Oxidative Defect Detection Within Free and Packed DNA Systems: A Quantum Mechanical/Molecular Mechanics (QM/ MM) Approach

Base excision repair enzymes (BERs) detect and repair oxidative DNA damage with efficacy despite the small size of the defects and their often only minor structural impact. A charge transfer (CT) model for rapid scanning of DNA stretches has been evoked to explain the high detection rate in the face of numerous, small lesions. The viability of CT DNA defect detection is explored via hybrid QM/MM computational studies that leverage the accuracy of quantum mechanics (QM) for a region of interest and the descriptive power of molecularmechanics (MM) for the remainder of the system. We find that the presence of an oxidative lesion lowers theredox free energy of oxidation by approximately 1.0 eV regardless of DNA compaction (free DNA versus packed DNA in nucleosome core particles) and damage location indicating the high feasibility of a CT-based process for defect detection in DNA

Distinct energetics and closing pathways for DNA polymerase β with 8-oxoG template and different incoming nucleotides

BACKGROUND: 8-Oxoguanine (8-oxoG) is a common oxidative lesion frequently encountered by DNA polymerases such as the repair enzyme DNA polymerase β (pol β). To interpret in atomic and energetic detail how pol β processes 8-oxoG, we apply transition path sampling to delineate closing pathways of pol β 8-oxoG complexes with dCTP and dATP incoming nucleotides and compare the results to those of the nonlesioned G:dCTP and G:dATPanalogues. RESULTS: Our analyses show that the closing pathways of the 8-oxoG complexes are different from one another and from the nonlesioned analogues in terms of the individual transition states along each pathway, associated energies, and the stability of each pathway's closed state relative to the corresponding open state. In particular, the closed-to-open state stability difference in each system establishes a hierarchy of stability (from high to low) as G:C > 8-oxoG:C > 8-oxoG:A > G:A, corresponding to -3, -2, 2, 9 k(B)T, respectively. This hierarchy of closed state stability parallels the experimentally observed processing efficiencies for the four pairs. Network models based on the calculated rate constants in each pathway indicate that the closed species are more populated than the open species for 8-oxoG:dCTP, whereas the opposite is true for 8-oxoG:dATP. CONCLUSION: These results suggest that the lower insertion efficiency (larger K(m)) for dATP compared to dCTP opposite 8-oxoG is caused by a less stable closed-form of pol β, destabilized by unfavorable interactions between Tyr271 and the mispair. This stability of the closed vs. open form can also explain the higher insertion efficiency for 8-oxoG:dATP compared to the nonlesioned G:dATP pair, which also has a higher overall conformational barrier. Our study offers atomic details of the complexes at different states, in addition to helping interpret the different insertion efficiencies of dATP and dCTP opposite 8-oxoG and G

The effect of oxidatively damaged DNA on the active site pre-organization during nucleotide incorporation in a high fidelity polymerase from \u3cem\u3eBacillus stearothermophilus\u3c/em\u3e

We study the effect of the oxidative lesion 8-oxoguanine (8oxoG) on the pre-organization of the active site for DNA replication in the closed (active) state of the Bacillus Fragment (BF), a Klenow analog from Bacillus stearothermophilus. Our molecular dynamics and free energy simulations of explicitly solvated model ternary complexes of BF bound to correct dCTP/incorrect dATP opposite guanine (G) and 8oxoG bases in DNA suggest that the lesion introduces structural and energetic changes at the catalytic site to favor dATP insertion. Despite the formation of a stable Watson-Crick pairing in the 8oxoG:dCTP system, the catalytic geometry is severely distorted to possibly slow down catalysis. Indeed, our calculated free energy landscapes associated with active site pre-organization suggest additional barriers to assemble an efficient catalytic site, which need to be overcome during dCTP incorporation opposite 8oxoG relative to that opposite undamaged G. In contrast, the catalytic geometry for the Hoogsteen pairing in the 8oxoG:dATP system is highly organized and poised for efficient nucleotide incorporation via the twometal- ion catalyzed phosphoryl transfer mechanism. However, the free energy calculations suggest that the catalytic geometry during dATP incorporation opposite 8oxoG is considerably less plastic than that during dCTP incorporation opposite G despite a very similar, well organized catalytic site for both systems. A correlation analysis of the dynamics trajectories suggests the presence of significant coupling between motions of the polymerase fingers and the primary distance for nucleophilic attack (i.e., between the terminal primer O3´ and the dNTP Pα atoms) during correct dCTP incorporation opposite undamaged G. This coupling is shown to be disrupted during nucleotide incorporation by the polymerase with oxidatively damaged DNA/dNTP substrates. We also suggest that the lesion affects DNA interactions with key polymerase residues, thereby affecting the enzymes ability to discriminate against noncomplementary DNA/dNTP substrates. Taken together, our results provide a unified structural, energetic, and dynamic platform to rationalize experimentally observed relative nucleotide incorporation rates for correct dCTP/incorrect dATP insertion opposite an undamaged/oxidatively damaged template G by BF

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

Structure and stereochemistry of the base excision repair glycosylase MutY reveal a mechanism similar to retaining glycosidases.

MutY adenine glycosylases prevent DNA mutations by excising adenine from promutagenic 8-oxo-7,8-dihydroguanine (OG):A mismatches. Here, we describe structural features of the MutY active site bound to an azaribose transition state analog which indicate a catalytic role for Tyr126 and approach of the water nucleophile on the same side as the departing adenine base. The idea that Tyr126 participates in catalysis, recently predicted by modeling calculations, is strongly supported by mutagenesis and by seeing close contact between the hydroxyl group of this residue and the azaribose moiety of the transition state analog. NMR analysis of MutY methanolysis products corroborates a mechanism for adenine removal with retention of stereochemistry. Based on these results, we propose a revised mechanism for MutY that involves two nucleophilic displacement steps akin to the mechanisms accepted for 'retaining' O-glycosidases. This new-for-MutY yet familiar mechanism may also be operative in related base excision repair glycosylases and provides a critical framework for analysis of human MutY (MUTYH) variants associated with inherited colorectal cancer

The Evolutionary Diversity of Uracil DNA Glycosylase Superfamily

Uracil DNA glycosylase (UDG) is a crucial member in the base excision (BER) pathway that is able to specially recognize and cleave the deaminated DNA bases, including uracil (U), hypoxanthine (inosine, I), xanthine (X) and oxanine (O). Currently, based on the sequence similarity of 3 functional motifs, the UDG superfamily is divided into 6 families. Each family has evolved distinct substrate specificity and properties. In this thesis, I broadened the UDG superfamily by characterization of three new groups of enzymes. In chapter 2, we identified a new subgroup of enzyme in family 3 SMUG1 from Listeria Innocua. This newly found SMUG1-like enzyme has distinct catalytic residues and exhibits strong preference on single-stranded DNA substrates. In chapter 3, we extensively investigated an untraditional family 1 UNG enzyme from Nitratifractor salsuginis (Nsa UNG). This enzyme is able to form a unique salt bridge network with uracil-containing DNA. In addition, this untraditional family 1 UNG can’t be inhibited by uracil DNA glycosylase inhibitor (Ugi). In chapter 4, a potential evolutionary immediate between family 1 UNG and family 4 UDGa was isolated from Janthinobacterium agaricidamnosum (Jag UNG). In the functional motifs, Jag UNG has evolved family 1 UNG unique features, but still keeps some features of family 4 UDGa. Through site-directed mutagenesis, molecular modeling and biophysical analysis, we estimated that QD in family 1 UNG might be coevolved from EG in family 4 UDGa. In addition, we found another two important sites (A82E and L245H) that may have special meaning in the evolutionary history. All these work reveal the evolutionary diversity of UDG superfamily

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

Molecular modelling studies of DNA damage recognition

How DNA repair proteins search and recognise the rare sites of damage from the massive numbers of normal DNA remains poorly understood. FapydG (2,6-diamino-4-hydroxy-5-formamidopyrimidine) is one of the most prevalent guanine derived lesions involving opening of the imidazole ring. It is typically repaired by formamidopyrimidine-DNA glycosylase (Fpg) as an initial step in base excision repair; if not repaired, the lesion generates a G: C -+ T: A transversion. Unfortunately, studies on the recognition of FapydG have been hindered by difficulties to synthesise and incorporate the FapydG residue into a DNA duplex. Crystal structures of Fpg-DNA complexes have demonstrated three common recognition events: the protein specifically binding to the extrahelical lesion, bending DNA centred on the damaged base, and flipping the damage into the pocket. Thus, molecular modelling and dynamics simulation have been used to gather dynamical information of those recognition events for damaged and undamaged DNA. The simulations were initially performed when FapydG or G occurs in several dodecamer B-DNA sequences in aqueous solution, then inside the lesion-recognition pocket of Fpg, and during the flipping pathway from the helical stack to an extrahelical position. The influence of the damage on DNA stability and flexibility was first investigated. Energetic analysis revealed that damage to DNA does appear to destabilise the duplex. DNA curvature analysis and a novel combined method of the principal component analysis (PCA) and the Mahalanobis distance (DM) indicated that damaged DNA can adopt the observed protein-bound conformation with lower energetic penalties than its normal counterpart. Results of these studies have provided the validation of DNA bending enhancement by the FapydG lesion. It also suggested that intrinsic DNA bending could be a principal element of how the repair protein locates the lesion from vast expanse of normal bases. Considering the specific recognition of FapydG by Fpg, the aF-/39 loop of the Fpg enzyme may function as a gatekeeping to accommodate the lesion while denying the normal base. Remarkably fluctuating movement of the flipped G residue and the aF-ß9 loop is due to the formation of the non-specific Fpg/G complex with a lower binding energy by 8.4 kcal/mol compared to the specific Fpg/FapydG complex. Free-energy profiles for both damaged and undamaged base flipping were generated from the umbrella sampling simulations and the Weight Histogram Analysis Method (WHAM). An energy barrier for flipping the damage out from the helix is 2.7 kcal/mol higher than its equivalent G and the lesion is highly stabilised inside the pocket. In contrast, G flipping seems to be rapidly rotated out and into the duplex without the formation of a specific complex. These studies could unravel a potentially comprehensive process of the repair protein to find and recognise the lesion through the slow kinetic pathway in which the more deformable damaged DNA is initially located by the protein; the protein subsequently compresses the duplex into an appropriate angle and direction to form a specific protein-DNA complex prior to being flipped and repaired

Modeling the influence of DNA lesion on the regulation of gene expression

Nucleic acids are organic macromolecules that result from the polymerization of nucleotides. These molecules are generally considered as the support of the genetic information. Two families of nucleic acids are currently known: DNA and RNA. From a structural point of view, the most popular form is the double helix of DNA. However, other forms exist and among them are the G-quadruplex. This is a folding of the DNA, or RNA, in an area rich in guanines. These form quadruplex of guanines, which are stacked on top of each other and are stabilized by a central cation. G-quadruplex structures are increasingly studied. This is not surprising since their biological role involves the regulation of genetic mechanisms. They are notably involved in the regulation of the cell cycle, but they also play a role in cancer, certain neurological or viral diseases. The aim of this PhD thesis is to study G-quadruplex using theoretical chemistry tools. The three years of work raise very important points for the research on G-quadruplex. First, the modeling of a theoretical G-quadruplex structure can be achieved by sequence homology and validated by calculations of a theoretical circular dichroism spectrum. Consequently, it is possible to use these tools to propose and use a G-quadruplex structure if it is not yet experimentally solved. Then, the work done shows that G-quadruplex form a very stable folding since they are globally conserved even when 8-oxo-guanine or strand breaks lesions are introduced at the quartets. Then, the paper focuses on the interaction between G-quadruplex and proteins. It highlights the important role of G-quadruplex RNA in the infection of the viral pathogen SARS-CoV-2. This RNA promotes the dimerization of the SUD protein of the virus, which in turn is responsible for the disruption of the immune system. Finally, this thesis provides a structural explanation for the specific interaction between the DARPin 2E4 protein and the G-quadruplex of the c-Myc promoter