Role of DNA structural dynamics in mutagenesis and oncogenesis

Project Summary Mutations are fundamental to evolution, yet they can also cause cancer and various genetic disorders. Despite their significance, our understanding of processes which generate or defend against mutations is incomplete. We don’t understand why the probability of a base substitution mutation varies by many orders of magnitude depending on the type of substitution and sequence context or why the efficiency of DNA damage repair differs significantly depending on the kind of damage and sequence. Neither the sequence of the DNA nor its three- dimensional structure can explain most mutational signatures and the biochemical origins of most signatures remain unknown. The central hypothesis in this project is that DNA dynamics, which leads to changes in the mode of base pairing, is a significant driver of mutational processes and mechanisms maintaining genomic stability. The project will develop and apply techniques for predicting and quantitatively measuring the propensities to form alternative conformational states of DNA base pairs in high throughput and in large protein- DNA assemblies and for mapping alternative base pair conformations at single nucleotide resolution genome- wide in vivo. Aim 1 will advance a thermodynamic ensemble model and determine the propensities of all mismatches to form mutagenic Watson-Crick-like conformations across sixteen trinucleotide sequence contexts, comprehensively measure replicative errors by proofreading-deficient human polymerase ε and b and use the results to test and refine a quantitative and predictive model for nucleotide misincorporation. Aim 2 will develop biophysical approaches to measure the propensities for base-opening, intra- and extra-helical flipping for A-8OG, A-G, and G-T in sixteen trinucleotide sequence contexts and use the results to test and refine a quantitative and predictive model for sequence-specific damage repair by MUTYH and Thymine DNA Glycosylase (TDG). Aim 3 will demonstrate that many DNA-binding proteins employ Hoogsteen base pairs to recognize DNA, determine the role of Hoogsteen base pairs in p53-DNA interactions in vitro and in vivo, and demonstrate the existence of Hoogsteen base pairs in nucleosomes across the yeast genome. If successful, the project will illuminate the role of DNA dynamics in mutation and genomic stability maintenance; deliver the first comprehensive database of DNA conformational propensities, which can aid the discovery of other mutational processes; advance quantitative and predictive models for fundamental processes driving oncogenesis; and potentially append the Watson-Crick paradigm by revealing Hoogsteen base pairs as an alternative building block of DNA in vivo.