Cohesins are required for meiotic DNA breakage and recombination in Schizosaccharomyces pombe

Abstract

In preparation for the unique segregation of homologs at the first meiotic division, chromosomes undergo dramatic changes. The meiosis-specific sister chromatid cohesins Rec8 and Rec11 of Schizosaccharomyces pombe are recruited around the time of premeiotic replication, and Rec10, a component of meiosis-specific linear elements, is subsequently added. Here we report that Rec10 is essential for meiosis-specific DNA breakage by Rec12 (Spo11 homolog) and for meiotic recombination. DNA breakage and recombination also depend on the Rec8 and Rec11 cohesins, strictly in some genomic intervals but less so in others. Thus, in addition to their previously recognized role in meiotic chromosome segregation, cohesins have a direct role, as do linear element components, in meiotic recombination by enabling double-strand DNA break formation by Rec12. Our results reveal a pathway, whose regulation is significantly different from that in the distantly related yeast Saccharomyces cerevisiae, for meiosis-specific chromosome differentiation and high-frequency recombination.

Fig. 1.

Recombination is strongly reduced in rec10Δ mutants throughout the genome and differentially reduced in rec8Δ and rec11Δ mutants. (Left) S. pombe chromosomes and genetic loci. The chromosomes and the positions of the loci used here are drawn to scale, based on the genome sequence (23). Thick lines indicate rDNA (≈0.5 Mb) at each end of Chr. III. Brackets indicate restriction fragments analyzed in Fig. 2 using radioactive probes at the positions of the open circles. (Right) Relative recombination rates in rec10Δ (black), rec8Δ (white), and rec11Δ (gray) mutants. Intragenic (gene conversion) intervals are underlined. The genetic distance in cM at the indicated locus or interval in each mutant is expressed relative to that of wild type. Data are from Tables 1, 2, and 4. Error bar, standard deviation based on the binomial distribution of the numbers of recombinants observed and total spore colonies tested.

Fig. 2.

Meiotic DNA breakage is strongly reduced or eliminated in rec10Δ, rec8Δ, and rec11Δ mutants. (A) DNA was prepared at the indicated time (hr) after meiotic induction of rad50S strains, digested with NotI, electrophoresed, and hybridized with a 1-kb probe from the left (top) end of the 501-kb NotI fragment J on Chr. I (see Fig. 1). A bullet (•) indicates the intact fragment, and asterisks (^*) indicate DNA from wild type (rec⁺) cells broken at meiosis-specific hotspots. Small open circle (o), irreproducible spot (see Fig. 4A). The approximate positions of genetic loci used to measure recombination (Fig. 1) are indicated. (B) DNA was digested with NotI, electrophoresed, and hybridized with a 1-kb probe from the right (bottom) end of Chr. II. (C) DNA was digested with PacI, electrophoresed, and hybridized with a 1-kb probe from the right (bottom) end of the 41-kb PacI fragment containing the ade6-3049 hotspot on Chr. III. (D) Whole chromosomal DNA was analyzed by pulsed-field gel electrophoresis and staining with ethidium bromide. Meiotically broken DNA is a smear (bracket) that migrates with or more rapidly than the three intact chromosomes (Chr. I-III). For details and complete time courses, see Fig. 4.

Fig. 3.

Proposed pathway for S. pombe meiotic chromosome differentiation, DSB formation, and recombination. During or shortly after premeiotic replication, the sister chromatid cohesins Rec8 and Rec11 are recruited to the chromosomes. Largely dependent upon their presence, the linear element component Rec10 is recruited, which in turn recruits or activates Rec12 and its partner proteins, Rec6, Rec7, Rec14, and Rec15. The Rec12 complex makes DNA breaks, which are resected by the Rad32•Rad50•Nbs1 nuclease complex or other proteins. DNA strand exchange between the resected DNA and an intact homolog is promoted by a combination of Rad51, Rhp55, Rhp57, Swi5, and Dmc1 proteins. Joint molecules include Holliday junctions (HJ), but it is not known whether these have double HJ, as illustrated, or a different structure; these molecules are resolved by the Mus81•Eme1 complex into crossover molecules. The crossovers facilitate reductional segregation of homologs at the first meiotic division. Red and blue lines are duplex DNA, except within the magnified oval, where they are single DNA strands. See text for references and further discussion.