Ctp1CtIP and Rad32Mre11 nuclease activity are required for Rec12Spo11 removal, but Rec12Spo11 removal is dispensable for other MRN-dependent meiotic functions

Abstract

The evolutionarily conserved Mre11/Rad50/Nbs1 (MRN) complex is involved in various aspects of meiosis. Whereas available evidence suggests that the Mre11 nuclease activity might be responsible for Spo11 removal in Saccharomyces cerevisiae, this has not been confirmed experimentally. This study demonstrates for the first time that Mre11 (Schizosaccharomyces pombe Rad32(Mre11)) nuclease activity is required for the removal of Rec12(Spo11). Furthermore, we show that the CtIP homologue Ctp1 is required for Rec12(Spo11) removal, confirming functional conservation between Ctp1(CtIP) and the more distantly related Sae2 protein from Saccharomyces cerevisiae. Finally, we show that the MRN complex is required for meiotic recombination, chromatin remodeling at the ade6-M26 recombination hot spot, and formation of linear elements (which are the equivalent of the synaptonemal complex found in other eukaryotes) but that all of these functions are proficient in a rad50S mutant, which is deficient for Rec12(Spo11) removal. These observations suggest that the conserved role of the MRN complex in these meiotic functions is independent of Rec12(Spo11) removal.

FIG. 1.

The rad50S mutant is temperature sensitive for meiotic spore viability and defective for meiotic DSB repair and Rec12^Spo11 removal. (a) Meiotic spore viability relative to that of the WT in different strains at 25°C and 34°C. Error bars show standard deviations, and values are averages for three independent experiments. (b) Pulsed-field gel electrophoresis of a synchronized meiotic pat1⁺ rad50S culture at 25°C and 34°C. The bands labeled I, II, and III correspond to the intact chromosomes, and the smears labeled DSB correspond to broken DNA fragments. (c) Meiotic progression of the time course presented in panel b, expressed as numbers of cells that have completed meiosis I at different time points. (d) Slot blot showing the presence of covalently bound Rec12^Spo11 on the DNA 6 h after meiotic induction at 34°C. At time point 0, no Rec12^Spo11 signals were visible (data not shown). The arrow indicates where the top and bottom fractions of the CsCl gradient were loaded. The bulk of the DNA was found in fractions 5, 6, and 7, which showed the strongest Rec12^Spo11 signals for rad50 mutants.

FIG. 2.

The rad50S mutant is proficient for meiotic recombination and meiosis-specific nucleosome remodeling, but both functions are defective in the rad50Δ mutant. (a) Meiotic intergenic recombination levels in surviving rad50Δ spores were strongly reduced at different genetic intervals at both 25°C and 34°C. However, the rad50S strain was proficient for recombination at both temperatures. (b) Meiosis-specific nucleosome remodeling at ade6-M26 (see black arrow for the WT at 3 h) was defective at 34°C in the rad50Δ strain (and the rad32^mre11Δ mutant), whereas the rad50S strain was proficient.

FIG. 3.

The rad50S mutant is proficient for linear element formation, whereas LEs are absent in the rad50Δ mutant. (a) Electron micrographs of lysed and spread meiotic nuclei. LEs in pat1-114 meiosis (used for its high degree of synchrony) were shorter than those in the WT (pat1⁺) (4), whereas networks and bundles were not detected. LEs in pat1-114 meiosis were classified into classes 1a (short LEs) and 1b (long LEs). LEs in rad50S cells were slightly longer and more abundant than those in the WT. LEs were absent in rad50Δ cells. The bottom two panels (rad50Δ) illustrate typical SPB orientations in mitotic (opposite the nucleolus [NLL]) and meiotic (next to the nucleolus) cells. This allows the distinction between mitotic and meiotic cells and confirms that rad50Δ cells underwent meiosis in the absence of LEs. (b) (Left top and middle) Quantification of LE classes 1a and 1b at different time points. Class 1b was more abundant in rad50S cells than in the WT. (Bottom left) Quantification of rad50Δ cells (without LEs) containing an SPB configuration indicative of meiosis. At later time points, cells started to form ascus and spore walls, making the cells resistant to lysis, which led to an artifactual underrepresentation of meiotic cells. (Right) Quantification of DAPI (4′,6-diamidino-2-phenylindole)-visualized elongated (horsetail) nuclei indicative of meiotic prophase. The percentage of cells with more than one nucleus indicates progression through the first and second meiotic divisions. All quantifications are based on at least 100 cells per time point.

FIG. 4.

The rad32^mre11-D65N mutant is defective for Rec12^Spo11 removal. (a) Analysis of spore viability epistasis between different rad32^mre11-D65N mutants. Note that the graph shows only the lower range (<0.15%) of spore viability. Error bars show standard deviations, and values are averages for three independent experiments. (b) Analysis of Rec12^Spo11 removal in different mutants at 34°C. Levels of covalently bound Rec12^Spo11 were increased in rad32^mre11-D65N mutant strains. The arrow indicates where the top and bottom fractions of the CsCl gradient were loaded. The bulk of the DNA was found in fractions 5, 6, and 7, which showed the strongest Rec12^Spo11 signals in rad50 mutants.

FIG. 5.

(a) A ctp1 deletion mutant is deficient in removing Rec12^Spo11 from the DNA in meiotic cells. The defect is comparable to that of rad50S and rad32^mre11-D65N strains. (b) Meiotic spore viability is strongly reduced in the ctp1Δ strain, similar to that of the rad32-D65N strain. (c) The ctp1Δ strain is proficient for ade6-M26 chromatin remodeling (black arrow).

FIG. 6.

Interpretation of the observed meiotic phenotypes of rad50Δ and rad50S mutants. Both rad50Δ and rad50S mutants (at a restrictive temperature) are deficient for the removal of covalently bound Rec12^Spo11 after meiotic DSB formation, leading to low spore viability. However, a small fraction of cells are able to remove Rec12^Spo11 (through an unknown mechanism), allowing repair of the DSBs and viable spore formation. For the rad50Δ mutant, these survivors show a strong reduction in recombination rates, suggesting that they survive through a nonrecombinogenic survival mechanism. The rad50S cells are proficient for meiotic recombination (once Rec12^Spo11 has been removed), and the surviving spores therefore show normal meiotic recombination levels.