Role of Msh2-Msh3 in determining outcomes in pathways of DNA metabolism

The DNA mismatch repair (MMR) pathway functions as a spellcheck when cells make copies of their genome, preventing errors and thereby reducing mutations and increasing the stability of the genome. However, in the presence of specific DNA sequences, particularly repetitive elements, mismatch repair activities get altered. These changes, which are poorly understood, promote mutations instead of preventing them and compromise genome stability. This project will identify key molecular changes in the behavior of mismatch repair proteins that lead to the switch from genome stability to instability. Undergraduate and graduate students will be trained in this project using cutting-edge genetics, molecular biology, and biochemical techniques. The project will also involve high school students from Buffalo Public Schools, with a student population that is ~80% black, indigenous or people of color. The activities will promote STEM education and encourage awareness of careers in science. The project will also engage K-12 students in classroom workshops and the general public in science-related events, such as Genome Day. Although MMR is well-characterized, there are key gaps in our mechanistic understanding of appropriate and aberrant Msh2-Msh3-mediated MMR, which result in replication error correction and triplet nucleotide repeat (TNR) expansion, respectively. This research will address unanswered questions about both pathways. Msh2-Msh3 binds to insertion/deletion loops in MMR and to DNA secondary structures in TNR expansions, indicating that distinct DNA structures lead to the differences in Msh2-Msh3-mediated outcomes and result in genome stability or instability. The aims include identifying allosteric changes within Msh2-Msh3 upon binding to MMR versus TNR DNA structures, using cross-linking mass spectrometry (XL-MS), and identifying interaction interfaces between Msh2-Msh3 and downstream MLH protein complexes in the presence of MMR or TNR DNA structures. Genetic approaches will also be used to determine the functional relevance of protein regions identified by XL-MS. The combination of in vivo and in vitro approaches will provide a fuller view of the context that influences the balance between Msh2-Msh3-mediated genome stability and instability. The outcomes will contribute to our understanding of MMR initiation and signaling via Msh2-Msh3 and MLH complexes and how it can go on the wrong path in the presence of alternative DNA structures. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.