| DNA mismatch repair (MMR) is a process that is responsible for repairing base-base mismatches and insertion/deletion loop errors incorporated during DNA replication. In humans, deficiencies in MMR are linked to cancers, including Lynch Syndrome. MMR is initiated by MutS in prokaryotes (or the MutS homologs in eukaryotes), which is responsible for recognizing the error in the DNA. Upon error recognition, MutS undergoes ATP-dependent conformational changes to form a sliding clamp state that moves along the length of the DNA. This state is thought to be important for downstream repair events, such as recruitment of the second protein in the pathway, MutL.;Recent single-molecule fluorescence studies have led to a refined model for error recognition and sliding clamp formation by Thermus aquaticus MutS. While it is well established that MutS bends DNA and that this DNA bending is dynamic in the absence of nucleotides, little evidence for the DNA bending status through MMR initiation exists in the presence of ATP.;In this work, the nucleotide dependence of Thermus aquaticus MutS-induced DNA bending throughout sliding clamp formation is characterized. The current model for MutS conformational changes is then modified to reflect the newfound DNA bending information. To this end, single-molecule fluorescence resonance energy transfer (smFRET) between two arms of specially designed DNA oligonucleotides is monitored. These substrates are designed such that changes in DNA bending would result in changes in FRET efficiency. Finally, a data analysis pipeline developed specifically for high throughput analysis of data of this type is presented. |
