Ensemble and single-molecule fluorescence studies of DNA mismatch repair initiation by MutS Public Deposited

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  • March 20, 2019
  • Sass, Lauryn E.
    • Affiliation: College of Arts and Sciences, Department of Chemistry
  • Single-molecule techniques have exploded in recent years and have become powerful in revealing the dynamic structure-function relationships of biological systems. In this dissertation, I have developed and applied ensemble and single-molecule fluorescence assays to observe the dynamics of mismatch repair initiation by repair enzyme MutS. Models of DNA mismatch repair initiation by MutS have been established, and the foundations of these models typically make comparisons between conformational changes in the DNA and the protein and mismatch repair signaling by MutS. The DNA bending model suggests that conformation changes induced in mismatched DNA serve as the method in which MutS recognizes a mismatch and signals repair. However, there was no evidence of a dynamic equilibrium of DNA bending and unbending induced by MutS. Single-molecule fluorescence assays were employed to follow this dynamic equilibrium between DNA bending and unbending induced by MutS. Results reveal that mismatched DNA-MutS complexes are, in fact, very dynamic, sampling a number of different DNA conformations with varied relative stabilities. These protein-DNA dynamics may be essential for mismatch recognition and repair signaling by MutS. MutS is able to induce many different DNA conformations in mismatched DNA, and the conformational dynamics of the complexes vary depending on the mismatch. These observations suggest that conformational dynamics helps MutS to perform all of its functions, including recognizing a number of different base-base mismatches and base insertions/deletions, recruiting mismatch repair cofactors to signal repair, and signaling cell cycle arrest in response to DNA damage. Increased DNA dynamics observed for MutS with a mutation in the conserved binding domain complement in vivo assays of mismatch repair and are consistent with observations that dynamic DNA conformational sampling serves as an important mechanism that MutS uses to signal repair. DNA conformational fluctuations were also induced by yeast MutSalpha, suggesting that eukaryotes employ similar protein-DNA dynamics in mismatch repair initiation by MutS. These results develop a correlation between protein-DNA dynamics and biological function and begin to demystify how MutS is capable of performing so many different tasks in the cell.
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  • Erie, Dorothy
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