English (United States)
DNA mismatch repair (MMR) is the highly conserved process responsible for identifying and correcting errors generated during DNA replication and is therefore essential for maintaining genome stability in nearly all living organisms. Consequently, MMR malfunctions result in increased spontaneous mutagenesis and the formation of cancer in mammals, highlighted by Lynch syndrome (LS), a hereditary predisposition to colorectal and other cancers. MMR is initiated by MutS, a protein responsible for recognizing post-replicative errors in DNA and signaling to downstream repair proteins. This study examines cancer-linked single amino acid mutants of the human MutS protein (using Thermus aquaticus MutS as a model system) in order to understand how these changes alter protein structure and function and thereby disrupt MMR. This work focuses on monitoring the timing and conformational dynamics of MutS as it utilizes ATP to work on DNA in order to construct a complete mechanism of action for each mutant protein. Preliminary kinetic analysis of seven T. aquaticus MutS mutants (T113R, G222D, F243S, Y244A, I400G, G434R, and L533R) homologous to human MutS variants commonly found in LS patients was carried out to assess their effect on the MutS mechanism. Subtle changes in activity could impact how well the mutant proteins recognize errors and initiate MMR. Several fluorescence-based assays were utilized to monitor specific steps in the MutS mechanism and identify any dissimilarities between the mutant proteins and wildtype. So far, a few key differences are discernible: Y244A and I400G are compromised in their ability to form the sliding clamp conformation, a critical intermediate in downstream signaling of repair, which indicates the uncoupling of their DNA binding and ATPase activity; T113R exhibits increased non-specific binding to DNA, potentially conferring reduced mismatch specificity; and G434R binds mismatched DNA tighter and with a higher affinity, possibly altering its initiation of MMR. This work advances our proof-of-principle study showing that detailed analysis of the structure, dynamics, and catalytic activities of individual MutS mutants is both feasible and can reveal critical information for understanding the molecular basis of Lynch syndrome.
Kessler, Emily Anne, "Investigating the Mechanistic Basis of Mutant MutS DNA Repair Protein Malfunction in Lynch Syndrome" (2018). Honors Theses - All. 1920.
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