Biochemical and Single-Molecule Fluorescence Characterization of MutS and MutS Homolog Protein-DNA Interactions

DeRocco, Vanessa Carolyn

Download PDF

Request Version for Screen Reader

Last Modified

March 21, 2019

Creator

DeRocco, Vanessa Carolyn
- Affiliation: College of Arts and Sciences, Department of Chemistry

Abstract

MutS and MutS homologs are the proteins within the prokaryote and eukaryote DNA mismatch repair pathways that are the responsible for recognizing single base-base mismatches or insertion/deletion errors in newly replicated DNA. Specific interactions between MutS and these DNA defects trigger a cascade of protein-protein interactions that ultimately results in repair of the DNA error. Mutations in the homologs of the MutS and MutL repair proteins involved the recognition and initiation of post replicative DNA mismatch repair are associated with ~80% of Hereditary Nonpolyposis Colorectal Cancer (HNPCC) occurrences. The mechanism by which MutS recognizes mismatch DNA and initiates of downstream repair is not well understood. In this dissertation, I present biochemical and single molecule fluorescence studies of Thermus aquaticus (Taq) MutS as well as human and yeast MutS heterodimer homologs (hMSH2-MSH6 and yMsh2-Msh6) protein-DNA interactions in an effort to better understand DNA mismatch recognition and repair. Single molecule fluorescence methodologies were employed to compare the MutS-DNA interactions of wild type Taq MutS with a mutant of Taq MutS, E41A. Previous work has shown that Taq MutS adopts a complex series of conformations when interaction with mismatch DNA with a bent state and an unbent state being the dominant states. In this work, the kinetics of interconversion among bending states was determined to vary widely for different mismatches. Further, the E41A mutant, which is known to have specific deficiencies in repair capability, demonstrates altered DNA bending kinetics on DNA mismatches that correlate with its repair deficiencies. Despite structural similarities, the biochemical properties of prokaryotic MutS and eukaryotic MSH2-MSH6 (MutSα) have been shown to vary. Characterization of MutSα-DNA interactions has been limited. In order to compare the prokaryote and eukaryote, the protein-DNA interactions of wild type human MutSα (hMutSα) protein as well as two HNPCC separation-of-function mutants, MSH2WT-MSH6T1219D and MSH2G674A-MSH6WT were characterized. In contrast to Taq MutS-DNA interactions, I observed few hMutα-DNA conformational changes suggesting a difference in the mechanism of MMR initiation between prokaryotes and eukaryotes. I further determined singular trapped conformational states for each HNPCC mutant that may be linked to MMR deficiency for each.

Date of publication