Replication Origin Selection Regulates the Distribution of Meiotic Recombination

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Variation in Genome-Wide Levels of Meiotic Recombination Is Established at the Onset of Prophase in Mammalian Males

Segregation of chromosomes during the first meiotic division relies on crossovers established during prophase. Although crossovers are strictly regulated so that at least one occurs per chromosome, individual variation in crossover levels is not uncommon. In an analysis of different inbred strains of male mice, we identified among-strain variation in the number of foci for the crossover-associated protein MLH1. We report studies of strains with “low” (CAST/EiJ), “medium” (C3H/HeJ), and “high” (C57BL/6J) genome-wide MLH1 values to define factors responsible for this variation. We utilized immunofluorescence to analyze the number and distribution of proteins that function at different stages in the recombination pathway: RAD51 and DMC1, strand invasion proteins acting shortly after double-strand break (DSB) formation, MSH4, part of the complex stabilizing double Holliday junctions, and the Bloom helicase BLM, thought to have anti-crossover activity. For each protein, we identified strain-specific differences that mirrored the results for MLH1; i.e., CAST/EiJ mice had the lowest values, C3H/HeJ mice intermediate values, and C57BL/6J mice the highest values. This indicates that differences in the numbers of DSBs (as identified by RAD51 and DMC1) are translated into differences in the number of crossovers, suggesting that variation in crossover levels is established by the time of DSB formation. However, DSBs per se are unlikely to be the primary determinant, since allelic variation for the DSB-inducing locus Spo11 resulted in differences in the numbers of DSBs but not the number of MLH1 foci. Instead, chromatin conformation appears to be a more important contributor, since analysis of synaptonemal complex length and DNA loop size also identified consistent strain-specific differences; i.e., crossover frequency increased with synaptonemal complex length and was inversely related to chromatin loop size. This indicates a relationship between recombination and chromatin compaction that may develop as DSBs form or earlier during establishment of the meiotic axis.     

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.     

Initiation of Meiotic Recombination in Ustilago maydis

A central feature of meiosis is the pairing and recombination of homologous chromosomes. Ustilago maydis, a biotrophic fungus that parasitizes maize, has long been utilized as an experimental system for studying recombination, but it has not been clear when in the life cycle meiotic recombination initiates. U. maydis forms dormant diploid teliospores as the end product of the infection process. Upon germination, teliospores complete meiosis to produce four haploid basidiospores. Here we asked whether the meiotic process begins when teliospores germinate or at an earlier stage in development. When teliospores homozygous for a cdc45 mutation temperature sensitive for DNA synthesis were germinated at the restrictive temperature, four nuclei became visible. This implies that teliospores have already undergone premeiotic DNA synthesis and suggests that meiotic recombination initiates at a stage of infection before teliospores mature. Determination of homologous recombination in plant tissue infected with U. maydis strains heteroallelic for the nar1 gene revealed that Nar⁺ recombinants were produced at a stage before teliospore maturation. Teliospores obtained from a spo11Δ cross were still able to germinate but the process was highly disturbed and the meiotic products were imbalanced in chromosomal complement. These results show that in U. maydis, homologous recombination initiates during the infection process and that meiosis can proceed even in the absence of Spo11, but with loss of genomic integrity.     

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.     

Sister Chromatid Cohesion Remodeling and Meiotic Recombination

Proper control of cohesion along the chromosome arms is essential for segregation of homologous chromosomes in meiosis. In a recent study we reported that Tid1p, a protein previously implicated in recombination, is required for resolution of Mcd1p-dependent cohesion in meiosis. Here we demonstrate that Pds5p and Dmc1p promote this cohesion. Pds5p is known to be required for maintenance of cohesion while Dmc1p is recognized as essential for meiotic recombination. Finding that the same defect in separation of sister chromatids could be suppressed by disrupting the functions of these proteins supports the emerging recognition that cohesion is remodeled during recombination and further indicates that cohesion is modified specifically to regulate meiotic recombination. We also find that overexpression of the regulatory subunit of Cdc7p kinase, Dbf4p, suppresses the tid1Δ sporulation defect, suggesting a role for Cdc7p/Dbf4p in regulating cohesion.     

Meiotic Recombination: An Affair of Two Recombinases

In E. coli, homologous recombination is catalyzed by the RecA recombinase. Two RecA-like factors, Rad51 and Dmc1, are found in eukaryotes. Whereas Rad51 is needed for homologous recombination reactions in both mitotic and meiotic cells, the role of Dmc1 is restricted to meiosis. Recent work has shown that, like RecA and Rad51, Dmc1 mediates the homologous DNA pairing strand exchange reaction via a filamentous intermediate assembled on single-stranded DNA. Emerging evidence suggests that the tumor suppressor BRCA2 functions in the assembly of nucleoprotein filaments of Rad51 and Dmc1. The manner in which Rad51 and Dmc1 functionally cooperate in meiotic recombination remains to be determined.     

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.     

OsHUS1 Facilitates Accurate Meiotic Recombination in Rice

Meiotic recombination normally takes place between allelic sequences on homologs. This process can also occur between non-allelic homologous sequences. Such ectopic interaction events can lead to chromosome rearrangements and are normally avoided. However, much remains unknown about how these ectopic interaction events are sensed and eliminated. In this study, using a screen in rice, we characterized a homolog of HUS1 and explored its function in meiotic recombination. In Oshus1 mutants, in conjunction with nearly normal homologous pairing and synapsis, vigorous, aberrant ectopic interactions occurred between nonhomologous chromosomes, leading to multivalent formation and subsequent chromosome fragmentation. These ectopic interactions relied on programed meiotic double strand breaks and were formed in a manner independent of the OsMER3-mediated interference-sensitive crossover pathway. Although early homologous recombination events occurred normally, the number of interference-sensitive crossovers was reduced in the absence of OsHUS1. Together, our results indicate that OsHUS1 might be involved in regulating ectopic interactions during meiosis, probably by forming the canonical RAD9-RAD1-HUS1 (9-1-1) complex.     

Trans-Regulation of Mouse Meiotic Recombination Hotspots by Rcr1

Meiotic recombination is required for the orderly segregation of chromosomes during meiosis and for providing genetic diversity among offspring. Among mammals, as well as yeast and higher plants, recombination preferentially occurs at highly delimited chromosomal sites 1–2 kb long known as hotspots. Although considerable progress has been made in understanding the roles various proteins play in carrying out the molecular events of the recombination process, relatively little is understood about the factors controlling the location and relative activity of mammalian recombination hotspots. To search for trans-acting factors controlling the positioning of recombination events, we compared the locations of crossovers arising in an 8-Mb segment of a 100-Mb region of mouse Chromosome 1 (Chr 1) when the longer region was heterozygous C57BL/6J (B6) × CAST/EiJ (CAST) and the remainder of the genome was either similarly heterozygous or entirely homozygous B6. The lack of CAST alleles in the remainder of the genome resulted in profound changes in hotspot activity in both females and males. Recombination activity was lost at several hotspots; new, previously undetected hotspots appeared; and still other hotspots remained unaffected, indicating the presence of distant trans-acting gene(s) whose CAST allele(s) activate or suppress the activity of specific hotspots. Testing the activity of three activated hotspots in sperm samples from individual male progeny of two genetic crosses, we identified a single trans-acting regulator of hotspot activity, designated Rcr1, that is located in a 5.30-Mb interval (11.74–17.04 Mb) on Chr 17. Using an Escherichia coli cloning assay to characterize the molecular products of recombination at two of these hotspots, we found that Rcr1 controls the appearance of both crossover and noncrossover gene conversion events, indicating that it likely controls the sites of the double-strand DNA breaks that initiate the recombination process.     

Mismatch Repair-Induced Meiotic Recombination Requires the Pms1 Gene Product

The presence of multiple heterologies in a 9-kilobase (kb) interval results in a decrease in meiotic crossovers from 26.0% to 10.1%. There is also an increase from 3.5% to 11.1% in gene conversions and ectopic recombinations between the flanking homologous MAT loci. The hypothesis that mismatch repair of heteroduplex DNA containing several heterologies would lead to a second round of recombination has now been tested by examining the effect of a mutation that reduces mismatch correction. The repair-defective pms1-1 allele restores the pattern of recombination to nearly that seen in congenic diploids without the heterologies. Mismatch repair-induced recombination causes a significant increase in MAT conversions and ectopic recombination events with as few as two heterozygosities separated by 0.3-0.7 kb, but not when the mismatches are separated by greater than 1 kb. The frequency of these events depends on both the number and position of the heterozygosities relative to the flanking homologous MAT loci used to detect the events. The creation of recombinogenic lesions by mismatch repair in yeast could be analogous to the creation of recombinogenic lesions in dam- Escherichia coli. We suggest that the repair of heteroduplex DNA containing multiple mismatches may produce chromosomal rearrangements and gamete inviability when naturally polymorphic chromosomes undergo meiotic recombination.     

A Genome-Wide Map of Mitochondrial DNA Recombination in Yeast

In eukaryotic cells, the production of cellular energy requires close interplay between nuclear and mitochondrial genomes. The mitochondrial genome is essential in that it encodes several genes involved in oxidative phosphorylation. Each cell contains several mitochondrial genome copies and mitochondrial DNA recombination is a widespread process occurring in plants, fungi, protists, and invertebrates. Saccharomyces cerevisiae has proved to be an excellent model to dissect mitochondrial biology. Several studies have focused on DNA recombination in this organelle, yet mostly relied on reporter genes or artificial systems. However, no complete mitochondrial recombination map has been released for any eukaryote so far. In the present work, we sequenced pools of diploids originating from a cross between two different S. cerevisiae strains to detect recombination events. This strategy allowed us to generate the first genome-wide map of recombination for yeast mitochondrial DNA. We demonstrated that recombination events are enriched in specific hotspots preferentially localized in non-protein-coding regions. Additionally, comparison of the recombination profiles of two different crosses showed that the genetic background affects hotspot localization and recombination rates. Finally, to gain insights into the mechanisms involved in mitochondrial recombination, we assessed the impact of individual depletion of four genes previously associated with this process. Deletion of NTG1 and MGT1 did not substantially influence the recombination landscape, alluding to the potential presence of additional regulatory factors. Our findings also revealed the loss of large mitochondrial DNA regions in the absence of MHR1, suggesting a pivotal role for Mhr1 in mitochondrial genome maintenance during mating. This study provides a comprehensive overview of mitochondrial DNA recombination in yeast and thus paves the way for future mechanistic studies of mitochondrial recombination and genome maintenance.     

Meiotic Recombination between Duplicated Genetic Elements in SACCHAROMYCES CEREVISIAE

We have studied the meiotic recombination behavior of strains carrying two types of duplications of an 18.6-kilobase HIS4 Bam HI fragment. The first type is a direct duplication of the HIS4 Bam HI fragment in which the repeated sequences are separated by Escherichia coli plasmid sequences. The second type, a tandem duplication, has no sequences intervening between the repeated yeast DNA. The HIS4 genes in each region were marked genetically so that recombination events between the duplicated segments could be identified. Meiotic progeny of the strains carrying the duplication were analyzed genetically and biochemically to determine the types of recombination events that had occurred. Analysis of the direct vs. tandem duplication suggests that the E. coli plasmid sequences are recombinogenic in yeast when homozygous. In both types of duplications recombination between the duplicated HIS4 regions occurs at high frequency and involves predominantly interchromosomal reciprocal exchanges (equal and unequal crossovers). The striking observation is that intrachromosomal reciprocal recombination is very rare in comparison with interchromosomal reciprocal recombination. However, intrachromosomal gene conversion occurs at about the same frequency as interchromosomal gene conversion. Reciprocal recombination events between regions on the same chromatid are the most infrequent exchanges. These data suggest that intrachromosomal reciprocal exchanges are suppressed.     

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.     

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.     

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.