Mechanisms and Dynamics of Oxidative DNA Damage Repair in Nucleosomes

DNA provides the blueprint for cell function and growth, as well as ensuring continuity from one cell generation to the next. In order to compact, protect, and regulate this vital information, DNA is packaged by histone proteins into nucleosomes, which are the fundamental subunits of chromatin. Reactive oxygen species, generated by both endogenous and exogenous agents, can react with DNA, altering base chemistry and generating DNA strand breaks. Left unrepaired, these oxidation products can result in mutations and/or cell death. The Base Excision Repair (BER) pathway exists to deal with damaged bases and single-stranded DNA breaks. However, the packaging of DNA into chromatin provides roadblocks to repair. Damaged DNA bases may be buried within nucleosomes, where they are inaccessible to repair enzymes and other DNA binding proteins. Previous in vitro studies by our lab have demonstrated that BER enzymes can function within this challenging environment, albeit in a reduced capacity. Exposure to ionizing radiation often results in multiple, clustered oxidative lesions. Near-simultaneous BER of two lesions located on opposing strands within a single helical turn of DNA of one another creates multiple DNA single-strand break intermediates. This, in turn, may create a potentially lethal double-strand break (DSB) that can no longer be repaired by BER. To determine if chromatin offers protection from this phenomenon, we incubated DNA glycosylases with nucleosomes containing clustered damages in an attempt to generate DSBs. We discovered that nucleosomes offer substantial protection from inadvertent DSB formation. Steric hindrance by the histone core in the nucleosome was a major factor in restricting DSB formation. As well, lesions positioned very close to one another were refractory to processing, with one lesion blocking or disrupting access to the second site. The nucleosome itself appears to remain intact during DSB formation, and in some cases, no DNA is released from the histones. Taken together, these results suggest that in vivo, DSBs generated by BER occur primarily in regions of the genome associated with elevated rates of nucleosome turnover or remodeling, and in the short linker DNA segments that lie between adjacent nucleosomes. DNA ligase IIIα (LigIIIα) catalyzes the final step in BER. In order to facilitate repair, DNA ligase must completely encircle the DNA helix. Thus, DNA ligase must at least transiently disrupt histone-DNA contacts. To determine how LigIIIα functions in nucleosomes, given this restraint, we incubated the enzyme with nick-containing nucleosomes. We found that a nick located further within the nucleosome was ligated at a lower rate than one located closer to the edge. This indicated that LigIIIα must wait for DNA to spontaneously, transiently unwrap from the histone octamer to expose the nick for recognition. Remarkably, the disruption that must occur for ligation is both limited and transient: the nucleosome remains resistant to enzymatic digest before and during ligation, and reforms completely once LigIIIα dissociates

Base excision repair processing of abasic site/single-strand break lesions within clustered damage sites associated with XRCC1 deficiency

Ionizing radiation induces clustered DNA damage, which presents a challenge to the cellular repair machinery. The repair efficiency of a single-strand break (SSB) is ∼4× less than that for repair of an abasic (AP) site when in a bistranded cluster containing 8-oxoG. To explore whether this difference in repair efficiency involves XRCC1 or other BER proteins, synthetic oligonucleotides containing either an AP site or HAP1-induced SSB (HAP1-SSB) 1 or 5 bp 5′ or 3′ to 8-oxoG on the opposite strand were synthesized and the repair investigated using either nuclear extracts from hamster cells proficient (AA8) or deficient (EM7) in XRCC1 or purified BER proteins. XRCC1 is important for efficient processing of an AP site in clustered damage containing 8-oxoG but does not affect the already low repair efficiency of a SSB. Ligase I partly compensates for the absence of the XRCC1/ligaseIII during short-patch BER of an AP site when in a cluster but only weakly if at all for a HAP1-SSB. The major difference between the repair of an AP site and a HAP1-SSB when in a 8-oxoG containing cluster is the greater efficiency of short-patch BER with the AP site compared with that for a HAP1-SSB

Clustering of Epigenetic Methylation and Oxidative Damage: Effects on Duplex DNA Solution Structure, Thermodynamic Stability and Local Dynamics

All known living organisms use DNA to store genetic templates used for development, proper function and reproduction. The structural integrity of DNA is therefore of extreme importance and cellular machinery continuously regulates our DNA either through addition of covalent molecules to regulate the transcription of genes or the removal of DNA lesions propagating from the exposure to reactive molecules. One of the most common DNA lesions, 8-oxoguanine (8OG), is a prominent, pro-mutagenic DNA adduct present at a baseline level from consistent generation of reactive oxygen species through oxidative metabolism or at greater concentrations through exposure to ionizing radiation and other toxins. Its mutagenic potential is attributed to its ability in the syn- conformation, to mimic thymine during DNA replication, resulting in a mispair with adenine. In contrast, 5-methylcytosine (5MC), occurs from the covalent addition of a methyl group to a cytosine base by a DNA methyltransferase. 5MC acts as an epigenetic gene regulator, often found densely packed within CpG islands upstream of transcriptionally inactive genes. It can be estimated that each human diploid cell contains hundreds of CpG dinucleotides undergoing active methylation while also harboring 8OG. Previous results obtained by Kasymov et al, showed reduced endonuclease activity by hOGG1 for substrates containing 5MC adjacent and cross strand from 8OG. In addition, the work presented by Maltseva et al, conveyed that the enzymatic methylation rates by maintenance DNA methyltransferases were severely impacted when 8OG is adjacent to the methylation target. These results prompted us to investigate the clustering of these two modifications in greater detail. We present the results of solution NMR structure determination, thermodynamic stability analysis and molecular dynamics simulations on the DNA sequence 5’-d(CGCGAATTCGCG)-3’ with clustered 5MC and 8OG in CpG dinucleotides. NMR spectroscopy and restrained molecular dynamics were used to refine the structure of 11 DNA duplexes containing different methylation and oxidation patterns. The results reveal that 8OG induces local unwinding 5’ to itself and 31P chemical shifts indicate an increase in the BII phosphate backbone conformation 3’ relative to 8OG. Melting temperatures of the duplexes was shown to decrease with the addition of 8OG in all contexts. Surprisingly, the addition of 5MC in two separate instances led to lower Tm values of already oxidized DNA samples. 1D-1H NMR linewidths indicate 8OG increases the base dynamics while incorporation of 5MC leads to a stabilizing effect. Our results indicate that addition of 8OG to a fully-methylated CpG induces a sequence dependent stabilizing effect. Molecular dynamics trajectories were analyzed for BI/BII phosphate conformation populations, conformational flexibility and local dynamics. Comparison of helical geometries and backbone angles indicated that our MD simulations accurately and reliably reproduced our NMR structures within one standard deviation. Principal component analysis was carried out to highlight the most dominant modes of motion for CpG sites with clustered 5MC and 8OG. Particularly, we report significant differences in concerted atomic displacements, with the 8OG:5MC base pair displaying the greatest dynamic effects

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

In What Ways Do Synthetic Nucleotides and Natural Base Lesions Alter the Structural Stability of G-Quadruplex Nucleic Acids?

Synthetic analogs of natural nucleotides have long been utilized for structural studies of canonical and noncanonical nucleic acids, including the extensively investigated polymorphic G-quadruplexes (GQs). Dependence on the sequence and nucleotide modifications of the folding landscape of GQs has been reviewed by several recent studies. Here, an overview is compiled on the thermodynamic stability of the modified GQ folds and on how the stereochemical preferences of more than 70 synthetic and natural derivatives of nucleotides substituting for natural ones determine the stability as well as the conformation. Groups of nucleotide analogs only stabilize or only destabilize the GQ, while the majority of analogs alter the GQ stability in both ways. This depends on the preferred syn or anti N-glycosidic linkage of the modified building blocks, the position of substitution, and the folding architecture of the native GQ. Natural base lesions and epigenetic modifications of GQs explored so far also stabilize or destabilize the GQ assemblies. Learning the effect of synthetic nucleotide analogs on the stability of GQs can assist in engineering a required stable GQ topology, and exploring the in vitro action of the single and clustered natural base damage on GQ architectures may provide indications for the cellular events

Oxidative DNA damage modulates genome and epigenome integrity via base excision repair

Oxidative DNA damage is one of the leading causes of genome instability, cell death, and diseases. It is repaired by DNA base excision repair (BER), during which repair and translesion DNA polymerases may incorporate damaged nucleotides and mediate RNA-guided DNA repair induced by DNA replication and gene transcription leading to the modulation of genome stability. On the other hand, oxidative DNA damage may result in cellular epigenetic responses to regulate DNA repair, altering genome stability and integrity. In this dissertation, we revealed the molecular mechanisms underlying the misincorporation of oxidized nucleotides, 5′,8-cyclo-2-cyclodeoxyadenosine (cdA) and RNA-guided base lesion repair mediated by repair and translesion DNA polymerases. We then explored how oxidative DNA damage induced cellular epigenetic responses by disrupting microRNA expression to regulate BER. We found that DNA polymerase β (pol β) and DNA polymerase η (pol η) incorporated cdA that basepaired with dC, resulting in an A:C mismatch. We further demonstrated that cdA lesions were readily extended and ligated in duplex DNA. We showed that the polymerases incorporated cdAs independent of their hydrogen bonding with a template nucleotide using molecular docking. Our study reveals a unique mechanism underlying the accumulation of cyclodeoxypurine lesions in the genome. We then explored the mechanisms by which DNA polymerases can utilize RNA as a template to synthesize DNA and repair a DNA base lesion. We found that translesion DNA polymerases, pol η, θ, and ν and repair DNA polymerases, pol β, λ and κ exhibited DNA synthesis activity, i.e., reverse transcriptase activity to mediate RNA-guided DNA base lesion repair. We further demonstrated that the completion of base lesion repair was accomplished by the RNA-guided translocation of a nick into duplex DNA via the strand displacement synthesis of the polymerases. We then explored the cellular mechanisms by which oxidative DNA damage modulates microRNA expression to regulate DNA repair. Our study revealed that oxidative DNA damage upregulated the expression of microRNA-499-5p (miR-499-5p) that subsequently downregulated the expression of the key BER enzyme, pol β, in human cells. Further analysis showed that the inhibition of 8-oxoG DNA glycosylase 1 (OGG1) activity significantly suppressed the upregulation of miR-499-5p, suggesting the epigenetic role of OGG1 in mediating the expression of miR-449-5p as cellular DNA damage response. Our study provides new insights into the crosstalk among oxidative DNA damage and repair, miRNAs, RNA-guided base lesion repair in modulating genome stability and integrity

FROM TEST TUBE TO THE HUMAN CELL NUCLEUS: THE NATURE OF “INVISIBLE” DAMAGE SEARCH TRANSITION STATES OF HUMAN DNA GLYCOSYLASES

The integrity of the genetic code is preserved by maintenance of DNA by glycosylases. These remarkable proteins efficiently repair infrequent lesions amongst a sea of nonspecific target sites. It is well accepted that glycosylases utilize DNA to accelerate their search through DNA chain translocation or “facilitated diffusion”, which reduces dimensionality of the search process. Although DNA translocation has been studied for over 50 years, little attention has been given to how the environment of the cell nucleus affects DNA translocation. It is therefore the goal of my thesis to determine the impact of physiological ions, macromolecular crowding and high concentrations of bystander DNA chains on this important aspect of DNA damage recognition in cells. It is essential to undertand the effects of these variables to interpret future in vivo experiments of damage recognition in cells. I began by investigating the effects of high ionic strength on the interactions of human uracil DNA glycosylase (hUNG) with DNA. One salient finding was that shielding of non-specific electrostatic interactions by physiological concentration of salt enhanced the specificity of hUNG for damaged DNA. Nevertheless, the fundamental aspects of DNA translocation by hUNG did not change in the presence of a physiological concentration of salt. I then explored the same question with another paradigm enzyme, human 8-oxoguanine DNA glycosylase (hOGG1). In contrast to hUNG, hOGG1’s non-specific itneractions with DNA were not electrostatic in nature, and accordingly, salt had no effect on its specificity for 8-oxoguainine lesions in DNA. These findings revealed that different DNA glycosylases can use entirely distinct non-specific DNA interactions during a damage search process that involves facilitated diffusion. I then moved to a comprehensive study of the effects of molecular crowding on the DNA interactions of both hUNG and hOGG1 using crowding conditions similar to that found in the cell nucleus. Although crowded solutions have high macroscopic viscosity, which is expected to slow translational diffusion, crowding had no effect on the rate of protein-DNA association. This is attributed to the caging effect of large crowders, which increases the efficiency of macromolecule association once both molecules enter the same caged environment. In addition, the cage provided by macromolecular crowders significantly increases the DNA chain translocation efficiency of both enzymes. Overall, the cage provided by crowders plays an important role in increasing DNA chain translocation under physiological conditions of high salt by introducing a barrier to escape of the enzyme to bulk solution. Finally, I probed the combined effects of salt, crowding, and high concentrations of bulk DNA chains in damage recognition and translocation. In the presence of excess DNA chains, the rate of repair by hOGG1 was insensitive to solution ionic strength, while the activity of hUNG was greatly stimulated by high salt. These opposite effects are directly related to the different contributions of electrostatics to the binding of non-specific DNA by both enzymes. The general effect of crowding was to promote chain translocation just as observed in the absence of bulk DNA. Overall these studies show that cellular crowding and ion concentrations have important effects on the rate and mechanism of DNA damage search and repair