The Mechanistic Contributions of Mismatch Repair (MMR) Proteins to Trinucleotide Repeat Expansion
Williams, Gregory Michael
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Mismatch repair (MMR) is a highly conserved DNA repair pathway that recognizes mispairs that occur spontaneously during DNA replication and coordinates their repair. It is thus an important pathway to maintain genome stability. In Saccharomyces cerevisiae , MutS homologues (Msh) Msh2-Msh6 or Msh2-Msh3 initiate MMR. Msh2-Msh6 initiates repair of base-base mismatches and Msh2-Msh3 initiates repair of insertion/deletion loops (IDLs). Both of these complexes have an overlapping specificity for small IDLs. In addition to MMR, both Msh2-Msh6 and Msh2-Msh3 are involved in other DNA repair pathways. Trinucleotide repeat (TNR) expansions are the underlying cause of over 40 neurodegenerative and neuromuscular diseases, including Huntington's disease and Myotonic Dystrophy. Genetic evidence has implicated the DNA mismatch repair (MMR) system as an important factor in promoting TNR expansions. Although the MMR factor Msh2-Msh3 typically promotes genome stability during DNA repair, it has an unexpected, noncanonical role that destabilizes CAG and CTG trinucleotide repeats, stimulating their expansion. Working in the model system Saccharomyces cerevisiae , we tested the role of Msh2-Msh3 as a driver of TNR expansion and demonstrated that Msh2-Msh3 promotes TNR expansions both in vivo and in vitro ( Chapter 2 ). We hypothesized that Msh2-Msh3 promotes expansions through binding and stabilization of TNR secondary structures that can form during Okazaki fragment processing in DNA replication. Modeling of human data has indicated that TNR tracts are increasingly likely to expand as they increase in size, but this has not been tested experimentally. I examined individual threshold-length TNR tracts in vivo as a function of time in the presence and the absence of Msh2-Msh3. I demonstrated, for the first time, that these TNR tracts are highly dynamic (lengthen) in the presence of Msh2-Msh3, but substantially stabilized in its absence, indicating that Msh2-Msh3 plays an important role in shifting the expansion-contraction equilibrium toward expansion in the early stages of TNR tract expansion ( Chapter 3 ). In order to uncover a better understanding of how MMR factors contribute mechanistically to TNR expansion, I am currently testing deletions of and mutations in Mlh1-Pms1 and Mlh1-Mlh3, two MMR factors that interact with Msh2-Msh3, as well as Msh2-Msh3 mutants that knockout specific functions of the complex such as nucleotide binding and hydrolysis. I have thus far shown that MLH complexes can promote the expansion of CAG and CTG repeat tracts. I have further demonstrated that the MLH complexes may mechanistically contribute to TNR expansions through their latent endonuclease activity. These experiments will further our mechanistic understanding of MutL and MutS complexes in TNR expansion pathway and test current models of replication-mediated TNR expansion ( Chapter 4 ).