Glycosylase enzymes illustrate one of the most remarkable examples of molecular recognition known as they are able to find and remove rare mutagenic DNA bases present within the vast background of nonspecific DNA in the genome. In order to accelerate the search process and efficiently find base lesions, glycosylases and other site specific DNA binding proteins are thought to use a reduced dimensionality search process and are able to stochastically slide and hop along DNA. Although many enzymes exhibit these properties, due to a lack of spatial and temporal resolution in current experimental approaches, mechanistic interpretations are often murky and inconsistent with other kinetic requirements in lesion recognition and catalysis. Therefore, in Chapter 2, using human Uracil DNA Glycosylase (hUNG), I have established a new approach that utilizes a small molecule to trap and time enzyme molecules that have ;;hopped’ off of the DNA providing novel quantitative insight into the lifetime and distance of hopping events as well as the speed and length of sliding on DNA. In Chapter 3, using DNA constructs containing neutrally charged methylphosphonate linkages as well as engineered hUNG variants with enhanced electrostatic properties, a model emerges that goes against the current dogma that facilitated diffusion involves isoenergetic movement along a smooth free energy landscape allowed by electrostatic interactions with the DNA backbone. Rather, sliding is surprisingly independent of the latter perturbations and combined with previous NMR measurements suggests that movement on DNA is achieved by dynamic motions of the protein and that the sliding form of the enzyme is similar to the transition state for DNA dissociation.In the next part of my thesis (Chapter 4), I investigate the effects of uracils present within densely spaced clusters and present within single stranded DNA.These two situations are relevant in the context of hUNG’s involvement in the generation of antibody diversity, where the processive single strand specific enzyme, Activation Induced Cytosine Deamaminase (AID), converts cytosines to uracils within the Ig locus. Notably I find that hUNG is more processive on single stranded DNA and shows a previously unobserved directional preference in the presence of neighboring abasic sites.Finally in Chapter 5, I incorporate experimental constraints for hUNG and another DNA glycosylase (hOGG1) into a complete model of facilitated diffusion using novel numerical simulations. Using this method, a data driven model consistent with the entire reaction coordinate is determined at unprecedented quantitative resolution. Further, these results lead to the surprising finding that despite these two enzymes divergence early in evolution, the search mechanism is nearly identical.
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