Rec25 and Rec27, novel linear-element components, link cohesin to meiotic DNA breakage and recombination

Meiosis is a specialized nuclear division by which sexually reproducing diploid organisms generate haploid gametes. Recombination between homologous chromosomes facilitates accurate meiotic chromosome segregation and is initiated by DNA double-strand breaks (DSBs) made by the conserved topoisomerase-like protein Spo11 (Rec12 in fission yeast), but DSBs are not evenly distributed across the genome. In Schizosaccharomyces pombe, proteinaceous structures known as linear elements (LinEs) are formed during meiotic prophase. The meiosis-specific cohesin subunits Rec8 and Rec11 are essential for DSB formation in some regions of the genome, as well as for formation of LinEs or the related synaptonemal complex (SC) in other eukaryotes. Proteins required for DSB formation decorate LinEs, and mutants lacking Rec10, a major component of LinEs, are completely defective for recombination. Although recombination may occur in the context of LinEs, it is not well understood how Rec10 is loaded onto chromosomes. We describe two novel components of LinEs in fission yeast, Rec25 and Rec27. Comparisons of rec25Delta, rec27Delta, and rec10Delta mutants suggest multiple pathways to load Rec10. In the major pathway, Rec10 is loaded, together with Rec25 and Rec27, in a Rec8-dependent manner with subsequent region-specific effects on recombination.     

Meiotic recombination proteins localize to linear elements in Schizosaccharomyces pombe

In fission yeast, meiotic prophase nuclei develop structures known as linear elements (LinEs), instead of a canonical synaptonemal complex. LinEs contain Rec10 protein. While Rec10 is essential for meiotic recombination, the precise role of LinEs in this process is unknown. Using in situ immunostaining, we show that Rec7 (which is required for meiosis-specific DNA double-strand break (DSB) formation) aggregates in foci on LinEs. The strand exchange protein Rad51, which is known to mark the sites of DSBs, also localizes to LinEs, although to a lesser degree. The number of Rec7 foci corresponds well with the average number of genetic recombination events per meiosis suggesting that Rec7 marks the sites of recombination. Rec7 and Rad51 foci do not co-localize, presumably because they act sequentially on recombination sites. The localization of Rec7 is dependent on Rec10 but independent of the DSB-inducing protein Rec12/Spo11. Neither Rec7 nor Rad51 localization depends on the LinE-associated proteins Hop1 and Mek1, but the formation of Rad51 foci depends on Rec10, Rec7, and, as expected, Rec12/Spo11. We propose that LinEs form around designated recombination sites before the induction of DSBs and that most, if not all, meiotic recombination initiates within the setting provided by LinEs.     

Rec8 Cohesin-mediated Axis-loop chromatin architecture is required for meiotic recombination

During meiotic prophase, cohesin-dependent axial structures are formed in the synaptonemal complex (SC). However, the functional correlation between these structures and cohesion remains elusive. Here, we examined the formation of cohesin-dependent axial structures in the fission yeast Schizosaccharomyces pombe. This organism forms atypical SCs composed of linear elements (LinEs) resembling the lateral elements of SC but lacking the transverse filaments. Hi-C analysis using a highly synchronous population of meiotic S. pombe cells revealed that the axis-loop chromatin structure formed in meiotic prophase was dependent on the Rec8 cohesin complex. In contrast, the Rec8-mediated formation of the axis-loop structure occurred in cells lacking components of LinEs. To dissect the functions of Rec8, we identified a rec8-F204S mutant that lost the ability to assemble the axis-loop structure without losing cohesion of sister chromatids. This mutant showed defects in the formation of the axis-loop structure and LinE assembly and thus exhibited reduced meiotic recombination. Collectively, our results demonstrate that the Rec8-dependent axis-loop structure provides a structural platform essential for LinE assembly, facilitating meiotic recombination of homologous chromosomes, independently of its role in sister chromatid cohesion.     

The Rec8 Gene of Schizosaccharomyces Pombe Is Involved in Linear Element Formation, Chromosome Pairing and Sister-Chromatid Cohesion during Meiosis

The fission yeast Schizosaccharomyces pombe does not form tripartite synaptonemal complexes during meiotic prophase, but axial core-like structures (linear elements). To probe the relationship between meiotic recombination and the structure, pairing, and segregation of meiotic chromosomes, we genetically and cytologically characterized the rec8-110 mutant, which is partially deficient in meiotic recombination. The pattern of spore viability indicates that chromosome segregation is affected in the mutant. A detailed segregational analysis in the rec8-110 mutant revealed more spores disomic for chromosome III than in a wild-type strain. Aberrant segregations are caused by precocious segregation of sister chromatids at meiosis I, rather than by nondisjunction as a consequence of lack of crossovers. In situ hybridization further showed that the sister chromatids are separated prematurely during meiotic prophase. Moreover, the mutant forms aberrant linear elements and shows a shortened meiotic prophase. Meiotic chromosome pairing in interstitial and centromeric regions is strongly impaired in rec8-110, whereas the chromosome ends are less deficient in pairing. We propose that the rec8 gene encodes a protein required for linear element formation and that the different phenotypes of rec8-110 reflect direct and indirect consequences of the absence of regular linear elements.     

SUMOylation is required for normal development of linear elements and wild-type meiotic recombination in Schizosaccharomyces pombe

In the fission yeast, Schizosaccharomyces pombe, synaptonemal complexes (SCs) are not formed during meiotic prophase. However, structures resembling the axial elements of SCs, the so-called linear elements (LinEs) appear. By in situ immunostaining, we found Pmt3 (S. pombe's SUMO protein) transiently along LinEs, suggesting that SUMOylation of some component(s) of LinEs occurs during meiosis. Mutation of the SUMO ligase Pli1 caused aberrant LinE formation and reduced genetic recombination indicating a role for SUMOylation of LinEs for the regulation of meiotic recombination. Western blot analysis of TAP-tagged Rec10 demonstrated that there is a Pli1-dependent posttranslational modification of this protein, which is a major LinE component and a distant homolog of the SC protein Red1. Mass spectrometry (MS) analysis revealed that Rec10 is both phosphorylated and ubiquitylated, but no evidence for SUMOylation of Rec10 was found. These findings indicate that the regulation of LinE and Rec10 function is modulated by Pli1-dependent SUMOylation of LinE protein(s) which directly or indirectly regulates Rec10 modification. On the side, MS analysis confirmed the interaction of Rec10 with the known LinE components Rec25, Rec27, and Hop1 and identified the meiotically upregulated protein Mug20 as a novel putative LinE-associated protein.     

Dynamics of chromosome organization and pairing during meiotic prophase in fission yeast

Interactions between homologous chromosomes (pairing, recombination) are of central importance for meiosis. We studied entire chromosomes and defined chromosomal subregions in synchronous meiotic cultures of Schizosaccharomyces pombe by fluorescence in situ hybridization. Probes of different complexity were applied to spread nuclei, to delineate whole chromosomes, to visualize repeated sequences of centromeres, telomeres, and ribosomal DNA, and to study unique sequences of different chromosomal regions. In diploid nuclei, homologous chromosomes share a joint territory even before entry into meiosis. The centromeres of all chromosomes are clustered in vegetative and meiotic prophase cells, whereas the telomeres cluster near the nucleolus early in meiosis and maintain this configuration throughout meiotic prophase. Telomeres and centromeres appear to play crucial roles for chromosome organization and pairing, both in vegetative cells and during meiosis. Homologous pairing of unique sequences shows regional differences and is most frequent near centromeres and telomeres. Multiple homologous interactions are formed independently of each other. Pairing increases during meiosis, but not all chromosomal regions become closely paired in every meiosis. There is no detectable axial compaction of chromosomes in meiotic prophase. S. pombe does not form mature synaptonemal complexes, but axial element-like structures (linear elements), which were analyzed in parallel. Their appearance coincides with pairing of interstitial chromosomal regions. Axial elements may define minimal structures required for efficient pairing and recombination of meiotic chromosomes.     

Nonrandom homolog segregation at meiosis I in Schizosaccharomyces pombe mutants lacking recombination

Physical connection between homologous chromosomes is normally required for their proper segregation to opposite poles at the first meiotic division (MI). This connection is generally provided by the combination of reciprocal recombination and sister-chromatid cohesion. In the absence of meiotic recombination, homologs are predicted to segregate randomly at MI. Here we demonstrate that in rec12 mutants of the fission yeast Schizosaccharomyces pombe, which are devoid of meiosis-induced recombination, homologs segregate to opposite poles at MI 63% of the time. Residual, Rec12-independent recombination appears insufficient to account for the observed nonrandom homolog segregation. Dyad asci are frequently produced by rec12 mutants. More than half of these dyad asci contain two viable homozygous-diploid spores, the products of a single reductional division. This set of phenotypes is shared by other S. pombe mutants that lack meiotic recombination, suggesting that nonrandom MI segregation and dyad formation are a general feature of meiosis in the absence of recombination and are not peculiar to rec12 mutants. Rec8, a meiosis-specific sister-chromatid cohesin, is required for the segregation phenotypes displayed by rec12 mutants. We propose that S. pombe possesses a system independent of recombination that promotes homolog segregation and discuss possible mechanisms.     

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.     

Casein Kinase 1 and Phosphorylation of Cohesin Subunit Rec11 (SA3) Promote Meiotic Recombination through Linear Element Formation

Proper meiotic chromosome segregation, essential for sexual reproduction, requires timely formation and removal of sister chromatid cohesion and crossing-over between homologs. Early in meiosis cohesins hold sisters together and also promote formation of DNA double-strand breaks, obligate precursors to crossovers. Later, cohesin cleavage allows chromosome segregation. We show that in fission yeast redundant casein kinase 1 homologs, Hhp1 and Hhp2, previously shown to regulate segregation via phosphorylation of the Rec8 cohesin subunit, are also required for high-level meiotic DNA breakage and recombination. Unexpectedly, these kinases also mediate phosphorylation of a different meiosis-specific cohesin subunit Rec11. This phosphorylation in turn leads to loading of linear element proteins Rec10 and Rec27, related to synaptonemal complex proteins of other species, and thereby promotes DNA breakage and recombination. Our results provide novel insights into the regulation of chromosomal features required for crossing-over and successful reproduction. The mammalian functional homolog of Rec11 (STAG3) is also phosphorylated during meiosis and appears to be required for fertility, indicating wide conservation of the meiotic events reported here.     

Meiotic chromosome dynamics dependent upon the rec8(+), rec10(+) and rec11(+) genes of the fission yeast Schizosaccharomyces pombe.

During meiosis homologous chromosomes replicate once, pair, experience recombination, and undergo two rounds of segregation to produce haploid meiotic products. The rec8(+), rec10(+), and rec11(+) genes of the fission yeast Schizosaccharomyces pombe exhibit similar specificities for meiotic recombination and rec8(+) is required for sister chromatid cohesion and homolog pairing. We applied cytological and genetic approaches to identify potential genetic interactions and to gauge the fidelity of meiotic chromosome segregation in the mutants. The rec8(+) gene was epistatic to rec10(+) and to rec11(+), but there was no clear epistatic relationship between rec10(+) and rec11(+). Reciprocal (crossover) recombination in the central regions of all three chromosomes was compromised in the rec mutants, but recombination near the telomeres was nearly normal. Each of the mutants also exhibited a high rate of aberrant segregation for all three chromosomes. The rec8 mutations affected mainly meiosis I segregation. Remarkably, the rec10 and rec11 mutations, which compromised recombination during meiosis I, affected mainly meiosis II segregation. We propose that these genes encode regulators or components of a "meiotic chromatid cohesion" pathway involved in establishing, maintaining, and appropriately releasing meiotic interactions between chromosomes. A model of synergistic interactions between sister chromatid cohesion and crossover position suggests how crossovers and cohesion help ensure the proper segregation of chromosomes in each of the meiotic divisions.     

Activation of an alternative, rec12 (spo11)-independent pathway of fission yeast meiotic recombination in the absence of a DNA flap endonuclease

Spo11 or a homologous protein appears to be essential for meiotic DNA double-strand break (DSB) formation and recombination in all organisms tested. We report here the first example of an alternative, mutationally activated pathway for meiotic recombination in the absence of Rec12, the Spo11 homolog of Schizosaccharomyces pombe. Rad2, a FEN-1 flap endonuclease homolog, is involved in processing Okazaki fragments. In its absence, meiotic recombination and proper segregation of chromosomes were restored in rec12Delta mutants to nearly wild-type levels. Although readily detectable in wild-type strains, meiosis-specific DSBs were undetectable in recombination-proficient rad2Delta rec12Delta strains. On the basis of the biochemical properties of Rad2, we propose that meiotic recombination by this alternative (Rec*) pathway can be initiated by non-DSB lesions, such as nicks and gaps, which accumulate during premeiotic DNA replication in the absence of Okazaki fragment processing. We compare the Rec* pathway to alternative pathways of homologous recombination in other organisms.     

Unusual nuclear structures in meiotic prophase of fission yeast: a cytological analysis

Earlier results from sectioned nuclei indicating that Schizosaccharomyces pombe does not develop a classical tripartite synaptonemal complex (SC) during meiotic prophase are confirmed by spreading of whole nuclei. The linear elements appearing during prophase I resemble the axial cores (SC precursors) of other organisms. The number of linear elements in haploid, diploid, and tetraploid strains is always higher than the chromosome number, implying that they are not formed continuously along the chromosomes. Time course experiments reveal that the elements appear after DNA replication and form networks and bundles. Later they separate and approximately 24 individual elements with a total length of 34 microns are observed before degradation and meiotic divisions. Parallel staining of DNA reveals changes in nuclear shape during meiotic prophase. Strains with a mei4 mutation are blocked at a late prophase stage. In serial sections we additionally observed a constant arrangement of the spindle pole body, the nucleolus, and the presumptive centromere cluster. Thus, S. pombe manages to recombine and segregate its chromosomes without SC. This might correlate with the absence of crossover interference. We propose a mechanism for chromosome pairing with initial recognition of the homologs at the centromeres and suggest functions of the linear elements in preparation of the chromosomes for meiosis I disjunction. With the spreading technique combined genetic, molecular, and cytological approaches become feasible in S. pombe. This provides an opportunity to study essential meiotic functions in the absence of SCs which may help to clarify the significance of the SC and its components for meiotic chromosome structure and function.     

S. pombe linear elements: the modest cousins of synaptonemal complexes

Synaptonemal complexes (SCs) are not formed during meiotic prophase in the fission yeast, Schizosaccharomyces pombe. Instead, so-called linear elements (LinEs) are formed at the corresponding stages. LinEs are remarkable in that their number does not correspond to the number of chromosomes or bivalents and that the changes in their organisation during prophase do not evidently reflect the pairing of chromosomes. Yet, LinEs are necessary for full meiotic pairing levels and for meiotic recombination. In this review, the composition of LinEs, their evolutionary relationship to SCs and their possible functions are discussed.     

Meiotically Induced Rec7 and Rec8 Genes of Schizosaccharomyces Pombe

The Schizosaccharomyces pombe rec7 and rec8 genes, which are required for meiotic intragenic recombination but not for mitotic recombination, have been cloned and their DNA sequences determined. Genetic and physical analyses demonstrated that the cloned fragments contained the rec genes rather than rec mutation suppressors. A 1.6-kb DNA fragment contained a functional rec7 gene, and a 2.1-kb fragment contained a functional rec8 gene. The nucleotide sequences of these fragments revealed open reading frames predicting 249 amino acids for the rec7 gene product and 393 amino acids for the rec8 gene product. Northern hybridization analysis showed that both rec gene mRNAs were detectable only at 2-3 hr after induction of meiosis. The absence of these mRNAs in mitosis and their disappearance at 4 hr and later in meiosis suggest that the rec7 and rec8 gene products may be involved primarily in the early steps of meiotic recombination in S. pombe.