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.     

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.     

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.     

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.     

Stimulation of Meiotic Recombination in Yeast by an Ars Element

In a previous study, meiotic recombination events were monitored in the 22-kb LEU2 to CEN3 region of chromosome III of Saccharomyces cerevisiae. One region (the hotspot) was shown to have an enhanced level of both gene conversion events and reciprocal crossovers, whereas a second region (the coldspot) was shown to have a depressed level of both types of recombination events. In this study we have analyzed the effects of a replication origin, ARS307, located about 2 kb centromere proximal to the hotspot region, on the distribution of meiotic recombination events. We find that a deletion of this origin results in a reduction of both gene conversions and reciprocal crossovers in the hotspot region, and that a 200-bp fragment of this ARS element can stimulate both types of recombination events when relocated to the coldspot region. Although the magnitude of stimulation of these events is similar in both orientations, whether the ARS is functional or not, the distribution of events is dependent upon the orientation of the element.     

Genetic Analysis of Variation in Human Meiotic Recombination

The number of recombination events per meiosis varies extensively among individuals. This recombination phenotype differs between female and male, and also among individuals of each gender. In this study, we used high-density SNP genotypes of over 2,300 individuals and their offspring in two datasets to characterize recombination landscape and to map the genetic variants that contribute to variation in recombination phenotypes. We found six genetic loci that are associated with recombination phenotypes. Two of these (RNF212 and an inversion on chromosome 17q21.31) were previously reported in the Icelandic population, and this is the first replication in any other population. Of the four newly identified loci (KIAA1462, PDZK1, UGCG, NUB1), results from expression studies provide support for their roles in meiosis. Each of the variants that we identified explains only a small fraction of the individual variation in recombination. Notably, we found different sequence variants associated with female and male recombination phenotypes, suggesting that they are regulated by different genes. Characterization of genetic variants that influence natural variation in meiotic recombination will lead to a better understanding of normal meiotic events as well as of non-disjunction, the primary cause of pregnancy loss.     

Analysis of Meiotic Recombination Pathways in the Yeast Saccharomyces Cerevisiae

In the yeast, Saccharomyces cerevisiae, several genes appear to act early in meiotic recombination. HOP1 and RED1 have been classified as such early genes. The data in this paper demonstrate that neither a red1 nor a hop1 mutation can rescue the inviable spores produced by a rad52 spo13 strain; this phenotype helps to distinguish these two genes from other early meiotic recombination genes such as SPO11, REC104, or MEI4. In contrast, either a red1 or a hop1 mutation can rescue a rad50S spo13 strain; this phenotype is similar to that conferred by mutations in the other early recombination genes (e.g., REC104). These two different results can be explained because the data presented here indicate that a rad50S mutation does not diminish meiotic intrachromosomal recombination, similar to the mutant phenotypes conferred by red1 or hop1. Of course, RED1 and HOP1 do act in the normal meiotic interchromosomal recombination pathway; they reduce interchromosomal recombination to ~10% of normal levels. We demonstrate that a mutation in a gene (REC104) required for initiation of exchange is completely epistatic to a mutation in RED1. Finally, mutations in either HOP1 or RED1 reduce the number of double-strand breaks observed at the HIS2 meiotic recombination hotspot.     

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.     

Meiotic Recombination and Sporulation in Repair-Deficient Strains of Yeast

A genetic system designed to monitor recombination and sporulation in various repair-deficient yeast strains was constructed. Variously heterozygous at seven or eight sites distributed across the genome, the system facilitated sensitive detection of changes in frequency or pattern of meiotic recombination. Ten rad mutants sensitive primarily to UV-irradiation and without terminal blocks in the sporulation process were studied. Seven were defective in excision repair (rad1, rad2, rad3, rad4, rad10, rad14 and rad16), and three were defective in mutagenic repair (rad5, rad9 and rad18). Individually, each mutant displayed behavior consistent with an orthodox meiosis including a wild-type meiotic recombination profile with respect to gene conversion, PMS and intergenic map distances. Accordingly, we conclude that these mutants are without major effect on meiotic heteroduplex formation or correction. However, certain combinations of excision-defective mutants with rad18 exhibited marked ascosporal inviability. Tetraploids homozygous for rad1 and rad18 produce a large proportion of diploid spores containing a recessive lethal.     

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.     

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.     

Genetic Analyses of Meiotic Recombination in Arabidopsis

Meiosis is essential for sexual reproduction and recombination is a critical step required for normal meiosis. Understanding the underlying molecular mechanisms that regulate recombination is important for medical, agricultural and ecological reasons. Readily available molecular and cytological tools make Arabidopsis an excellent system to study meiosis. Here we review recent developments in molecular genetic analyses on meiotic recombination. These include studies on plant homologs of yeast and animal genes, as well as novel genes that were first identified in plants. The characterizations of these genes have demonstrated essential functions from the initiation of recombination by double-strand breaks to repair of such breaks, from the formation of doubie-HoUiday junctions to possible resolution of these junctions, both of which are critical for crossover formation. The recent advances have ushered a new era in plant meiosis, in which the combination of genetics, genomics, and molecular cytology can uncover important gene functions.     

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.     

Identification of Functionally Related Genes That Stimulate Early Meiotic Gene Expression in Yeast

Meiosis and spore formation in the yeast Saccharomyces cerevisiae are associated with increased expression of sporulation-specific genes. One of these genes, IME2, encodes a putative protein kinase that is a positive regulator of other sporulation-specific genes. We have isolated mutations that cause reduced expression of an ime2-lacZ fusion gene. We found mutations in IME1, a known positive regulator of IME2, and MCK1, a known positive regulator of IME1. We also isolated recessive mutations in 12 other genes, which we designate RIM (Regulator of IME2) genes. Our analysis indicates that the defects in rim1, rim8, rim9 and rim13 mutants are a consequence of diminished IME1 expression and can be suppressed by expression of IME1 from the heterologous ACT1 promoter. These rim mutations also reduced expression of an ime1-HIS3 fusion, in which the HIS3 gene is expressed from the IME1 promoter, and caused reduced levels of IME1 RNA. Although the rim1, rim8, rim9 and rim13 mutant phenotypes are similar to those of mck1 mutants, we found that the defects in ime2-lacZ expression and sporulation of the mck1 rim double mutants were more severe than either single mutant. In contrast, the defects of the rim rim double mutants were similar to either single mutant. The rim1, rim8, rim9 and rim13 mutants also display slow growth at 17° and share a smooth colony morphology that is not evident in mck1 mutants or isogenic wild-type strains. We suggest that RIM1, RIM8, RIM9 and RIM13 encode functionally related products that act in parallel to MCK1 to stimulate IME1 expression.     

Meiotic association between Spo11 regulated by Rec102, Rec104 and Rec114

Meiotic recombination is initiated by DNA double-stranded break (DSB) formation catalyzed by Spo11, a type-II topoisomerase-like transesterificase, presumably via a dimerization-mediated mechanism. We demonstrate the existence of in vivo interactions between Spo11 proteins carrying distinct tags, and the chromatin-binding and DSB activity of tagged Spo11at innate and targeted DSB sites upon fusion to the Gal4 DNA-binding domain. First we identified the interaction between Spo11-3FLAG and Gal4BD-Spo11 proteins, and established that this interaction specifically occurs at the time of DSB formation. We then observed that presence of the Gal4BD-spo11Y135F (nuclease-deficient) protein allows Spo11-3FLAG recruitment at the GAL2 locus, indicative of the formation of a hetero-complex near the GAL2 UAS sites, but no formation of double- or single-strand breaks. Spo11 self-interaction around the GAL2 DSB site depends on other proteins for DSB formation, in particular Rec102, Rec104 and Rec114. Together, these results suggest that in vivo self-association of Spo11 during meiosis is genetically regulated. The results are discussed in relation to possible roles of Spo11 self-interaction in the control of the cleavage activity.     

Selection for Early Meiotic Mutants in Yeast

In the yeast Saccharomyces cerevisiae, only a/alpha cells can enter meiosis; a and alpha cells cannot. Because a/alpha cells are typically diploid and a and alpha cells are typically haploid, this cell type restriction ensures that only diploid cells enter meiosis. Entry into meiosis is accompanied by an increase in expression of the IME1 gene; the IME1 product (IME1) then activates IME2 and other meiotic genes. We have found that IME1 expression is toxic to starved haploid cells, presumably because IME1 directs them into meiosis. IME1 toxicity is greater in rad52 mutants, in which meiotic recombination causes lethal damage. Suppressors of IME1 toxicity include recessive mutations in two genes, RIM11 and RIM16 (Regulator of Inducer of Meiosis), that are required for IME1 to activate IME2 expression. RIM11 maps near CIN4 on chromosome XIII.     

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.