DISSECTING THE SEARCH PATHWAY OF DNA GLYCOSYLASES

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

Direct experimental evidence for quadruplex–quadruplex interaction within the human ILPR

Here we report the analysis of dual G-quadruplexes formed in the four repeats of the consensus sequence from the insulin-linked polymorphic region (ACAGGGGTGTGGGG; ILPRn=4). Mobilities of ILPRn=4 in nondenaturing gel and circular dichroism (CD) studies confirmed the formation of two intramolecular G-quadruplexes in the sequence. Both CD and single molecule studies using optical tweezers showed that the two quadruplexes in the ILPRn=4 most likely adopt a hybrid G-quadruplex structure that was entirely different from the mixture of parallel and antiparallel conformers previously observed in the single G-quadruplex forming sequence (ILPRn=2). These results indicate that the structural knowledge of a single G-quadruplex cannot be automatically extrapolated to predict the conformation of multiple quadruplexes in tandem. Furthermore, mechanical pulling of the ILPRn=4 at the single molecule level suggests that the two quadruplexes are unfolded cooperatively, perhaps due to a quadruplex–quadruplex interaction (QQI) between them. Additional evidence for the QQI was provided by DMS footprinting on the ILPRn=4 that identified specific guanines only protected in the presence of a neighboring G-quadruplex. There have been very few experimental reports on multiple G-quadruplex-forming sequences and this report provides direct experimental evidence for the existence of a QQI between two contiguous G-quadruplexes in the ILPR

DNA Translocation by Human Uracil DNA Glycosylase: The Case of Single-Stranded DNA and Clustered Uracils

Human uracil DNA glycosylase (hUNG) plays a central role in DNA repair and programmed mutagenesis of Ig genes, requiring it to act on sparsely or densely spaced uracil bases located in a variety of contexts, including U/A and U/G base pairs, and potentially uracils within single-stranded DNA (ssDNA). An interesting question is whether the facilitated search mode of hUNG, which includes both DNA sliding and hopping, changes in these different contexts. Here we find that hUNG uses an enhanced local search mode when it acts on uracils in ssDNA, and also, in a context where uracils are densely clustered in duplex DNA. In the context of ssDNA, hUNG performs an enhanced local search by sliding with a mean sliding length larger than that of double-stranded DNA (dsDNA). In the context of duplex DNA, insertion of high-affinity abasic product sites between two uracil lesions serves to significantly extend the apparent sliding length on dsDNA from 4 to 20 bp and, in some cases, leads to directionally biased 3′ → 5′ sliding. The presence of intervening abasic product sites mimics the situation where hUNG acts iteratively on densely spaced uracils. The findings suggest that intervening product sites serve to increase the amount of time the enzyme remains associated with DNA as compared to nonspecific DNA, which in turn increases the likelihood of sliding as opposed to falling off the DNA. These findings illustrate how the search mechanism of hUNG is not predetermined but, instead, depends on the context in which the uracils are located

DNA Translocation by Human Uracil DNA Glycosylase: Role of DNA Phosphate Charge

Human DNA repair glycosylases must encounter and inspect each DNA base in the genome to discover damaged bases that may be present at a density of <1 in 10 million normal base pairs. This remarkable example of specific molecular recognition requires a reduced dimensionality search process (facilitated diffusion) that involves both hopping and sliding along the DNA chain. Despite the widely accepted importance of facilitated diffusion in protein–DNA interactions, the molecular features of DNA that influence hopping and sliding are poorly understood. Here we explore the role of the charged DNA phosphate backbone in sliding and hopping by human uracil DNA glycosylase (hUNG), which is an exemplar that efficiently locates rare uracil bases in both double-stranded DNA and single-stranded DNA. Substitution of neutral methylphosphonate groups for anionic DNA phosphate groups weakened nonspecific DNA binding affinity by 0.4–0.5 kcal/mol per substitution. In contrast, sliding of hUNG between uracil sites embedded in duplex and single-stranded DNA substrates persisted unabated when multiple methylphosphonate linkages were inserted between the sites. Thus, a continuous phosphodiester backbone negative charge is not essential for sliding over nonspecific DNA binding sites. We consider several alternative mechanisms for these results. A model consistent with previous structural and nuclear magnetic resonance dynamic results invokes the presence of open and closed conformational states of hUNG. The open state is short-lived and has weak or nonexistent interactions with the DNA backbone that are conducive for sliding, and the populated closed state has stronger interactions with the phosphate backbone. These data suggest that the fleeting sliding form of hUNG is a distinct weakly interacting state that facilitates rapid movement along the DNA chain and resembles the transition state for DNA dissociation

Microscopic mechanism of DNA damage searching by hOGG1

The DNA backbone is often considered a track that allows long-range sliding of DNA repair enzymes in their search for rare damage sites in DNA. A proposed exemplar of DNA sliding is human 8-oxoguanine ((o)G) DNA glycosylase 1 (hOGG1), which repairs mutagenic (o)G lesions in DNA. Here we use our high-resolution molecular clock method to show that macroscopic 1D DNA sliding of hOGG1 occurs by microscopic 2D and 3D steps that masquerade as sliding in resolution-limited single-molecule images. Strand sliding was limited to distances shorter than seven phosphate linkages because attaching a covalent chemical road block to a single DNA phosphate located between two closely spaced damage sites had little effect on transfers. The microscopic parameters describing the DNA search of hOGG1 were derived from numerical simulations constrained by the experimental data. These findings support a general mechanism where DNA glycosylases use highly dynamic multidimensional diffusion paths to scan DNA

Microscopic mechanism of DNA damage searching by hOGG1

The DNA backbone is often considered a track that allows long-range sliding of DNA repair enzymes in their search for rare damage sites in DNA. A proposed exemplar of DNA sliding is human 8-oxoguanine (oG) DNA glycosylase 1 (hOGG1), which repairs mutagenic oG lesions in DNA. Here we use our high-resolution molecular clock method to show that macroscopic 1D DNA sliding of hOGG1 occurs by microscopic 2D and 3D steps that masquerade as sliding in resolution-limited single-molecule images. Strand sliding was limited to distances shorter than seven phosphate linkages because attaching a cova-lent chemical road block to a single DNA phosphate located between two closely spaced damage sites had little effect on transfers. The microscopic param-eters describing the DNA search of hOGG1 were de-rived from numerical simulations constrained by the experimental data. These findings support a general mechanism where DNA glycosylases use highly dy-namic multidimensional diffusion paths to scan DNA