A Mutation in the Putative MLH3 Endonuclease Domain Confers a Defect in Both Mismatch Repair and Meiosis in Saccharomyces cerevisiae

摘要

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)2E(X)4E that is found in a subset of MLH proteins predicted to have endonuclease activities ([Kadyrov et al . 2006][1]). 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][1], [2007][2]; [Erdeniz et al . 2007][3]). 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 mlh3 Δ. 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. [1]: #ref-25 [2]: #ref-26 [3]: #ref-10