Measuring Hoogsteen Base-Pairs : From Test Tubes to Cells

Abstract

Nucleic acids dynamic in nature and are constantly interconverting between multiple conformations. The ensemble defines the population of all conformations at any given instance. Duplex DNA ensembles tend to be dominated by one conformation - the Watson-Crick base-pair. As a result, the vast majority of studies investigating DNA transactions have focused primarily on Watson-Crick base pairs. However, an alternative base-pair conformation which is only ~2-5 kcal/mol destabilized relative to the Watson-Crick conformation called the Hoogsteen has been shown to be widespread in ensembles of a variety of DNA sequences. Hoogsteen base-pairs have distinct physicochemical properties relative to Watson-Crick base-pairs and can therefore allow for functions that are inaccessible to the Watson-Crick geometry. A long standing goal of biochemistry is to gain a predictive understanding of sequence specificity of DNA interactions. In this work, I hypothesized that Hoogsteen dynamics could provide a mechanism for sequence specificity in DNA-protein interactions as well as DNA damage. I utilized NMR spectroscopy to test whether Hoogsteen dynamics are sequence dependent. I built upon on a prior UV melting based approach to measure Hoogsteen bps to further validate the sequence dependence of Hoogsteen. I then investigated the impact of Hoogsteen bps on the susceptibility of DNA to cytotoxic damage. Upon discovering enhanced methylation damage of Hoogsteen bps, I used it as a molecular marker of Hoogsteen bps and developed a high throughput sequencing based method to probe Hoogsteen bps inside cells at single nucleotide resolution. To further understand the origins of damage susceptibility of double-stranded DNA, I compared it to the damage susceptibility of A-RNA and discovered the greater protection conferred by the A-RNA helix against chemical damage. This work describes multiple biochemical approaches to investigate DNA dynamics. While I focused on A-T Hoogsteen base-pairs, the techniques described here can be applied to investigate a plethora of nucleic acid dynamic processes. Future studies can then investigate the roles of these dynamics in biological phenomena and lead us towards a quantitative era of biochemistry.