Saccharomyces cerevisiae MutLalpha is a mismatch repair endonuclease

Area of Science:

• Molecular Biology
• Genetics
• Biochemistry

Background:

• MutL homologs are essential for DNA mismatch repair and maintaining genetic stability.
• The endonuclease activity of human MutLalpha (MLH1-PMS2) depends on a specific PMS2 motif, conserved in yeast but absent in bacteria like E. coli.
• Bacterial MutL proteins utilize DNA methylation for strand directionality, unlike eukaryotic counterparts.

Purpose of the Study:

• To investigate the strand-directed endonuclease activity of yeast MutLalpha.
• To determine the role of the conserved DQHA(X)2E(X)4E motif in yeast MutLalpha function.
• To elucidate the mechanism of DNA incision by yeast MutLalpha in the context of mismatch repair.

Main Methods:

• In vitro assays to assess endonuclease activity of wild-type and mutant yeast MutLalpha.
• Genetic analysis in yeast to evaluate the in vivo consequences of mutations in the conserved motif.
• Biochemical experiments to examine ATPase activity and complex formation with other DNA repair proteins.

Main Results:

• Yeast MutLalpha functions as a strand-directed endonuclease, dependent on mismatch, yMutSalpha, yRFC, yPCNA, ATP, and a strand break.
• Amino acid substitutions in the PMS1 DQHA(X)2E(X)4E motif abolished in vitro endonuclease activity and caused significant genetic instability in vivo.
• These mutations did not affect yeast MutLalpha ATPase activity or its ability to form a ternary complex with yMutSalpha.

Conclusions:

• The conserved DQHA(X)2E(X)4E motif is critical for yeast MutLalpha endonuclease activity and genetic stability.
• Yeast MutLalpha employs a strand-directed incision mechanism distinct from bacterial MutL.
• Loaded yPCNA may direct yeast MutLalpha incision to the discontinuous strand of nicked DNA heteroduplexes.