Functional organization of protein determinants of meiotic DNA break hotspots

Abstract

During Schizosaccharomyces pombe meiotic prophase, homologous chromosomes are co-aligned by linear elements (LinEs) analogous to the axial elements of the synaptonemal complex (SC) in other organisms. LinE proteins also promote the formation of meiotic DNA double-strand breaks (DSBs), the precursors of cross-overs. Rec10 is required for essentially all DSBs and recombination, and three others (Rec25, Rec27, and Mug20) are protein determinants of DSB hotspots - they bind DSB hotspots with high specificity and are required for DSB formation there. These four LinE proteins co-localize in the nucleus in an interdependent way, suggesting they form a complex. We used random mutagenesis to uncover recombination-deficient missense mutants with novel properties. Some missense mutations changed essential residues conserved among Schizosaccharomyces species. DSB formation, gene conversion, and crossing-over were coordinately reduced in the mutants tested. Based on our mutant analysis, we revised the rec27 open reading frame: the new start codon is in the previously annotated first intron. Genetic and fluorescence-microscopy assays indicated that the Rec10 N- and C-terminal regions have complex interactions with Rec25. These mutants are a valuable resource to elucidate further how LinE proteins and the related SCs of other species regulate meiotic DSB formation to form crossovers crucial for meiosis.

Figure 1

Schematic of the screen to isolate LinE mutants. A library of plasmids containing a randomly (PCR) mutagenized rec25, rec27 or mug20 gene was transferred to diploid cells homozygous for rec25Δ, rec27Δ or mug20Δ; linE* means rec25, rec27 or mug20. After sporulation on EMM2+Ade plates, Ade⁺ recombinant frequencies were estimated by spot test (bottom right), in which recombination-deficient mutants produce a low frequency of white (Ade⁺) papillae on a background of red (Ade⁻) cells.

Figure 2

Meiotic recombination of newly isolated LinE mutants. Meiotic gene conversion at ade6 (two allele pairs) and crossing-over between ade6 and arg1 in LinE mutants were measured as described in Methods. Data (mean ± SEM) are from Supplementary Table S3; n ≥ 3 for each mutant. NS (not significant; P > 0.05), **(P < 0.01), and ***(P < 0.001) indicate significance of the difference from wild type by unpaired t-test.

Figure 3

Revised Rec27 ORF starts within the first formerly designated intron. (A) Meiotic recombination between ade6-M26 and ade6-52 in rec27 mutants. Data (mean ± SEM) are from Supplementary Table S3; n = 4 for each strain. NS (non-significant) and * (P < 0.05) indicate significance of the difference from rec27 ⁺ by unpaired t-test. (B) Map of revised rec27 ORF (lower line) compared to the previous annotation (upper line) (http://www.pombase.org/spombe/result/SPBC577.05c). Pink boxes are exons.

Figure 4

Meiotic DSBs of LinE mutants. LinE mutants were meiotically induced; DNA was extracted at the indicated times, digested with BsrG1 and analyzed for DSBs at the ade6-3049 hotspot by Southern blot hybridization. Unbroken DNA (10.1 kb) migrates at the top of the blots. Meiotically broken DNA (3–4 kb) migrates as multiple bands near the middle of the blots. The percentage of total radioactivity in the DSB position was quantified with ImageQuant and is shown below each lane and in the graph on the right. Data are individual values or (for wild-type and rec27-238 cells) the range for two independent experiments. Full-length blots are presented in Suppl. Figure S7.

Figure 5

Rec25-GFP localization in rec10 missense mutants. Cells containing Rec25-GFP and the indicated rec10 mutations were induced for meiosis and fixed for fluorescence microscopy. The upper panels show Rec25-GFP, while the lower panels show DAPI-stained DNA. The figures show only the time points when GFP signal was detected (from 1.5 hr to 4 hr) after meiotic induction.