A mutation in the putative MLH3 endonuclease domain confers a defect in both mismatch repair and meiosis in Saccharomyces cerevisiae.
Interference-dependent crossing over in yeast and mammalian meioses involves the mismatch repair protein homologs MSH4-MSH5 and MLH1-MLH3. The MLH3 protein contains a highly conserved metal-binding motif DQHA(X)(2)E(X)(4)E that is found in a subset of MLH proteins predicted to have endonuclease activities (Kadyrov et al. 2006). Mutations within this motif in human PMS2 and Saccharomyces cerevisiae PMS1 disrupted the endonuclease and mismatch repair activities of MLH1-PMS2 and MLH1-PMS1, respectively (Kadyrov et al. 2006, 2007; Erdeniz et al. 2007). As a first step in determining whether such an activity is required during meiosis, we made mutations in the MLH3 putative endonuclease domain motif (-D523N, -E529K) and found that single and double mutations conferred mlh3-null-like defects with respect to meiotic spore viability and crossing over. Yeast two-hybrid and chromatography analyses showed that the interaction between MLH1 and mlh3-D523N was maintained, suggesting that the mlh3-D523N mutation did not disrupt the stability of MLH3. The mlh3-D523N mutant also displayed a mutator phenotype in vegetative growth that was similar to mlh3Delta. Overexpression of this allele conferred a dominant-negative phenotype with respect to mismatch repair. These studies suggest that the putative endonuclease domain of MLH3 plays an important role in facilitating mismatch repair and meiotic crossing over.
Nishant
K T
KT
Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853-2703, USA.
Plys
Aaron J
AJ
Alani
Eric
E
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000595:Amino Acid Sequence; D003434:Crossing Over, Genetic; D053843:DNA Mismatch Repair; D004720:Endonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016368:Frameshift Mutation; D015870:Gene Expression; D005800:Genes, Fungal; D008540:Meiosis / Q000235:genetics / Q000502:physiology; D008969:Molecular Sequence Data; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins; D009154:Mutation; D010641:Phenotype; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013172:Spores, Fungal / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
D048868:Adaptor Proteins, Signal Transducing; C088859:MLH1 protein, S cerevisiae; C492408:MLH3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D004720:Endonucleases; D000070957:MutL Protein Homolog 1; D000070956:MutL Proteins