Presentation Time: 9:00-9:20
Home University: UNC-Chapel Hill
Research Mentor: Dorothy Erie, Chemistry
Program: CSS
Research Title: DNA Mismatch Repair Proteins
DNA mismatch repair is essential for maintenance of genome stability. Mismatch repair (MMR) corrects errors made during DNA replication, including mismatches and insertion-deletion loops. This process enhances DNA replication by about 100 to 1000-fold by improving the accuracy of the incorporated base pairs. We used the MMR protein MutS from Thermus aquaticus as our model. Previous studies have shown that a glutamate in the DNA binding domain of MutS hydrogen bonds with a mismatched nucleotide leading to a change of shape in the DNA, determinant of repair. In this study, we were specifically interested in MutS’s interaction with thymine inserts (T-bulges) and GT mismatches. Previous studies have shown that wild type MutS can repair T-bulges and GT mismatches whereas mutating glutamate to alanine, MutS can only repair T-bulges and not GT mismatches. We have mutated the glutamate to alanine to inhibit MutS causing conformational changes in the DNA with GT mismatches leading to MMR so we could study how MutS is interacting with the DNA. To visualize the protein-DNA interaction, a Cys mutation was also introduced into the MutS protein so it could be fluorescently labeled. Single molecule fluorescence resonance energy transfer (smFRET) experiments were performed to visualize measure conformational changes of the mutated protein and DNA. FRET is a function of the distance between the donor and acceptor fluorescent dyes.