Ctp1 and the MRN-complex are required for endonucleolytic Rec12 removal with release of a single class of oligonucleotides in fission yeast.

DNA double-strand breaks (DSBs) are formed during meiosis by the action of the topoisomerase-like Spo11/Rec12 protein, which remains covalently bound to the 5' ends of the broken DNA. Spo11/Rec12 removal is required for resection and initiation of strand invasion for DSB repair. It was previously shown that budding yeast Spo11, the homolog of fission yeast Rec12, is removed from DNA by endonucleolytic cleavage. The release of two Spo11 bound oligonucleotide classes, heterogeneous in length, led to the conjecture of asymmetric cleavage. In fission yeast, we found only one class of oligonucleotides bound to Rec12 ranging in length from 17 to 27 nucleotides. Ctp1, Rad50, and the nuclease activity of Rad32, the fission yeast homolog of Mre11, are required for endonucleolytic Rec12 removal. Further, we detected no Rec12 removal in a rad50S mutant. However, strains with additional loss of components localizing to the linear elements, Hop1 or Mek1, showed some Rec12 removal, a restoration depending on Ctp1 and Rad32 nuclease activity. But, deletion of hop1 or mek1 did not suppress the phenotypes of ctp1Delta and the nuclease dead mutant (rad32-D65N). We discuss what consequences for subsequent repair a single class of Rec12-oligonucleotides may have during meiotic recombination in fission yeast in comparison to two classes of Spo11-oligonucleotides in budding yeast. Furthermore, we hypothesize on the participation of Hop1 and Mek1 in Rec12 removal.

Rec10- and Rec12-independent recombination in meiosis of Schizosaccharomyces pombe.

The Rec10 protein, a component of the linear elements forming along sister chromatids in meiotic prophase of Schizosaccharomyces pombe, plays an important role in the activation of Rec12 for double-strand break formation, and thus the initiation of recombination between homologous chromosomes. Recombination between homologous chromosomes was moderately reduced in homozygous crosses of the C-terminal truncation mutant rec10-155 and strongly in the full deletion allele rec10-175. Both alleles were also tested in two assays for intrachromosomal recombination (PS1 and VL1) and showed only slight reductions, while deletion of rec12 led to a 13-fold reduction. The even stronger reductions in rec10 rec12 double deletion crosses indicate partially redundant functions of Rec10 and Rec12 in the initiation of intrachromosomal recombination. A low level of double-strand breaks has been detected in rec10-175 meiosis at the mbs1 hotspot of recombination, and spore viability in the double mutant was also lower than in the single-deletion mutants. Low levels of apparent crossover and conversion between homologous chromosomes in the absence of Rec12 have been quantified using a newly developed assay. The results also indicate that the functions of Rec10 differ in several respects from those of its distant homologue Red1 in Saccharomyces cerevisiae, including interactions with Hop1 and Mek1 for promotion of recombination between homologues at the expense of sister chromatid recombination.

Functional interactions among members of the meiotic initiation complex in fission yeast.

DNA double-strand breaks (DSBs) initiate meiotic recombination in Schizosaccharomyces pombe and in other organisms. The Rec12 protein catalyzes the formation of these DSBs in concert with a multitude of accessory proteins the role of which in this process remains to be discovered. In an all-to-all yeast two-hybrid matrix analysis, we discovered new interactions among putative members of the meiotic recombination initiation complex. We found that Rec7, an axial-element associated protein with homologies to Saccharomyces cerevisiae Rec114, is interacting with Rec24. Rec7 and Rec24 also co-immunoprecipitate in S. pombe during meiosis. An amino acid change in a conserved, C-terminal phenylalanine in Rec7, F325A interrupts the interaction with Rec24. Moreover, rec7F325A shows a recombination deficiency comparable to rec7Delta. Another interaction was detected between Rec12 and Rec14, the orthologs of which in S. cerevisiae Spo11 and Ski8 interact accordingly. Amino acid changes Rec12Q308A and Rec12R309A disrupt the interaction with Rec14, like the according amino acid changes Spo11Q376A and Spo11RE377AA loose the interaction with Ski8. Both amino acid changes in Rec12 reveal a recombination deficient rec12 (-) phenotype. We propose that both Rec7-Rec24 and Rec12-Rec14 form subcomplexes of the meiotic recombination initiation complex.

Sites of strong Rec12/Spo11 binding in the fission yeast genome are associated with meiotic recombination and with centromeres.

Meiotic recombination arises from Rec12/Spo11-dependent formation of DNA double-strand breaks (DSBs) and their subsequent repair. We identified Rec12-binding peaks across the Schizosaccharomyces pombe genome using chromatin immunoprecipitation after reversible formaldehyde cross-linking combined with whole-genome DNA microarrays. Strong Rec12 binding coincided with previously identified DSBs at the recombination hotspots ura4A, mbs1, and mbs2 and correlated with DSB formation at a new site. In addition, Rec12 binding corresponded to eight novel conversion hotspots and correlated with crossover density in segments of chromosome I. Notably, Rec12 binding inversely correlated with guanine-cytosine (GC) content, contrary to findings in Saccharomyces cerevisiae. Although both replication origins and Rec12-binding sites preferred AT-rich gene-free regions, they seemed to exclude each other. We also uncovered a connection between binding sites of Rec12 and meiotic cohesin Rec8. Rec12-binding peaks lay often within 2.5 kb of a Rec8-binding peak. Rec12 binding showed preference for large intergenic regions and was found to bind preferentially near to genes expressed strongly in meiosis. Surprisingly, Rec12 binding was also detected in centromeric core regions, which raises the intriguing possibility that Rec12 plays additional roles in meiotic chromosome dynamics.

The Rad52 homologs Rad22 and Rti1 of Schizosaccharomyces pombe are not essential for meiotic interhomolog recombination, but are required for meiotic intrachromosomal recombination and mating-type-related DNA repair.

Proteins of the RAD52 epistasis group play an essential role in repair of some types of DNA damage and genetic recombination. In Schizosaccharomyces pombe, Rad22 (a Rad52 ortholog) has been shown to be as necessary for repair and recombination events during vegetative growth as its Saccharomyces cerevisiae counterpart. This finding contrasts with previous reports where, due to suppressor mutations in the fbh1 gene, rad22 mutants did not display a severe defect. We have analyzed the roles of Rad22 and Rti1, another Rad52 homolog, during meiotic recombination and meiosis in general. Both proteins play an important role in spore viability. During meiotic prophase I, they partially colocalize and partially localize to Rad51 foci and linear elements. Genetic analysis showed that meiotic interchromosomal crossover and conversion events were unexpectedly not much affected by deletion of either or both genes. A strong decrease of intrachromosomal recombination assayed by a gene duplication construct was observed. Therefore, we propose that the most important function of Rad22 and Rti1 in S. pombe meiosis is repair of double-strand breaks with involvement of the sister chromatids. In addition, a novel mating-type-related repair function of Rad22 specific to meiosis and spore germination is described.

Cohesin and recombination proteins influence the G1-to-S transition in azygotic meiosis in Schizosaccharomyces pombe.

To determine whether recombination and/or sister-chromatid cohesion affect the timing of meiotic prophase events, the horsetail stage and S phase were analyzed in Schizosaccharomyces pombe strains carrying mutations in the cohesin genes rec8 or rec11, the linear element gene rec10, the pairing gene meu13, the double-strand-break formation genes rec6, rec7, rec12, rec14, rec15, and mde2, and the recombination gene dmc1. The double-mutant strains rec8 rec11 and rec8 rec12 were also assayed. Most of the single and both double mutants showed advancement of bulk DNA synthesis, start of nuclear movement (horsetail stage), and meiotic divisions by up to 2 hr. Only mde2 and dmc1 deletion strains showed wild-type timing. Contrasting behavior was observed for rec8 deletions (delayed by 1 hr) compared to a rec8 point mutation (advanced by 1 hr). An hypothesis for the role of cohesin and recombination proteins in the control of the G(1)-to-S transition is proposed. Finally, differences between azygotic meiosis and two other types of fission yeast meiosis (zygotic and pat1-114 meiosis) are discussed with respect to possible control steps in meiotic G(1).

Roles of Hop1 and Mek1 in meiotic chromosome pairing and recombination partner choice in Schizosaccharomyces pombe.

Synaptonemal complex (SC) proteins Hop1 and Mek1 have been proposed to promote homologous recombination in meiosis of Saccharomyces cerevisiae by establishment of a barrier against sister chromatid recombination. Therefore, it is interesting to know whether the homologous proteins play a similar role in Schizosaccharomyces pombe. Unequal sister chromatid recombination (USCR) was found to be increased in hop1 and mek1 single and double deletion mutants in assays for intrachromosomal recombination (ICR). Meiotic intergenic (crossover) and intragenic (conversion) recombination between homologous chromosomes was reduced. Double-strand break (DSB) levels were also lowered. Notably, deletion of hop1 restored DSB repair in rad50S meiosis. This may indicate altered DSB repair kinetics in hop1 and mek1 deletion strains. A hypothesis is advanced proposing transient inhibition of DSB processing by Hop1 and Mek1 and thus providing more time for repair by interaction with the homologous chromosome. Loss of Hop1 and Mek1 would then result in faster repair and more interaction with the sister chromatid. Thus, in S. pombe meiosis, where an excess of sister Holliday junction over homologous Holliday junction formation has been demonstrated, Hop1 and Mek1 possibly enhance homolog interactions to ensure wild-type level of crossover formation rather than inhibiting sister chromatid interactions.

The end1 gene of Schizosaccharomyces pombe coding for a DNase is identical with the pnu1 gene coding for an RNase.

The DNA nuclease activity encoded by the end1 gene, and its inactivation by mutation, was described in connection with the characterization of DNA topoisomerases in the fission yeast Schizosaccharomyces pombe (Uemura and Yanagida, 1984). Subsequently, end1 mutant strains were used for the preparation of cell extracts for the study of enzymes and intermediates involved in DNA metabolism. The molecular identification of the end1 gene and its identity with the pnu1 gene is presented. The end1-458 mutation alters glycine to glutamate in the conserved motif TGPYLP. The pnu1 gene codes for an RNase that is induced by nitrogen starvation (Nakashima et al., 2002b). Thus, the End1/Pnu1 protein, like related mitochondrial proteins in other organisms, is an example of a sugar-non-specific nuclease. The analysis of strains carrying a pnu1 deletion revealed no defects in meiotic recombination and spore viability.

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.

Characterization of rec15, an early meiotic recombination gene in Schizosaccharomyces pombe.

In S. pombe strains mutant for rec15 aberrant ascus morphology, reduced spore viability and severe reduction of meiotic recombination was detected. Genetic and cytological analysis identified frequent interruption of meiosis after the first division, and nondisjunction I, as the main segregation errors in the mutant. Chromosome segregation at meiosis I was not random in rec15, suggesting the presence of a backup system for correct segregation of achiasmate chromosomes. The analysis of meiotic progression in time-course experiments revealed that the major meiotic events, such as the onset of premeiotic DNA synthesis, of horse-tail nuclear movement, and of the first meiotic division occurred earlier in rec15 than in wild-type. The early onset of meiotic events is a novel observation for an early recombination mutant and implies a function of rec15 protein already at or before DNA synthesis.

Linear element formation and their role in meiotic sister chromatid cohesion and chromosome pairing.

Fission yeast does not form synaptonemal complexes in meiotic prophase. Instead, linear elements appear that resemble the axial cores of other eukaryotes. They have been proposed to be minimal structures necessary for proper meiotic chromosome functions. We examined linear element formation in meiotic recombination deficient mutants. The rec12, rec14 and meu13 mutants showed altered linear element formation. Examination of rec12 and other mutants deficient in the initiation of meiotic recombination revealed that occurrence of meiosis-specific DNA breaks is not a precondition for the formation of linear elements. The rec11 and rec8 mutants exhibited strongly impaired linear elements with morphologies specific for these meiotic cohesin mutants. The rec10 and rec16/rep1 mutants lack linear elements completely. The region specificity of loss of recombination in the rec8, rec10 and rec11 mutants can be explained by their defects in linear element formation. Investigation of the rec10 mutant showed that linear elements are basically dispensable for sister chromatid cohesion, but contribute to full level pairing of homologous chromosomes.