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 rec7Δ. 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 comple

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 2.5kb 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 dynamic

New insights into the performance of human whole-exome capture platforms

Whole exome sequencing (WES) is increasingly used in research and diagnostics. WES users expect coverage of the entire coding region of known genes as well as sufficient read depth for the covered regions. It is, however, unknown which recent WES platform is most suitable to meet these expectations. We present insights into the performance of the most recent standard exome enrichment platforms from Agilent, NimbleGen and Illumina applied to six different DNA samples by two sequencing vendors per platform. Our results suggest that both Agilent and NimbleGen overall perform better than Illumina and that the high enrichment performance of Agilent is stable among samples and between vendors, whereas NimbleGen is only able to achieve vendor- and sample-specific best exome coverage. Moreover, the recent Agilent platform overall captures more coding exons with sufficient read depth than NimbleGen and Illumina. Due to considerable gaps in effective exome coverage, however, the three platforms cannot capture all known coding exons alone or in combination, requiring improvement. Our data emphasize the importance of evaluation of updated platform versions and suggest that enrichment-free whole genome sequencing can overcome the limitations of WES in sufficiently covering coding exons, especially GC-rich regions, and in characterizing structural variant

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 ctp1Δ 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

The Mating-Type-Related Bias of Gene Conversion in Schizosaccharomyces pombe

The mating-type bias (mat-bias) of gene conversion was previously described as a phenomenon in which the number of prototrophic recombinants in an ura4A heteroallelic two-factor cross relates to the mating types of the parents. We show now that the mat-bias is restricted neither to ura4A nor to recombination hotspots, but occurs at other genomic loci, too. It is specific for gene conversion and absent in azygotic meiosis. Thus, the mat-bias must originate from mating-type-specific “imprinting” events before karyogamy takes place. Structural variations of the mating-type locus, such as h+N, h+S, h−S, h+smtΔ, or h−smtΔ, showed mat-bias manifestation. Mutations in genes coding for histone acetylase (gcn5, ada2) and histone deacetylase (hos2, clr6) activities smooth or abolish the mat-bias. In addition, the mat-bias depends on the presence of Swi5. We propose a new role for Swi5 and the histone acetylation status in mat-bias establishment through directionality of repair from the intact chromatid to the broken chromatid

The meiotic recombination hot-spot ura4A in Schizosaccharomyces pombe

The meiotic recombination hot spot ura4A (formerly ura4-aim) of Schizosaccharomyces pombe was observed at the insertion of the ura4 gene 15 kb centromere-proximal to ade6 on chromosome III. Crosses heterozygous for the insertion showed frequent conversion at the heterology with preferential loss of the insertion. This report concerns the characterization of 12 spontaneous ura4A mutants. A gradient of conversion ranging from 18% at the 5 end to 6% at the 3 end was detected. A novel phenomenon also was discovered: a matingtype-related bias of conversion. The allele entering with the h parent acts preferentially as the acceptor for conversion (ratio of 3:2). Tetrad analysis of two-factor crosses showed that heteroduplex DNA is predominantly asymmetrical, enters from the 5 end, and more often than not covers the entire gene. Restoration repair of markers at the 5 end was inferred. Random spore analyses of two-factor crosses and normalization of prototroph-recombinant frequencies to physical distance led to the demonstration of map expansion: Crosses involving distant markers yielded recombinant frequencies higher than the sum of the frequencies measured in the subintervals. Finally, marker effects on recombination were defined for two of the ura4A mutations

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)

New insights into the performance of human whole-exome capture platforms

Whole exome sequencing (WES) is increasingly used in research and diagnostics. WES users expect coverage of the entire coding region of known genes as well as sufficient read depth for the covered regions. It is, however, unknown which recent WES platform is most suitable to meet these expectations. We present insights into the performance of the most recent standard exome enrichment platforms from Agilent, NimbleGen and Illumina applied to six different DNA samples by two sequencing vendors per platform. Our results suggest that both Agilent and NimbleGen overall perform better than Illumina and that the high enrichment performance of Agilent is stable among samples and between vendors, whereas NimbleGen is only able to achieve vendor- and sample-specific best exome coverage. Moreover, the recent Agilent platform overall captures more coding exons with sufficient read depth than NimbleGen and Illumina. Due to considerable gaps in effective exome coverage, however, the three platforms cannot capture all known coding exons alone or in combination, requiring improvement. Our data emphasize the importance of evaluation of updated platform versions and suggest that enrichment-free whole genome sequencing can overcome the limitations of WES in sufficiently covering coding exons, especially GC-rich regions, and in characterizing structural variants.ISSN:1362-4962ISSN:0301-561