Activation of meiotic recombination by nuclear import of the DNA break hotspot-determining complex in fission yeast

Abstract

Meiotic recombination forms crossovers important for proper chromosome segregation and offspring viability. This complex process involves many proteins acting at each of the multiple steps of recombination. Recombination initiates by formation of DNA double-strand breaks (DSBs), which in the several species examined occur with high frequency at special sites (DSB hotspots). In Schizosaccharomyces pombe, DSB hotspots are bound with high specificity and strongly activated by linear element (LinE) proteins Rec25, Rec27 and Mug20, which form colocalized nuclear foci with Rec10, essential for all DSB formation and recombination. Here, we test the hypothesis that the nuclear localization signal (NLS) of Rec10 is crucial for coordinated nuclear entry after forming a complex with other LinE proteins. In NLS mutants, all LinE proteins were abundant in the cytoplasm, not the nucleus; DSB formation and recombination were much reduced but not eliminated. Nuclear entry of limited amounts of Rec10, apparently small enough for passive nuclear entry, can account for residual recombination. LinE proteins are related to synaptonemal complex proteins of other species, suggesting that they also share an NLS, not yet identified, and undergo protein complex formation before nuclear entry.This article has an associated First Person interview with Mélody Wintrebert, joint first author of the paper.

Keywords: DSB hotspot determinants; Linear element proteins; Meiotic recombination; Nuclear localization signal; S. pombe; Synaptonemal complex proteins.

Fig. 1.

Schematic of Rec10 protein, showing NLS sites A and B defined and analyzed here. The solid blue line shows the full-length (791 amino acids) of Rec10. Positions of the NLS sites and their amino acid sequences are shown below; the position of the GFP fusion to the C-terminus of Rec10 is shown above.

Fig. 2.

Plasmid-borne rec10 NLS mutations block entry of other LinE proteins into the nucleus. Strains bearing rec25–GFP and a complete rec10 deletion on the chromosome and carrying a plasmid with rec10⁺ (WT), with the indicated rec10 NLS mutation, or without rec10 (rec10Δ) were harvested during asynchronous (h⁹⁰) meiosis and examined by light microscopy. In the merged images, green indicates Rec25–GFP, and red indicates chromatin in the nucleus; yellow is their overlap. The dashed line outlines the cell. ΔA indicates deletion of Rec10 NLS site A, and AlaA indicates substitution of alanine for each of the four amino acids of site A; site B was similarly deleted or mutated, indicated as ΔB and AlaB, respectively. Combinations of these mutations are indicated by ΔA ΔB or AlaA AlaB. Images are representative of >50 cells from three separate cultures analyzed on different days. Cells are in the horsetail stage, during which the nucleus moves repeatedly from one end of the cell to the other; nuclei are longer when in the middle of the cell than when at either end, when the nucleus becomes nearly round (Robinow, 1977; Bähler et al., 1993; Chikashige et al., 1994). Scale bars: 5 μm.

Fig. 3.

Chromosomal rec10 NLS mutations block entry of Rec10–GFP into the nucleus. Strains without (WT) or with a rec10 NLS mutation were harvested during asynchronous (h⁹⁰) meiosis and examined by light microscopy. Green indicates Rec10, and red indicates chromatin in the nucleus; yellow is their overlap. The dashed line outlines the cell. NLS mutations are designated as in Fig. 2. Images are representative of >50 cells from three separate cultures analyzed on different days. Cells are in the horsetail stage, as in Fig. 2. Scale bars: 5 μm.

Fig. 4.

Chromosomal rec10 NLS mutations block entry of Rec25–GFP, Rec27–GFP and Mug20–GFP into the nucleus. (A) Strains bearing rec25–GFP and without (WT) or with the indicated rec10 NLS mutations were harvested during asynchronous (h⁹⁰) meiosis and examined by light microscopy. (B) Strains bearing rec27–GFP were analyzed as in panel A. (C) Strains bearing mug20–GFP were analyzed as in panel A. In the merged images, green indicates GFP, and red indicates chromatin in the nucleus; yellow is their overlap. See Fig. 2 for examples of separate images for Rec25–GFP and chromatin. The dashed line outlines the cell. NLS mutations are designated as in Fig. 2. Images are representative of >50 cells from three separate cultures analyzed on different days. Cells are in the horsetail stage, as in Fig. 2. Scale bars: 5 μm.

Fig. 5.

Rec10–NLS mutations reduce DSB formation at DSB hotspots. Strains (pat1-as1 rad50S) with the indicated chromosomal rec10 genotype were induced for meiosis and harvested at the indicated times. DNA was analyzed by Southern blotting for DSBs at the ade6-3049 hotspot on the 74 kb PmeI fragment of chromosome 3 (A) and at multiple hotspots (hotspot A, mbs1, mbs2) on the 501 kb NotI fragment of chromosome 1 (B). NLS mutations are designated as in Fig. 2. See Fig. S2 for additional data.

Fig. 6.

Rec10 protein is present at wild-type levels in rec10-NLS mutants. Strains with chromosomal rec10-NLS–GFP fusions were induced for meiosis (pat1-as1) and analyzed by western blotting for Rec10 abundance in cell extracts at the indicated times. NLS mutations are designated as in Fig. 2. M, protein markers with the indicated masses.