The fission yeast BLM homolog Rqh1 promotes meiotic recombination

Abstract

RecQ helicases are found in organisms as diverse as bacteria, fungi, and mammals. These proteins promote genome stability, and mutations affecting human RecQ proteins underlie premature aging and cancer predisposition syndromes, including Bloom syndrome, caused by mutations affecting the BLM protein. In this study we show that mutants lacking the Rqh1 protein of the fission yeast Schizosaccharomyces pombe, a RecQ and BLM homolog, have substantially reduced meiotic recombination, both gene conversions and crossovers. The relative proportion of gene conversions having associated crossovers is unchanged from that in wild type. In rqh1 mutants, meiotic DNA double-strand breaks are formed and disappear with wild-type frequency and kinetics, and spore viability is only moderately reduced. Genetic analyses and the wild-type frequency of both intersister and interhomolog joint molecules argue against these phenotypes being explained by an increase in intersister recombination at the expense of interhomolog recombination. We suggest that Rqh1 extends hybrid DNA and biases the recombination outcome toward crossing over. Our results contrast dramatically with those from the budding yeast ortholog, Sgs1, which has a meiotic antirecombination function that suppresses recombination events involving more than two DNA duplexes. These observations underscore the multiple recombination functions of RecQ homologs and emphasize that even conserved proteins can be adapted to play different roles in different organisms.

Figure 1.—

srs2 and rqh1 null mutants have reduced crossover and gene conversion frequencies. (A) Crossover frequencies were measured in three intergenic intervals among the products of meiotic crosses and converted to centimorgans by the method of Haldane (1919). Data represent combined numbers from at least two independent experiments (537–600 spore colonies, total, for each cross), normalized to the wt genetic distance. Error bars indicate 95% binomial proportion confidence intervals calculated by the Wilson score interval method (Wilson 1927). The strains used for the lys3–ura1 measurements were: GP2 and GP850 (wt), GP5348 and GP5352 (srs2Δ), GP5350 and GP5356 (rqh1Δ). The strains used for both the ura4A⁺–tps16 and the tps16–arg1 measurements were: GP1040 and GP5495 (wt), GP5494 and GP5559 (srs2Δ), GP5493 and GP5557 (rqh1Δ). The wt genetic distances for lys3–ura1, ura4A⁺–tps16, and tps16–arg1 were 25, 17, and 55 cM, respectively. (B) Gene conversion frequencies at ade6 and ura1 were measured as the frequency of prototrophs in meiotic crosses involving point mutations of each gene (ade6-52 × ade6-M26 and ura1-61 × ura1-171). Data are the average of three independent experiments, normalized to the prototroph frequency observed in the wt crosses. Error bars indicate SEM (n = 3). The strains used in crosses for the ade6 measurements were: GP1040 and GP5495 (wt), GP5494 and GP5559 (srs2Δ), GP5493 and GP5557 (rqh1Δ). The strains used in crosses for the ura1 measurements were: GP850 and GP747 (wt), GP5348 and GP5354 (srs2Δ), GP5350 and GP5357 (rqh1Δ). The wt prototroph frequencies, per viable spore, for ura1 and ade6 were 0.00020 and 0.0033, respectively.

Figure 1.—

srs2 and rqh1 null mutants have reduced crossover and gene conversion frequencies. (A) Crossover frequencies were measured in three intergenic intervals among the products of meiotic crosses and converted to centimorgans by the method of Haldane (1919). Data represent combined numbers from at least two independent experiments (537–600 spore colonies, total, for each cross), normalized to the wt genetic distance. Error bars indicate 95% binomial proportion confidence intervals calculated by the Wilson score interval method (Wilson 1927). The strains used for the lys3–ura1 measurements were: GP2 and GP850 (wt), GP5348 and GP5352 (srs2Δ), GP5350 and GP5356 (rqh1Δ). The strains used for both the ura4A⁺–tps16 and the tps16–arg1 measurements were: GP1040 and GP5495 (wt), GP5494 and GP5559 (srs2Δ), GP5493 and GP5557 (rqh1Δ). The wt genetic distances for lys3–ura1, ura4A⁺–tps16, and tps16–arg1 were 25, 17, and 55 cM, respectively. (B) Gene conversion frequencies at ade6 and ura1 were measured as the frequency of prototrophs in meiotic crosses involving point mutations of each gene (ade6-52 × ade6-M26 and ura1-61 × ura1-171). Data are the average of three independent experiments, normalized to the prototroph frequency observed in the wt crosses. Error bars indicate SEM (n = 3). The strains used in crosses for the ade6 measurements were: GP1040 and GP5495 (wt), GP5494 and GP5559 (srs2Δ), GP5493 and GP5557 (rqh1Δ). The strains used in crosses for the ura1 measurements were: GP850 and GP747 (wt), GP5348 and GP5354 (srs2Δ), GP5350 and GP5357 (rqh1Δ). The wt prototroph frequencies, per viable spore, for ura1 and ade6 were 0.00020 and 0.0033, respectively.

Figure 2.—

The srs2 and rqh1 null mutations have no effect on the frequency with which gene conversions at ade6 are accompanied by crossovers between flanking markers. Gene convertants were selected as prototrophs arising in meiotic crosses involving the ade6-52 and ade6-M26 point mutations. Among these gene convertants (589–605 spore colonies, total, for each cross), the percentage of spores displaying crossovers between the markers ura4A⁺ and tps16, flanking ade6, was measured. Percentages represent combined data from two independent experiments. Error bars indicate 95% binomial proportion confidence intervals calculated by the Wilson score interval method. The strains used for the crosses were: GP1040 and GP5495 (wt), GP5494 and GP5559 (srs2Δ), GP5493 and GP5557 (rqh1Δ).

Figure 3.—

The rqh1 null mutation has moderately reduced spore viability and viable spore yield, while there is no significant effect of the srs2 null mutation on spore viability. Left: spore viability was measured as the percentage of spores, relative to wt, successfully germinating into a visible colony after gridding by micromanipulation on YEA-5S plates. The wt spore viability was 78%. From each of two independent crosses 152 spores were micromanipulated for each genotype. Error bars indicate 95% binomial proportion confidence intervals calculated by the Wilson score interval method using the combined data. Crosses were GP2 × GP19 (wt), GP5352 × GP5353 (srs2Δ), GP5355 × GP5356 (rqh1Δ), and GP5868 × GP5869 (rec12Δ). Right: viable spore yield was measured as the number of viable spores produced per number of cells of the less numerous parent in the cross. The strains used were GP2 × GP19 (wt) and GP5355 × GP5356 (rqh1Δ). Data, expressed as a percentage of rqh1⁺, which produced 7.2 spores per less numerous cell, are the mean of at least four independent experiments ± SEM.

Figure 4.—

DSB formation and repair are normal in the rqh1 null mutant. (A) Southern blots of NotI fragment J on chromosome I, probed from the right side, showing formation and repair of DSBs at the mbs1 and mbs2 meiotic DSB hotspots in the wt and rqh1Δ backgrounds. Synchronous meiosis was induced by temperature-shifting pat1-114 cultures, and DNA, isolated at the times indicated, was analyzed as described by Young et al. (2002). The strains used were GP65 (wt) and GP5263 (rqh1Δ). The asterisk indicates an additional break site introduced by the kan^R substitution at the rqh1 locus ∼70 kb from mbs1. The markers are MidRange I PFG (right) and Lambda Ladder PFG marker (left) (New England Biolabs). (B) DSB frequencies at the mbs1 and mbs2 hotspot sites, from DNA samples harvested 4 hr after meiotic induction (when breaks are close to maximal) and probed as in Figure 4A. Quantitation was done using a phosphorimager. For each genetic background, values are from one h⁺ and one h⁻ induction, each assayed on multiple Southern blots. The mean ± SEM is shown; n = 5 for wt and 7 for rqh1Δ. The strains used were: GP65 and GP64 (wt); GP5263 and GP5489 (rqh1Δ).

Figure 5.—

Frequency of meiotic recombination between tandemly duplicated copies of the ade6 gene, one marked with M26 and the other with 469, to give Ade⁺ spores. In this assay, recombination cannot use the homologous chromosome as a template, since ade6 is deleted from one parent. The tandem 1.9-kb duplications of ade6 are separated by ∼4.4 kb of vector DNA and the ura4 gene, cloned into the pUC18 vector (Schuchert and Kohli 1988). The strains used were GP5837 and GP5860 (wt) and GP5835 and GP5859 (rqh1Δ). Data are the mean of four independent experiments ± SEM.

Figure 6.—

The rqh1Δ mutation does not affect the frequency of intersister vs. interhomolog joint molecules at mbs1 but does substantially reduce the frequency of crossovers at mbs1. (A) The mbs1 region of chromosome I from diploids heterozygous for PmlI and XbaI restriction sites, flanking the mbs1 hotspot. PvuII, PmlI (“L”), and XbaI (“R”) digestion and probing as shown reveal two parental (9.2- and 6.8-kb) and two recombinant (11.2- and 4.8-kb) fragments. Solid arrows indicate DSB sites. (B) Predicted migration during 2-D gel electrophoresis of mbs1-probed DNA digested with PvuII, PmlI, and XbaI (Figure 6A). X-shaped molecules arise during recombination and can be distinguished on the basis of their sizes. Intersister joint molecules (IS) arising from either parent (P1 X or P2 X) migrate as spikes, whereas interhomolog JMs (IH X) migrate as two spots connected by an arc and run at a position between the two intersister spikes (Cromie et al. 2006). (C) DNA isolated 5 hr after meiotic induction of strains GP5086 (rqh1⁺) and GP6489 (rqh1Δ) was analyzed as in Figure 6B. Solid arrowheads indicate X-form JM species. The thin arrow indicates a partial digestion product or a crossover product. The mean intersister (IS) and interhomolog (IH) JM frequencies are indicated. Values are means of at least two independent experiments ± SEM (n = 3, GP5086) or with the range shown (n = 2, GP6489). The GP5086 data were previously published in Cromie et al. (2006). (D) DNA was isolated at the indicated times after meiotic induction of strains GP5086 (rqh1⁺) and GP6489 (rqh1Δ) and analyzed by gel electrophoresis and Southern blotting with digestion and probing at mbs1 as in Figure 6A. Asterisks indicate cross-hybridizing DNA not specific to meiosis. The mean crossover frequency among DNA species at 6 hr was calculated as 2 × R2/total. Values are means of at least two independent experiments ± SEM (n = 3, GP5086) or with the range shown (n = 2, GP6489). The GP5086 data were previously published in Cromie et al. (2006).