Recruitment and Function of Mlh1-Pms1 in DNA Mismatch Repair

Mismatch Repair (MMR) is a highly conserved DNA repair pathway that repairs base-base mispairs and small insertion/deletion loops that frequently arise during DNA replication. Defects in MMR cause cancer, and studies in S. cerevisiae and E. coli have helped elucidate the function of MMR in humans. In E. coli, homodimeric MutS initially recognizes a DNA mispair, which then recruits homodimeric MutL and further downstream proteins. In S. cerevisiae, MMR is initiated by MutS-homologous complexes Msh2-Msh6 or Msh2-Msh3 binding a DNA mispair, which then recruit the MutL-homologous complex Mlh1-Pms1 to DNA. This interaction between these complexes is essential for MMR, though due to its dynamic and transient nature it has been difficult to study. Using Deuterium Exchange/Mass Spectrometry (DXMS) of E.coli MutS and MutL, I identified a putative interaction interface in the N-terminal hetero-dimer region of Mlh1-Pms1, though further biochemical analysis indicated that this region was likely not the interface region. While performing these studies, a published low-resolution crosslinked structure of the E. coli MutS and MutL identified a new putative interface region between MutS and MutL. I used this information to elucidate the interface between Msh2-Msh6 or Msh2-Msh3 and Mlh1-Pms1. Genetic assays determined mutations in this interface region of MLH1, but not PMS1, caused a null MMR phenotype, and biochemical assays concluded that it is the Mlh1 subunit that interacts with Msh2 and functions in the initiation of MMR. I also characterized mutations affecting the flexible linker region of Mlh1. I found point mutations and deletions that lead to a complete loss of MMR, and found that purified Mlh1-Pms1 mutants containing amino acid substitutions in this region are defective for endonuclease activity, revealing an unknown essential functional for this region of Mlh1 in MMR. Together, my work increases our understanding of the initiation of eukaryotic MMR, as well as how Mlh1-Pms1 leads to downstream repair