Crossover invariance determined by partner choice for meiotic DNA break repair

Abstract

Crossovers between meiotic homologs are crucial for their proper segregation, and crossover number and position are carefully controlled. Crossover homeostasis in budding yeast maintains crossovers at the expense of noncrossovers when double-strand DNA break (DSB) frequency is reduced. The mechanism of maintaining constant crossover levels in other species has been unknown. Here we investigate in fission yeast a different aspect of crossover control--the near invariance of crossover frequency per kb of DNA despite large variations in DSB intensity across the genome. Crossover invariance involves the choice of sister chromatid versus homolog for DSB repair. At strong DSB hotspots, intersister repair outnumbers interhomolog repair approximately 3:1, but our genetic and physical data indicate the converse in DSB-cold regions. This unanticipated mechanism of crossover control may operate in many species and explain, for example, the large excess of DSBs over crossovers and the repair of DSBs on unpaired chromosomes in diverse species.

Figure 1. Model for Meiotic Recombination in S. pombe

Meiotic replication (not shown) produces sister chromatids, each a DNA duplex (thick lines, red and blue distinguishing the homologs). 1. A DSB is made in one duplex by Rec12 (with assistance by other proteins), and Rec12 (green ball) remains covalently linked to the 5’ ends of each DNA strand (thinner lines). 2. The MRN complex (Rad32-Rad50-Nbs1) with Ctp1 clips off Rec12 and resects one DNA strand to form long ss DNA with a 3’-end. 3. This ss DNA forms a nucleoprotein filament with Rad51, and strand invasion, aided by Rhp55-Rhp57, is promoted in three possible ways to form single Holliday junctions (HJs). At strong DSB hotspots Rad22-Rti1 promotes intersister HJ formation, and Swi5-Sfr1 promotes interhomolog HJ formation; both reactions are independent of Dmc1. Rad22-Rti1 plays a minor role in interhomolog gene conversion, perhaps by SDSA (Octobre et al., 2008). In DSB-poor regions, Swi5-Sfr1 and Dmc1 promote interhomolog HJ formation. 4. The HJs are resolved by Mus81-Eme1 into crossovers as shown or non-crossovers (not shown). The crossovers aid chromosome segregation at the first meiotic division and promote genetic diversification. See Cromie and Smith (2008) and Milman et al. (2009) for references and further discussion.

Figure 2. Holliday Junction-formation at the DSB Hotspot mbs1 Is Dependent on Rad51 and Its Mediators But Is Independent of Dmc1

DNA of meiotically induced cells with the indicated mutations was digested with PvuII, separated by two-dimensional gel electrophoresis, and Southern blot-hybridized with a ds DNA probe specific for mbs1 (see Figure 3, upper left panel for diagram). Images of Southern blots of DNA from HJ resolvase-proficient (mus81⁺) strains show the formation and repair of HJs from the start of meiotic induction (0 hr). The corresponding graph shows the quantification of branched DNA recombination intermediates indicated by the dashed lines (4 hr panel, top row); these recombination intermediates migrate above the linear DNA arc and are formed after 3 hr, when replication is complete. The quantification of replication intermediates (dashed lines in 2.5 hr panel, top row) is omitted here for clarity (see Figure S2A for the complete time-course). Replication and recombination intermediates are inferred from the timing of DNA replication (Figures S1), dependence on Rec12, and accumulation in mus81Δ mutants (Cromie et al. 2006). The half-hour delay in maximal HJ abundance in the dmc1Δ mutants is within our experimental error. Each measurement is the mean of two independent meiotic inductions, and nearly all values are within 20% of their respective mean; error bars are omitted for clarity. See also Figures S1, S2, and S3.

Figure 3. Swi5-Sfr1 Is Necessary for the Formation of Interhomolog, but Not Intersister, HJs at the DSB Hotspot mbs1

The relative amounts of IH and IS HJs in the indicated mutants were determined as in Figure 2 using diploids with heterozygous restriction sites as indicated in the diagram in the upper left panel. The black bars at mbs1 indicate the ds DNA probe. IH and IS HJs were determined by differences in their masses, 18.4 and 13.6 kb for IS HJs, and an intermediate mass of 16 kb for IH HJs. Parental fragments are 9.2 kb (P1) and 6.8 kb (P2). Gel images from 4.5 or 5 hr (the time of maximal HJs) for the indicated mutants are shown. Red arrows indicate IS HJs; blue arrows, IH HJs. Quantification of HJs in 2 – 5 experiments (a single experiment for sfr1Δ) is displayed on the bar graph; data are the mean, and the error bars indicate the range or SEM. The ratio of IS:IH HJs is given below for comparison. See also Figures S1, S2, and S4.

Figure 4. Crossover DNA at the DSB Hotspot mbs1 Is Dependent on Rad51 and Its Mediators, but Not on Dmc1

The level of crossover DNA at mbs1 was measured by the accumulation of the R2 recombinant DNA fragment (black arrowhead; see Figure 3, upper left panel for diagram). Crossover frequency is 2 × (R2 DNA)/total DNA. Each measurement is the average of the crossover DNA fragment at 6 or 7 hr in two independent meiotic inductions (one for sfr1Δ); the error bars indicate the range. Based on tetrad analyses, the residual level of crossover DNA in rad51Δ, swi5Δ rhp57Δ, and mus81Δ mutants can be accounted for by gene conversion of the right-hand marker (Cromie et al., 2005). See also Figures S1, S2, and S5.

Figure 5. HJ Formation in DSB-poor Regions Requires Dmc1

(A) DNA from the indicated mutants extracted 5 hr after meiotic induction was digested with PvuII, separated by two-dimensional gel electrophoresis, and probed for HJs at the positions shown in (C). (B) The fraction (%) of total probed DNA in the position of HJs was determined for blots in (A) or similar blots. Data are the means of 2 – 3 determinations; SEM is <20% of the mean. (C) Map of the left portion of NotI fragment J on chromosome I shows genes used for crosses in Table 1B, the positions (indicated by horizontal brackets labeled a, b, and c) of the restriction fragments analyzed in (A), and the level of Rec12-DNA covalent linkages (relative DSB frequency) in a rad50S strain 5 hr after meiotic induction, normalized to the genome median (Cromie et al., 2007; Hyppa et al., 2008). See also Figure S1.

Figure 6. Model for Crossover Invariance by Differential Choice of Homolog vs. Sister Chromatid for DSB Repair

DSB repair at strong DSB hotspots is predominantly with the sister chromatid and therefore yields few crossovers per DSB. At weaker DSB sites repair is predominantly with the homolog and yields more crossovers per DSB. The result is a more uniform distribution of crossovers (nearly constant cM/kb; crossover invariance) than of DSBs, as observed (Young et al., 2002; Table 1B). The proteins required for DSB repair are also differential, as indicated (see Figure 1).