The DNA mismatch repair system is responsible for reducing post replicative errors in the newly synthesized DNA strand. Repair is initiated by the MutS homolog Msh2-Msh6 in eukaryotes which recognizes small insertion/deletion loops, and single base pair mismatches. This study focuses on characterizing the binding interaction between different high binding affinity DNA substrates and Msh2-Msh6 in terms of affinity, DNA and protein structure to better understand the mechanisms of recognition and function. Previously, a linear correlation between the mismatched base dynamics and binding affinity was observed, in which highly mobile mismatched bases were well recognized by the protein. The G:G mismatch was anomalous; however, as it exhibited high dynamic motion, as measured by our 6-MI probe, a fluorescent guanosine analog, but was poorly recognized by Msh2-Msh6. We used fluorescence spectroscopy to measure the binding affinity of the 6-MI:G mismatch, which was found to be approximately 10nM and in line with the linear correlation. Additionally, a spectral peak shift of 6-MI fluorescence to bluer wavelengths was observed with some mismatched substrates upon protein binding. We interpret this blue shift to arise from hydrophobic stacking interactions between a phenylalanine residue (F432) and the mismatched base pair as observed in the X-ray crystal structure. We are also exploring the high affinity binding of Msh2-Msh6 to another DNA substrate, the Holliday Junction. Our investigation aims to understand the functionality of this Msh2-Msh6-junction binding interaction by studying nucleotide utilization upon binding. Malachite green phosphate release assays were conducted in order to obtain the Msh2-Msh6 ATP hydrolysis rate alone and in the presence of Holliday junction and mismatched DNA. Msh2-Msh6 alone has a Kcat = 0.27s-1, with the Kcat increasing in the presence of G:T mismatched 34-mer duplex and the junction to 0.47s-1 and 0.6s-1 respectively. In the future, binding experiments will be performed, in which a fluorescent probe is incorporated into the junction substrate and changes in binding affinity between Msh2-Msh6 and the junction will be determined in the presence of ATP and ADP to understand how DNA binding and ATP hydrolysis are functionally coupled.
"},{"label":"Degree","value":["Bachelor of Arts with Departmental Honors"]},{"label":"Author-supplied Keywords","value":["Msh2-Msh6","Dynamics","ATPase"]},{"label":"Administrative Note","value":"Drupal Node ID: 24046