The pch2Delta mutation in baker's yeast alters meiotic crossover levels and confers a defect in crossover interference

Abstract

Pch2 is a widely conserved protein that is required in baker's yeast for the organization of meiotic chromosome axes into specific domains. We provide four lines of evidence suggesting that it regulates the formation and distribution of crossover events required to promote chromosome segregation at Meiosis I. First, pch2Delta mutants display wild-type crossover levels on a small (III) chromosome, but increased levels on larger (VII, VIII, XV) chromosomes. Second, pch2Delta mutants show defects in crossover interference. Third, crossovers observed in pch2Delta require both Msh4-Msh5 and Mms4-Mus81 functions. Lastly, the pch2Delta mutation decreases spore viability and disrupts crossover interference in spo11 hypomorph strains that have reduced levels of meiosis-induced double-strand breaks. Based on these and previous observations, we propose a model in which Pch2 functions at an early step in crossover control to ensure that every homolog pair receives an obligate crossover.

Figure 1. pch2Δ has increased levels of meiotic COs on the large chromosome XV.

Recombination levels in four genetic intervals were analyzed on chromosome XV in the EAY1108/EAY1112 strain background (A). CO frequencies were calculated in cM from tetrads (B) and as recombination frequencies in spores (C). See Table 1 and Table S3 for raw data and statistical analyses.

Figure 2. pch2Δ has increased meiotic CO levels on chromosomes VII and VIII, but not the small chromosome III.

(A) The organization of genetic markers assayed on a small (III), medium (VIII), and large (VII) chromosome in the NHY942/NHY943 strain background is shown. (B) CO frequencies in cM were calculated from four-spore viable tetrads in wild-type, pch2Δ/pch2Δ, spo11-HA/spo11-HA and pch2Δ spo11-HA/spo11-HA strains. See Table 1 for raw data and statistical analyses.

Figure 3. Pch2 promotes spore viability in spo11 hypomorphs.

(A) Spore viability distributions of wild-type and mutant strains in the NHY942/NHY943 strain background are displayed. The X-axes represent the number of viable spores per tetrad and the Y-axes represent the percent of tetrads comprising each class. The total number of tetrads dissected (n) and the overall percent spore viability (% SV) are shown. (B) Bar graph showing spore viability in wild-type (gray) and pch2Δ (black) mutants containing the indicated spo11 mutations. The SPO11/SPO11, spo11-HA/spo11-HA, spo11-HA/spo11yf-HA, and spo11da-HA/spo11da-HA alleles confer 100, 80, 30 and 20% total DSB levels, respectively, in the PCH2 background .

Figure 4. CO interference is reduced in absence of Pch2.

Interference was measured using the method of Malkova et al. , for strains in the EAY (A) and NHY (B) strain backgrounds. For each interval, the map distance was separately calculated for tetrads in which the adjacent interval had (+) or did not have (−) a CO event. If the two map distances are significantly different (G-test), then CO interference is present between the two intervals. The ratio of the CO+ to CO− map distances gives the strength of the interference, with values nearer to zero indicating stronger interference. The average interference ratio between adjacent genetic intervals is shown above the intervals. Solid lines indicate interference was statistically significant when either interval was used as the reference. The broken lines indicate interference was not statistically significant when one or both intervals were used as the reference. See Table S2 for raw data and statistical analyses.

Figure 5. pch2 does not appear to have increased levels of meiotic DSBs.

Southern blots were performed to measure meiotic DSBs in dmc1Δ, pch2Δ dmc1Δ, spo11-HA dmc1Δ, and pch2Δ spo11-HA dmc1Δ strains. For the YCR048w hotspot on chromosome III (A) DNA was digested with BglII and probed with a chromosome III fragment (SGD coordinates 215,422-216,703, [63]). For the CEN15 hotspot (B), DNA was digested with SphI and NheI and probed with a chromosome XV fragment (SGD coordinates 331,713-332,402, [42]). The parental bands are marked with asterisks and arrows show bands that form due to DSB formation. Approximate sizes for all bands are shown ,. The lanes on the CEN15 blot have been reordered for easy comparison of the two strains. (C) Analysis of DSBs at the YCR048W hotspot at T = 3.5 and 7 hrs in spo11-HA dmc1Δ (EAY2562/EAY2563) and pch2Δ spo11-HA dmc1Δ (EAY2564/EAY2565) strains. A representative blot is shown. In side-by-side experiments the DSB levels at T = 7 hrs in pch2Δ spo11-HA dmc1Δ ranged from 30-90% (30, 61, 72, 76, 80, 89, and 90%) of the levels observed in spo11-HA dmc1Δ.

Figure 6. The pch2 Δ MI delay is suppressed by the spo11-HA hypomorph.

Synchronous cultures were induced to undergo meiosis and progression past MI (MI±MII) was assayed in the NHY background for wild-type (diamonds), spo11-HA (open squares), pch2Δ (triangles), and pch2Δ spo11-HA (stars; Materials and Methods). A representative time course is shown.

Figure 7. Model for interference-regulation of the CO vs. NCO decision.

We propose that Pch2 acts to inhibit CO designation in a chromosomal region in response to a prior crossover-designated recombination event in that region. See text for details.