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

Abstract

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.

Figure 1. Rec25 and Rec27 Are Required for Efficient Homologous Chromosome Pairing

h⁹⁰ ade3⁺::lacO his7⁺::GFP-LacI-NLS mei4Δcells with the indicated rec deletions (strains S1899, S1901, S1903, and S1904) were grown on sporulation medium (MEA) at 25°C for more than 29 hr to ensure that cells were blocked in meiotic prophase and examined under a fluorescence microscope. The percentage of cells with unpaired chromosomes (grey bar) and paired chromosomes (black bar) is shown. Previously described criteria for pairing were used [16]. Homologous chromosomes were scored as paired when their GFP signals touched each other or only a single signal was visible, or as unpaired when two independent GFP signals were observed even if they were in close proximity. The data are the mean of six independent experiments in which 200–300 asci were counted for each genotype in each experiment. Standard deviation (SD) and p values based on Student’s t-test are shown.

Figure 2. Rec10 Structures (LinEs) Are Not Formed in rec25Δ and rec27Δ Mutants

Diploid pat1-114 cells with the indicated deletions (strains S964, S1628, S1624, S1554, and S1572) were induced for meiosis, and cells in prophase were collected for preparation of nuclear spreads. The spreads were stained with DAPI (DNA; blue) and anti-Rec10 antibodies (green), and photographed under a fluorescence microscope. Results at 3.5 hr after meiotic induction are shown. The fraction of nuclei with Rec10 structures (LinEs) is indicated. 100 nuclei were counted in a well-stained area of the preparation, except for rec25Δand rec27Δ, in which the entire preparation (more than 5 × 10³ nuclei examined) was screened. Similar results were obtained with 3 hr chromosome spreads and in an independent experiment (unpublished data).

Figure 3. Rec25-GFP and Rec10 Co-localize during LinE Formation

Chromosome spreads of diploid pat1-114 rec25-GFP (strain S1702) cells were prepared at the indicated times after meiotic induction, stained with DAPI (DNA; blue), anti-GFP (Rec25; green) and anti-Rec10 (red) antibodies, and photographed under a fluorescence microscope. The fraction of nuclei with structures (LinEs or precursors) is indicated. 200 nuclei, 100 in each of two different well-stained areas of the same preparation, were counted. Nuclear signals were positive for both GFP and Rec10 or for neither. Staining was done twice with duplicate spreads of the same meiotic induction.

Figure 4. Loading of LinE Components and Cohesins

(A) Rec25-GFP Is Defectively Loaded in rec8Δand Not Loaded in rec10Δand rec27ΔMutants. Chromosome spreads, prepared from strains S1702, S1812, S1816, and S1814 as in Figure 2, were stained with DAPI (DNA; blue) and anti-GFP antibodies (Rec25; green), and photographed under a fluorescence microscope. The fraction of nuclei with Rec25 structures is indicated. Results at 3.5 hr after meiotic induction are shown. 200 nuclei, 100 in each of two different well-stained areas of the same preparation, were counted, except for rec10Δand rec27Δ, in which the entire preparation (more than 5 × 10³ nuclei examined) was screened. Similar results were obtained with spreads at 3 hr after meiotic induction (unpublished data). (B) Rec8-GFP Is Normally Loaded in rec10Δ, rec25Δ, and rec27ΔMutants. Chromosome spreads, prepared from strains S1855, S1809, S1815, and S1854 as in Figure 2, were stained with DAPI (DNA; blue) and anti-GFP antibodies (Rec8; green), and photographed under a fluorescence microscope. Results at 3.5 hr after meiotic induction are shown. The fluorescent signal was too weak to quantify, but in every genotype positive Rec8-GFP nuclei were found.