Genetic analysis of mlh3 mutations reveals interactions between crossover promoting factors during meiosis in baker's yeast

Abstract

Crossing over between homologous chromosomes occurs during the prophase of meiosis I and is critical for chromosome segregation. In baker's yeast, two heterodimeric complexes, Msh4-Msh5 and Mlh1-Mlh3, act in meiosis to promote interference-dependent crossing over. Mlh1-Mlh3 also plays a role in DNA mismatch repair (MMR) by interacting with Msh2-Msh3 to repair insertion and deletion mutations. Mlh3 contains an ATP-binding domain that is highly conserved among MLH proteins. To explore roles for Mlh3 in meiosis and MMR, we performed a structure-function analysis of eight mlh3 ATPase mutants. In contrast to previous work, our data suggest that ATP hydrolysis by both Mlh1 and Mlh3 is important for both meiotic and MMR functions. In meiotic assays, these mutants showed a roughly linear relationship between spore viability and genetic map distance. To further understand the relationship between crossing over and meiotic viability, we analyzed crossing over on four chromosomes of varying lengths in mlh3Δ mms4Δ strains and observed strong decreases (6- to 17-fold) in crossing over in all intervals. Curiously, mlh3Δ mms4Δ double mutants displayed spore viability levels that were greater than observed in mms4Δ strains that show modest defects in crossing over. The viability in double mutants also appeared greater than would be expected for strains that show such severe defects in crossing over. Together, these observations provide insights for how Mlh1-Mlh3 acts in crossover resolution and MMR and for how chromosome segregation in Meiosis I can occur in the absence of crossing over.

Keywords: DNA mismatch repair; Mlh1-Mlh3; Msh4-Msh5; crossing over; meiotic recombination.

Figure 1

The ATPase domain of Mlh3 is highly conserved across eukaryotic species and within the MLH protein family. (A) Location of the mlh3 mutations analyzed in this study with respect to Homo sapiens, S. cerevisiae, and Mus musculus protein sequences. Conserved residues are highlighted in bold. (B) Location of the mlh3 mutations created with respect to the conserved ATPase domains in the Saccharomyces cerevisiae MLH family of proteins (Ban and Yang 1998; Tran and Liskay 2000). ATPase domain IV is not shown.•, locations of mlh3 alleles analyzed in this study.

Figure 2

Cumulative genetic distances for wild type, mlh3Δ, mms4Δ, and mlh3Δ mms4Δ on four chromosomes. (A) Location of genetic markers used to determine map distances in the NHY942/NHY943 background for chromosomes III, VII, VIII, and the EAY1108/EAY1112 background for chromosome XV. (B) The cumulative genetic distance for each chromosome is shown for both complete tetrad data (black bars) and single spore data (white bars). Raw data are shown in Table 7. Data for wild type for chromosomes III, VII, and VIII are from Zanders and Alani (2009). Data for wild type and mms4Δ for chromosome XV are from Argueso et al. (2004). Data for mlh3Δ and mlh3Δ mms4Δ on chromosome XV are from Nishant et al. (2008). For chromosome III, the physical distances (end of the marker gene to the beginning of the next, in KB) are: HIS4-LEU2, 23; LEU2-CEN3, 22; CEN3-MAT, 90. For chromosome VII, the physical distances are: LYS5-MET13, 56, MET13-CYH2, 36; CYH2-TRP5, 135. For chromosome VIII, the physical distances are: CEN8-THR1, 54; THR1-CUP1, 52. For chromosome XV, the physical distances are: URA3-LEU2, 136; LEU2-LYS2, 43; LYS2-ADE2, 59; ADE2-HIS3, 157.

Figure 3

mlh3 strains show a roughly linear relationship between crossing over and spore viability. Spore viabilities are plotted vs. genetic map distances on chromosome XV for eight mlh3 ATP binding domain mutations, wild type (open triangle), and mlh3Δ (open circle).

Figure 4

Spore viability profile of wild-type and select mutants. The horizontal axis shows the number of viable spores per tetrad, and the vertical axis shows the percentage of tetrads in each class. n, the total number of tetrads dissected, and percent spore viability are shown. Data for wild-type, mlh3Δ, mms4Δ, and mlh3Δ mms4Δ are from the NHY942/943 background (Tables 6 and 7; the remaining data are from the EAY1108/1112 background (Tables 4 and 5).

Figure 5

Model of crossover pathways during meiosis. A summary of the crossover pathways are shown. In wild-type cells (left), DSBs are made and resected, and initial single-end invasion intermediates can be dissolved by Sgs1−dependent mechanisms, leading to noncrossovers. Single-end invasion intermediates that are not resolved as noncrossovers can proceed through the Mus81-Mms4 interference-independent pathway, leading to crossovers, or Msh4-Msh5 can stabilize the SEI in an interference-dependent mechanism. These stabilized joint molecules undergo crossover placement decisions, and are subsequently resolved in an Mlh1-Mlh3-dependent manner. In the absence of Mlh3 and Mms4 (right), initial recombination events occur as in wild type. However, due to the lack of the major Mlh1-Mlh3 and Mus81-Mms4 resolvase functions, other pathways are activated, including Sgs1-dependent resolution to form noncrossovers and other resolution activities (e.g., Slx-Slx4, Yen1), resulting in a larger number of events being resolved into noncrossovers.