Mismatch repair MLH complexes make distinct contributions to post-replicative mismatch repair versus trinucleotide repeat expansions

Mismatch repair (MMR) is a highly conserved DNA repair pathway that promotes genome stability by directing the repair of errors in DNA replication. In <i>Saccharomyces cerevisiae</i>, MMR is initiated by either Msh2-Msh3 or Msh2-Msh6, via recognition of insertion deletion loops (IDLs; up to ~ 17 nucleotides) and misincorporation events, respectively. Both complexes recognize and bind small (1-2 nucleotide) IDLs. Once bound, MSH complexes recruit one or more downstream MLH complexes to continue repair: Mlh1-Pms1, Mlh1-Mlh2 and/or Mlh1-Mlh3. Msh2-Msh3 also promotes <i>CAG</i> trinucleotide repeat (TNR) expansions through specific DNAbinding to TNR DNA structures, followed by recruitment of MLH complexes. These expansions lead to genome instability that causes neurodegenerative diseases such as Huntington's Disease in humans. Here, we defined a hierarchy of MLH function in these Msh2-Msh3mediated pathways <i>in vivo</i> in <i>S. cerevisiae</i>. We determined that Mlh1-Pms1 is the primary MLH complex required in Msh2-Msh3-mediated MMR. In contrast, all three MLH complexes were required to promote <i>CAG</i> expansions, with loss of Mlh1-Pms1 or Mlh1-Mlh2 exhibiting the strongest effects. Mutations in <i>PMS1</i> and <i>MLH3</i> were synergistic. We propose a model in which Mlh1-Pms1 is primarily responsible for "appropriate" Msh2-Msh3-mediated MMR, while all three MLH complexes collaborate specifically in the presence of <i>CAG</i> structure, to promote a "pathogenic" Msh2-Msh3-mediated pathway that leads to expansions. Our model highlights the importance of DNA structure-dependent conformations in modulating MLH function.