Al-Hashimi, Hashim MSchumacher, Maria AGu, Stephanie2024-06-062024-06-062024https://hdl.handle.net/10161/30961<p>Replicative errors contribute to the genetic diversity needed for evolution but in high frequency can lead to genomic instability. The Watson-Crick base pairs that ensure the fidelity of the genome generation after generation adopt a Watson-Crick geometry that is defined by their shape complementarity as a function of their hydrogen bonding patterns and their inter-nucleotide distance. The ability of a base pair to adopt the Watson-Crick geometry can determine its ability to be incorporated by the polymerase. Conversely, mismatches which can adopt a Watson-Crick-like geometry can also be incorporated by the polymerases. These Watson-Crick-like mismatches have been hypothesized to be lowly-populated and short-lived alternative conformations, which are difficult to characterize and visualize with most biophysical techniques. In this thesis, using NMR spectroscopy and kinetic simulations, we determined the ground state conformation of the A•G mismatch, characterized the formation of Watson-Crick-like Hoogsteen excited state conformations in DNA duplexes and investigated their role in DNA replication errors. By including a DNA-dynamics-driven step into a minimal kinetic mechanism for correct incorporation after the initial nucleotide binding step, we were able to accurately recapitulate the rate with which A•dGTP mismatches are misincorporated for polymerase β, across different pH conditions, and for the most damaged form of the mismatch involving 8-oxoguanine. We further find that the introduction of 8-oxoguanine to A•G does not stabilize the mismatch to only one conformation but rather redistributes the dynamic ensemble of the mismatch to favor the mutagenic conformation as the ground state. Through direct and indirect targeting of the 8-oxoguanine and adenine, respectively, the lesion not only affects the mismatch itself but also rescues long-range dynamics of the DNA duplex that was previously repressed by the unmodified mismatch. We also characterize the sequence-dependent dynamics of the A•G mismatch and find that the nearest-neighbor base changes can significantly alter the dynamic ensemble. These sequence-dependent modulations to the conformational landscape can explain the sequence-dependent behavior of MutY repair enzyme. </p>BiochemistryDissertation