the duplex form of the bacteriophage DNA at the origin of DNA replication. The gene II protein was expressed in yeast in an attempt to determine the role of nicked DNA in the initiation of recombination. Stimulation of recombination in yeast by the gene II protein was dependent on the presence of a recognition site for gene II enzyme in the region being assayed. Recombination was stimulated in both directions from the gene II recognition site but showed a directional bias. The distribution of alleles among the recombinants indicated that the chromosome with the gene II recognition site acted as the recipient in gene conversion events.

The RAD50 gene, a member of the double strand break repair epistasis group, is not required for spontaneous mitotic recombination in yeast

Mutations in the RAD50 gene of Saccharomyces cerevisiae have been shown to reduce double strand break repair, meiotic recombination, and radiation-inducible mitotic recombination. Several different point mutations (including ochre and amber alleles) have been previously examined for effects on spontaneous mitotic recombination and did not reduce the frequency of recombination. Instead, the rad50 mutations conferred a moderate hyper-rec phenotype. This paper examines a deletion/interruption allele of RAD50 that removes 998 of 1312 amino acids and adds 1.1 kb of foreign DNA. The results clearly indicate that spontaneous mitotic recombination can occur in the absence of RAD50; in fact, the frequency of recombination is elevated over the wild-type cell. One possible interpretation of these observations is that the initiating lesion in spontaneous recombination events in mitosis might not be a double strand break.

Spontaneous mitotic recombination in yeast: the hyper-recombinational rem1 mutations are alleles of the RAD3 gene.

The RAD3 gene of Saccharomyces cerevisiae is required for UV excision-repair and is essential for cell viability. We have identified the rem1 mutations (enhanced spontaneous mitotic recombination and mutation) of Saccharomyces cerevisiae as alleles of RAD3 by genetic mapping, complementation with the cloned wild-type gene, and DNA hybridization. The high levels of spontaneous mitotic gene conversion, crossing over, and mutation conferred upon cells by the rem1 mutations are distinct from the effects of all other alleles of RAD3. We present preliminary data on the localization of the rem1 mutations within the RAD3 gene. The interaction of the rem1 mutant alleles with a number of radiation-sensitive mutations is also different than the interactions reported for previously described (UV-sensitive) alleles of RAD3. Double mutants of rem1 and a defect in the recombination-repair pathway are inviable, while double mutants containing UV-sensitive alleles of RAD3 are viable. The data presented here demonstrate that: (1) rem1 strains containing additional mutations in other excision-repair genes do not exhibit elevated gene conversion; (2) triple mutants containing rem1 and mutations in both excision-repair and recombination-repair are viable; (3) such triple mutants containing rad52 have reduced levels of gene conversion but wild-type frequencies of crossing over. We have interpreted these observations in a model to explain the effects of rem1. Consistent with the predictions of the model, we find that the size of DNA from rem1 strains, as measured by neutral sucrose gradients, is smaller than wild type.

The mutagenicity and carcinogenicity of hair dyes

Synopsis A number of the constituents of semi-permanent and permanent hair dyes have been shown to be mutagenic in bacteria, to induce mutation, chromosome breakage, sister chromatid exchanges and malignant transformation in mammalian cells, to cause mutation in Drosophila, to induce mitotic recombination in yeast and to induce tumours in rodents. Some hair dye ingredients and commercial dyes have been shown to give rise, in the urine of treated rats, to metabolites which are also mutagenic in bacteria. The test systems used are briefly described and the data generated are reviewed. In addition, attempts to identify whether the hazards predicted from the laboratory studies present a risk to man either by epidemiological studies or monitoring of exposed populations are also discussed.

Induction of mitotic recombination in yeast by starvation for thymine nucleotides.

The biosynthesis of thymine nucleotides in Saccharomyces cerevisiae can be inhibited either by genetic lesions in the structural gene for thymidylate synthetase (TMP1) or by drugs that prevent the methylation of dUMP to dTMP. This methylation can be blocked by folate antagonists. We find that 5-fluoro-dUMP (FdUMP) is also an effective inhibitor in vivo. Inhibition of dTMP biosynthesis by these three different routes causes thymineless death. In addition to being cytotoxic, we find that FdUMP is highly recombinagenic in yeast but does not induce nuclear gene mutations. Provision of exogenous dTMP eliminates this induced mitotic recombination and cell killing. Similar results were obtained when a thymineless condition was provoked in cells by antifolate drugs or by dTMP deprivation in strains auxotrophic for this nucleotide. These findings show that, in contrast to the situation in prokaryotes, starvation for thymine nucleotides in yeast induces genetic recombination but is not mutagenic.

The RAD52 gene is required for homothallic interconversion of mating types and spontaneous mitotic recombination in yeast.

The rad52-1 mutation prevents homothallic mating type interconversion and reduces mitotic recombination in yeast. It has been previously reported that rad52-1 abolishes meiotic recombination. These data suggest either that a generalized recombination function(s) is required for mating type switching or that generalized recombination and specific homothallic functions are jointly controlled by the RAD52 gene. The rad52-1 mutation affects the interconversion of the two yeast mating types (a and alpha) differently, suggesting that the interconversion process is not equivalent for both mating types. This type of asymmetry is not predicted by current models of homothallic switching.

The influence of pH on the effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) on Saccharomyces cerevisiae and Salmonella typhimurium

The genetic effects of 2,4-D (2,4-dichlorophenoxyacetic acid, Na salt) have been investigated in cells of the yeast Saccharomyces cerevisiae and of the bacterium Salmonella typhimurium in experiments in vitro and in vivo. Experiments in vitro showed that the killing of both yeast and bacteria is dependent on the pH in the treatment solution of 2,4-D. A dose-dependent increase of the frequency of mitotic gene conversion and mitotic recombination in yeast was observed at pH 4.50 and 4.30. In experiments in vitro with two strains of Salmonella no significant increase of the number of revertants to prototrophy was obtained. The positive correlation between survival of cells and dissociation of 2,4-D in the pH region 2.8-5.0 indicates that the cells are unable to take up dissociated 2,4-D. Therefore the survival is high at a high pH when most 2,4-D is in dissociated form, and the survival is low at a relatively low pH when more of the 2,4-D is in its undissociated form. No genetic effects were induced by oral administration of tolerable doses of 2,4-D in host-mediated assays using mice as hosts and yeast or Salmonella as indicator cells.

Genetic activity of niridazole in yeast

Niridazole, one of several drugs presently known to be of value in the treatment of human schistosomiasis, was tested for its activity in inducing mitotic recombination in yeast. It was found that niridazole is genetically active when the treatment of yeast cells is performed in a rich medium (YPG-medium) under growing conditions, but not when treatment is carried out in a non-nutrient suspension (phosphate buffer). The data suggest that niridazole might be converted to an active compound by yeast metabolism. The results of the experiments with niridazole in the non-nutrient medium were compared with those of AF-2 and SQ18, 506, two agents which have been shown to be genetically active in the present assay system.

Genetic activity of niridazole in yeast

Niridazole, one of several drugs presently known to be of value in the treatment of human schistosomiasis, was tested for its activity in inducing mitotic recombination in yeast. It was found that niridazole is genetically active when the treatment of yeast cells is performed in a rich medium (YPG-medium) under growing conditions, but not when treatment is carried out in a non-nutrient suspension (phosphate buffer). The data suggest that niridazole might be converted to an active compound by yeast metabolism. The results of the experiments with niridazole in the non-nutrient medium were compared with those of AF-2 and SQ18, 506, two agents which have been shown to be genetically active in the present assay system.

Genetic activity of niridazole in yeast

Niridazole, one of several drugs presently known to be of value in the treatment of human schistosomiasis, was tested for its activity in inducing mitotic recombination in yeast. It was found that niridazole is genetically active when the treatment of yeast cells is performed in a rich medium (YPG-medium) under growing conditions, but not when treatment is carried out in a non-nutrient suspension (phosphate buffer). The data suggest that niridazole might be converted to an active compound by yeast metabolism. The results of the experiments with niridazole in the non-nutrient medium were compared with those of AF-2 and SQ18, 506, two agents which have been shown to be genetically active in the present assay system.

Induction of intragenic mitotic recombination in yeast by hydroxylamine and its mutagenic specificity.

Hydroxylamine in 1 M concentration is capable of inducing mutations (Table 1) and recombination (Figs. 1–3) in yeast. It is postulated that HA-produced lesions in DNA are repaired and thus HA-induced mutations in yeast cannot be specific with respect to the type of base substitution; the recombinogenic ability of a mutagen can be considered as an indication that the lesions in DNA produced by this mutagen are repaired.

Comparison of the effects of ultraviolet light and ethylmethanesulphonate upon the frequency of mitotic recombination in yeast.

Diploid cultures of the yeast Saccharomyces cerevisiae were treated with UV light and ethylmethanesulphonate and scored for mitotic recombination and cell survival. Intergenic recombination was induced to the same frequency by both treatments at the same survival level whereas EMS induced more intragenic recombinants than UV treatment at the same survival.