Developing New AFM Imaging Technique and Software for DNA Mismatch Repair

Atomic Force Microscopy (AFM) is a powerful technique to study the assembly and function of multiprotein-DNA complexes, such as the MutS-MutL-DNA complex in DNA mismatch repair. As a high-resolution, single-molecule imaging technique, AFM has the advantage of directly visualizing individual protein-DNA complexes in their native conformations, but it has two severe limitations. First, it is unable to resolve the location of the DNA inside the protein. Second, it lacks a comprehensive software that is tailored for single-molecule studies and high-throughput analyses. To tackle these issues, we developed DREEM (Dual-Resonance-frequency-Enhanced Electrostatic Force Microscopy). DREEM is a new AFM imaging method that is capable of resolving DNA path inside the protein-DNA complex. With DREEM, we can reveal the path of the DNA wrapping around histones in nucleosomes and the path of DNA through multiprotein mismatch repair complexes. We also developed Image Metrics, a full-featured AFM software package that excels in high-throughput shape analysis and single-molecule analysis. Built using MATLAB, Image Metrics is uniquely positioned in single-molecule studies with its specially designed single-molecule analysis and shape analysis modules. It also blends unique flexibility into a user-friendly interface that allows for easy customization and automation of user workflows. Finally, as a case study, we used AFM and Image Metrics to study DNA mismatch repair (MMR) in the context of trinucleotide repeat (TNR) expansion diseases. Our data show that when interacting with heteroduplex DNAs with the TNR context, MMR proteins adopt conformations that are different from a repair signaling capable species, suggesting the altered conformations may not be competent for repair.Doctor of Philosoph