The Role of the SPO11 Gene in Meiotic Recombination in Yeast

Several complementary experimental approaches were used to demonstrate that the SPO11 gene is specifically required for meiotic recombination. First, sporulating cultures of spo11-1 mutant diploids were examined for landmark biochemical, cytological and genetic events of meiosis and ascosporogenesis. Cells entered sporulation with high efficiency and showed a near-doubling of DNA content. Synaptonemal complexes, hallmarks of intimate homologous pairing, and polycomplex structures appeared during meiotic prophase. Although spontaneous mitotic intra- and intergenic recombination occurred at normal levels, no meiotic recombination was observed. Whereas greater than 50% of cells completed both meiotic divisions, packaging of the four meiotic products into mature ascospores took place in only a small subset of asci. Haploidization occurred in less than 1% of viable colony-forming units. Second, the Rec- meiotic defect conferred by spo11-1 was confirmed by dyad analysis of spores derived from spo13-1 single-division meiosis in which recombination is not a requirement for viable ascospore production. Diploids homozygous for the spo13-1 mutation undergo meiotic levels of exchange followed by a single predominantly equational division and form asci containing two near-diploid spores. With the introduction of the spo11-1 mutation, high spore viability was retained, whereas intergenic recombination was reduced by more than 100-fold.     

Chromosomal Translocations Generated by High-Frequency Meiotic Recombination between Repeated Yeast Genes

We have examined meiotic and mitotic recombination between repeated genes on nonhomologous chromosomes in the yeast Saccharomyces cerevisiae. The results of these experiments can be summarized in three statements. First, gene conversion events between repeats on nonhomologous chromosomes occur frequently in meiosis. The frequency of such conversion events is only 17-fold less than the analogous frequency of conversion between genes at allelic positions on homologous chromosomes. Second, meiotic and mitotic conversion events between repeated genes on nonhomologous chromosomes are associated with reciprocal recombination to the same extent as conversion between allelic sequences. The reciprocal exchanges between the repeated genes result in chromosomal translocations. Finally, recombination between repeated genes on nonhomologous chromosomes occurs much more frequently in meiosis than in mitosis.     

Meiotic Recombination on Artificial Chromosomes in Yeast

We have examined the meiotic recombination characteristics of artificial chromosomes in Saccharomyces cerevisiae. Our experiments were carried out using minichromosome derivatives of yeast chromosome III and yeast artificial chromosomes composed primarily of bacteriophage lambda DNA. Tetrad analysis revealed that the artificial chromosomes exhibit very low levels of meiotic recombination. However, when a 12.5-kbp fragment from yeast chromosome VIII was inserted into the right arm of the artificial chromosome, recombination within that arm mimicked the recombination characteristics of the fragment in its natural context including the ability of crossovers to ensure meiotic disjunction. Both crossing over and gene conversion (within the ARG4 gene contained within the fragment) were measured in the experiments. Similarly, a 55-kbp region from chromosome III carried on a minichromosome showed crossover behavior indistinguishable from that seen when it is carried on chromosome III. We discuss the notion that, in yeast, meiotic recombination behavior is determined locally by small chromosomal regions that function free of the influence of the chromosome as a whole.     

Indistinguishable Landscapes of Meiotic DNA Breaks in rad50+ and rad50S Strains of Fission Yeast Revealed by a Novel rad50+ Recombination Intermediate

The fission yeast Schizosaccharomyces pombe Rec12 protein, the homolog of Spo11 in other organisms, initiates meiotic recombination by creating DNA double-strand breaks (DSBs) and becoming covalently linked to the DNA ends of the break. This protein–DNA linkage has previously been detected only in mutants such as rad50S in which break repair is impeded and DSBs accumulate. In the budding yeast Saccharomyces cerevisiae, the DSB distribution in a rad50S mutant is markedly different from that in wild-type (RAD50) meiosis, and it was suggested that this might also be true for other organisms. Here, we show that we can detect Rec12-DNA linkages in Sc. pombe rad50⁺ cells, which are proficient for DSB repair. In contrast to the results from Sa. cerevisiae, genome-wide microarray analysis of Rec12-DNA reveals indistinguishable meiotic DSB distributions in rad50⁺ and rad50S strains of Sc. pombe. These results confirm our earlier findings describing the occurrence of widely spaced DSBs primarily in large intergenic regions of DNA and demonstrate the relevance and usefulness of fission yeast studies employing rad50S. We propose that the differential behavior of rad50S strains reflects a major difference in DSB regulation between the two species—specifically, the requirement for the Rad50-containing complex for DSB formation in budding yeast but not in fission yeast. Use of rad50S and related mutations may be a useful method for DSB analysis in other species.     

The Role of DNA Repair Genes in Recombination between Repeated Sequences in Yeast

The presence of repeated sequences in the genome represents a potential source of karyotypic instability. Genetic control of recombination is thus important to preserve the integrity of the genome. To investigate the genetic control of recombination between repeated sequences, we have created a series of isogenic strains in which we could assess the role of genes involved in DNA repair in two types of recombination: direct repeat recombination and ectopic gene conversion. Naturally occurring (Ty elements) and artificially constructed repeats could be compared in the same cell population. We have found that direct repeat recombination and gene conversion have different genetic requirements. The role of the RAD51, RAD52, RAD54, RAD55, and RAD57 genes, which are involved in recombinational repair, was investigated. Based on the phenotypes of single and double mutants, these genes can be divided into three functional subgroups: one composed of RAD52, a second one composed of RAD51 and RAD54, and a third one that includes the RAD55 and RAD57 genes. Among seven genes involved in excision repair tested, only RAD1 and RAD10 played a role in the types of recombination studied. We did not detect a differential effect of any rad mutation on Ty elements as compared to artificially constructed repeats.     

Recombination Among Three Mitochondrial Genes in Yeast (Saccharomyces cerevisiae)

Eight crosses involving three different mitochondrial genes were made in Saccharomyces cerevisiae. All three genes were linked, but an unambiguous linkage map could not be constructed.     

Temperature-Sensitive Yeast Mutants Defective in Meiotic Recombination and Replication

A system is described for isolating temperature-sensitive mutants of Saccharomyces cerevisiae with defects in early meiotic events. We used an otherwise haploid strain disomic (n+1) for chromosome III, and heteroallelic at the leucine-2 locus. Meiotic development was initiated by exposure of the strain to acetate sporulation medium, and monitored by the appearance of leucine-independent intragenic recombinants. Mutant isolation was based on the recovery of thermally induced defects in recombination. The temperature-sensitive characteristic was included to allow eventual characterizations of the temporal period during meiosis when each gene performs its essential function. Following mutagenesis with either ethyl methane sulfonate or nitrosoguanidine individual clones were tested at 34° and 24° for acetate-induced recombination. Starting with 2700 clones, derived from cells that survived mutagenic treatment, we isolated 48 strains with thermally induced lesions in recombination. In the majority of mutants premeiotic replication occurred normally, or nearly normally, at the restrictive temperature, indicating that the meiotic cycle was initiated and that there was a defect in an event required for intragenic recombination. We also detected mutants where the thermally induced lesion in recombination resulted from temperature-sensitive premeiotic DNA synthesis.     

Yeast Mer1 Mutants Display Reduced Levels of Meiotic Recombination

Mutations at the MER1 locus were identified in a search for meiotic mutants defective in chromosome segregation. mer1 strains show decreased levels of inter- and intrachromosomal meiotic recombination and produce inviable spores. The MER1 gene was cloned by complementation of the spore inviability phenotype. Strains carrying disruptions of the MER1 gene are mitotically viable. The epistatic relationships between MER1 and previously characterized meiotic genes are described.     

Evolution of Mutational Robustness in the Yeast Genome: A Link to Essential Genes and Meiotic Recombination Hotspots

Deleterious mutations inevitably emerge in any evolutionary process and are speculated to decisively influence the structure of the genome. Meiosis, which is thought to play a major role in handling mutations on the population level, recombines chromosomes via non-randomly distributed hot spots for meiotic recombination. In many genomes, various types of genetic elements are distributed in patterns that are currently not well understood. In particular, important (essential) genes are arranged in clusters, which often cannot be explained by a functional relationship of the involved genes. Here we show by computer simulation that essential gene (EG) clustering provides a fitness benefit in handling deleterious mutations in sexual populations with variable levels of inbreeding and outbreeding. We find that recessive lethal mutations enforce a selective pressure towards clustered genome architectures. Our simulations correctly predict (i) the evolution of non-random distributions of meiotic crossovers, (ii) the genome-wide anti-correlation of meiotic crossovers and EG clustering, (iii) the evolution of EG enrichment in pericentromeric regions and (iv) the associated absence of meiotic crossovers (cold centromeres). Our results furthermore predict optimal crossover rates for yeast chromosomes, which match the experimentally determined rates. Using a Saccharomyces cerevisiae conditional mutator strain, we show that haploid lethal phenotypes result predominantly from mutation of single loci and generally do not impair mating, which leads to an accumulation of mutational load following meiosis and mating. We hypothesize that purging of deleterious mutations in essential genes constitutes an important factor driving meiotic crossover. Therefore, the increased robustness of populations to deleterious mutations, which arises from clustered genome architectures, may provide a significant selective force shaping crossover distribution. Our analysis reveals a new aspect of the evolution of genome architectures that complements insights about molecular constraints, such as the interference of pericentromeric crossovers with chromosome segregation.     

Extensive Recombination of a Yeast Diploid Hybrid through Meiotic Reversion

In somatic cells, recombination between the homologous chromosomes followed by equational segregation leads to loss of heterozygosity events (LOH), allowing the expression of recessive alleles and the production of novel allele combinations that are potentially beneficial upon Darwinian selection. However, inter-homolog recombination in somatic cells is rare, thus reducing potential genetic variation. Here, we explored the property of S. cerevisiae to enter the meiotic developmental program, induce meiotic Spo11-dependent double-strand breaks genome-wide and return to mitotic growth, a process known as Return To Growth (RTG). Whole genome sequencing of 36 RTG strains derived from the hybrid S288c/SK1 diploid strain demonstrates that the RTGs are bona fide diploids with mosaic recombined genome, derived from either parental origin. Individual RTG genome-wide genotypes are comprised of 5 to 87 homozygous regions due to the loss of heterozygous (LOH) events of various lengths, varying between a few nucleotides up to several hundred kilobases. Furthermore, we show that reiteration of the RTG process shows incremental increases of homozygosity. Phenotype/genotype analysis of the RTG strains for the auxotrophic and arsenate resistance traits validates the potential of this procedure of genome diversification to rapidly map complex traits loci (QTLs) in diploid strains without undergoing sexual reproduction.     

Me14, a Yeast Gene Required for Meiotic Recombination

Mutants at the MEI4 locus were detected in a search for mutants defective in meiotic gene conversion. mei4 mutants exhibit decreased sporulation and produce inviable spores. The spore inviability phenotype is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The MEI4 gene has been cloned from a yeast genomic library by complementation of the recombination defect and has been mapped to chromosome V near gln3. Strains carrying a deletion/insertion mutation of the MEI4 gene display no meiotically induced gene conversion but normal mitotic conversion frequencies. Both meiotic interchromosomal and intrachromosomal crossing over are completely abolished in mei4 strains. The mei4 mutation is able to rescue the spore-inviability phenotype of spo13 and 52 strains (i.e., mei4 spo13 rad52 mutants produce viable spores), indicating that MEI4 acts before RAD52 in the meiotic recombination pathway.     

Competition between Adjacent Meiotic Recombination Hotspots in the Yeast Saccharomyces Cerevisiae

In a wild-type strain of Saccharomyces cerevisiae, a hotspot for meiotic recombination is located upstream of the HIS4 gene. An insertion of a 49-bp telomeric sequence into the coding region of HIS4 strongly stimulates meiotic recombination and the local formation of meiosis-specific double-strand DNA breaks (DSBs). When strains are constructed in which both hotspots are heterozygous, hotspot activity is substantially less when the hotspots are on the same chromosome than when they are on opposite chromosomes.     

The Rec102 Mutant of Yeast Is Defective in Meiotic Recombination and Chromosome Synapsis

A mutation at the REC102 locus was identified in a screen for yeast mutants that produce inviable spores. rec102 spore lethality is rescued by a spo13 mutation, which causes cells to bypass the meiosis I division. The rec102 mutation completely eliminates meiotically induced gene conversion and crossing over but has no effect on mitotic recombination frequencies. Cytological studies indicate that the rec102 mutant makes axial elements (precursors to the synaptonemal complex), but homologous chromosomes fail to synapse. In addition, meiotic chromosome segregation is significantly delayed in rec102 strains. Studies of double and triple mutants indicate that the REC102 protein acts before the RAD52 gene product in the meiotic recombination pathway. The REC102 gene was cloned based on complementation of the mutant defect and the gene was mapped to chromosome XII between CDC25 and STE11.     

Genetic Evidence for Preferential Strand Transfer during Meiotic Recombination in Yeast

During meiotic recombination in the yeast Saccharomyces cerevisiae, heteroduplexes are formed as an intermediate in the exchange process. In the formation of an asymmetric heteroduplex, one chromosome acts as a donor of a single DNA strand and the other acts as a recipient. We present genetic evidence that the nontranscribed strand is donated more frequently than the transcribed strand in spores that have an unrepaired mismatch at the HIS4 locus.     

Effects of Glucose Repression on the Transmission and Recombination of Mitochondrial Genes in Yeast (SACCHAROMYCES CEREVISIAE)

Matings of a number of Saccharomyces cerevisiae stocks give different output ratios of mitochondrial genotypes depending on whether the cells are glucose-repressed or derepressed. The effects of glucose repression are independent of cellular mating type and mitochondrial genotype, and take place at least in part after zygotes are formed. An explanation is proposed in terms of changes in the relative numbers of mitochondrial DNA molecules contributed by the a and α parents, modified by selective replication or destruction of molecules inside the zygote.     

The Yeast Mer2 Gene Is Required for Chromosome Synapsis and the Initiation of Meiotic Recombination

Mutation of the MER2 gene of Saccharomyces cerevisiae confers meiotic lethality. To gain insight into the function of the Mer2 protein, we have carried out a detailed characterization of the mer2 null mutant. Genetic analysis indicates that mer2 completely eliminates meiotic interchromosomal gene conversion and crossing over. In addition, mer2 abolishes intrachromosomal meiotic recombination, both in the ribosomal DNA array and in an artificial duplication. The results of a physical assay demonstrate that the mer2 mutation prevents the formation of meiosis-specific, double-strand breaks, indicating that the Mer2 protein acts at or before the initiation of meiotic recombination. Electron microscopic analysis reveals that the mer2 mutant makes axial elements, which are precursors to the synaptonemal complex, but homologous chromosomes fail to synapse. Fluorescence in situ hybridization of chromosome-specific DNA probes to spread meiotic chromosomes demonstrates that homolog alignment is also significantly reduced in the mer2 mutant. Although the MER2 gene is transcribed during vegetative growth, deletion or overexpression of the MER2 gene has no apparent effect on mitotic recombination or DNA damage repair. We suggest that the primary defect in the mer2 mutant is in the initiation of meiotic genetic exchange.     

Isolation of Mutants Defective in Early Steps of Meiotic Recombination in the Yeast Saccharomyces Cerevisiae

Using a selection based upon the ability of early Rec- mutations (e.g., rad50) to rescue the meiotic lethality of a rad52 spo13 strain, we have isolated 177 mutants. Analysis of 56 of these has generated alleles of the known Rec genes SPO11, ME14 and MER1, as well as defining five new genes: REC102, REC104, REC107, REC113 and REC114. Mutations in all of the new genes appear to specifically affect meiosis; they do not have any detectable mitotic phenotype. Mutations in REC102, REC104 and REC107 reduce meiotic recombination several hundred fold. No alleles of RED1 or HOP1 were isolated, consistent with the proposal that these genes may be primarily involved with chromosome pairing and not exchange.     

Meiotic Recombination in Human Oocytes

Studies of human trisomies indicate a remarkable relationship between abnormal meiotic recombination and subsequent nondisjunction at maternal meiosis I or II. Specifically, failure to recombine or recombination events located either too near to or too far from the centromere have been linked to the origin of human trisomies. It should be possible to identify these abnormal crossover configurations by using immunofluorescence methodology to directly examine the meiotic recombination process in the human female. Accordingly, we initiated studies of crossover-associated proteins (e.g., MLH1) in human fetal oocytes to analyze their number and distribution on nondisjunction-prone human chromosomes and, more generally, to characterize genome-wide levels of recombination in the human female. Our analyses indicate that the number of MLH1 foci is lower than predicted from genetic linkage analysis, but its localization pattern conforms to that expected for a crossover-associated protein. In studies of individual chromosomes, our observations provide evidence for the presence of “vulnerable” crossover configurations in the fetal oocyte, consistent with the idea that these are subsequently translated into nondisjunctional events in the adult oocyte.