Genetic analysis of baker's yeast Msh4-Msh5 reveals a threshold crossover level for meiotic viability

Abstract

During meiosis, the Msh4-Msh5 complex is thought to stabilize single-end invasion intermediates that form during early stages of recombination and subsequently bind to Holliday junctions to facilitate crossover formation. To analyze Msh4-Msh5 function, we mutagenized 57 residues in Saccharomyces cerevisiae Msh4 and Msh5 that are either conserved across all Msh4/5 family members or are specific to Msh4 and Msh5. The Msh5 subunit appeared more sensitive to mutagenesis. We identified msh4 and msh5 threshold (msh4/5-t) mutants that showed wild-type spore viability and crossover interference but displayed, compared to wild-type, up to a two-fold decrease in crossing over on large and medium sized chromosomes (XV, VII, VIII). Crossing over on a small chromosome, however, approached wild-type levels. The msh4/5-t mutants also displayed synaptonemal complex assembly defects. A triple mutant containing a msh4/5-t allele and mutations that decreased meiotic double-strand break levels (spo11-HA) and crossover interference (pch2Δ) showed synergistic defects in spore viability. Together these results indicate that the baker's yeast meiotic cell does not require the ∼90 crossovers maintained by crossover homeostasis to form viable spores. They also show that Pch2-mediated crossover interference is important to maintain meiotic viability when crossovers become limiting.

Figure 1. Structure-function analysis of msh4, msh5 alleles.

(A) Comparison of domain organization of yeast Msh proteins with the Thermus aquaticus (Taq) MutS protein. The five domains (I–V) identified in yeast Msh proteins based on structural homology to Taq MutS are shown to scale . (B) Sequence alignment of Msh4 protein sequences from S. cerevisiae (YFL003C), H. sapiens (NM_002440), M. musculus (BC145838), A. thaliana (NM_117842) and C. elegans (AF178755) and Msh5 protein sequences from S. cerevisiae (YDL154W), H. sapiens (BC002498), M. musculus (NM_013600), A. thaliana (EF471448) and C. elegans (NM_070130). Representative residues from four different classes used for structure-function analysis are shown; Class 1 (Msh4, Msh5 specific); Class 2 (Msh4 specific); Class 3 (Msh5 specific) and Class 4 (Msh family specific). (C) Spore viability profiles of 57 msh4, msh5 mutations in the EAY background are shown with reference to the domain organization of the Msh4, Msh5 proteins. The height of each line corresponds to the spore viability of each mutant relative to wild-type and null. Four domains (II–V) in the Msh4, Msh5 proteins based on structural homology to MutS are shown .

Figure 2. The Msh5 subunit is more sensitive to mutagenesis.

(A) Comparison of spore viability of 29 msh4 and 28 msh5 mutants in ascending order in the EAY background. (B) Spore viability of conserved pairs of residues in Msh4, Msh5 ATP binding domain and DNA binding domain. msh4-G639A and msh5-G648A contain mutations analogous to ATP binding mutations in Msh2 while msh4-R676W and msh5-R685W contain mutations analogous to ATP hydrolysis mutations in Msh2. msh4-N532A, msh4-Y485A, msh4-L493A, msh4-L553A, and their matched mutations in Msh5 (msh5-D527A, msh5-Y480A, msh5-V488A, msh5-L548A) are conserved within the DNA binding domain (IV). The number of tetrads dissected for each strain is presented in Table 1.

Figure 3. Crossovers can be reduced to a threshold level without affecting spore viability.

Plot of spore viability versus genetic map distance on chromosome XV in 57 msh4, msh5 mutants in the EAY strain background. Wild-type, msh4Δ, and msh5Δ data were also plotted. The msh4/5-t (green font) and msh4/5-bt (blue font) alleles analyzed in greater depth are shown. Raw data are shown in Table 1.

Figure 4. Spore viability profile of wild-type and mutant strains in the NHY942/943 strain background.

The vertical axis shows the percentage of each tetrad class and the horizontal axis represents the number of viable spores in a tetrad. n: total number of tetrads dissected, SV: percentage spore viability. Data for wild-type, pch2Δ, spo11-HA and pch2Δ spo11-HA are from Zanders and Alani .

Figure 5. Cumulative genetic map distance in msh4/5 hypomorphs and double and triple mutations with pch2Δ and spo11-HA.

(A) Location of genetic markers assayed on chromosomes III, VII and VIII in the NHY strain background. Solid circle indicates the centromere. (B) Sum of the genetic map distance (from total spores and complete tetrads) over chromosomes III, VII and VIII in the NHY942/NHY943 strain background. Raw data are shown in Table 2. Data for wild-type, pch2Δ, spo11-HA, and pch2Δ spo11-HA are from Zanders and Alani .

Figure 6. Chromosome size-dependent loss of the meiotic crossover buffer in msh4/5-t mutants.

Cumulative genetic map distances for chromosomes III, VII, and VIII are shown separately for msh4/5 hypomorphs as well as their double and triple mutations with pch2Δ and spo11-HA.

Figure 7. msh4/5 hypomorphs are defective in Zip1 polymerization.

Meiotic chromosome spreads isolated from cells at 4 hr after induction into meiosis were incubated with antibodies to Zip1 and Msh5 and counterstained with DAPI. (A) Localization of Zip1 and Msh5 in wild-type, msh4-E276A, msh4-R676W and msh4Δ mutants. (B) Zip1, Msh5 localization in msh5-S416A, msh5-D532A, msh5-D539A and msh5Δ mutants.