Hydrolysis of the damaged deoxythymidine glycol nucleoside and comparison to canonical DNA.

Genomic integrity is continually under attack by both endogenous and exogenous sources. One of the most common forms of damage is oxidation of the thymine nucleobase to form (5R,6S)-dihydroxy-5,6-dihydro-thymine (thymine glycol or Tg), which stops DNA polymerases and is thus cytotoxic. Thymine glycol damage is repaired through a variety of mechanisms, including the multi-step base excision repair (BER) pathway. In the first BER step, the glycosidic bond of the dTg nucleotide is hydrolyzed by a DNA glycosylase. In order to understand the catalytic effect of the glycosylases, the corresponding uncatalyzed mechanisms and barriers are required, as well as an appreciation of the relative reactivity of the glycosidic bond with respect to the corresponding canonical nucleoside. To this end, the PCM-B3LYP/6-31+G(d) reaction potential energy surfaces (PES) for deoxythymidine (dT) and dTg hydrolysis are characterized in the present study using solvent-phase optimizations and a model containing three explicit water molecules. The surfaces are comparable to those generated using functionals that account for dispersion interactions (B3LYP-D3 and M06-2X). Mapping the PES as a function of the glycosidic bond length and nucleophile-sugar distance reveals a synchronous S(N)2 mechanism as the lowest energy pathway for damaged dTg hydrolysis, which contrasts the preferred dissociative S(N)1 mechanism isolated for the deglycosylation of natural dT. As proposed for other enzymes, the difference in excision pathway may at least in part help the enzyme selectively target the damaged base and discriminate against the natural counterpart. Interestingly, the barrier to dTg deglycosylation (ΔG(‡) = 138.0 kJ mol(-1)) is much higher than for dT deglycosylation (ΔG(‡) = 112.7 kJ mol(-1)), which supports the stability of this lesion and clarifies the catalytic feat presented to DNA repair enzymes that remove this detrimental damage from the genome. Although nucleotide excision repair (NER) typically targets bulky DNA lesions, the large calculated barrier for dTg deglycosylation rationalizes why the NER mechanism also excises this non-bulky lesion from cellular DNA.

Combined effects of π-π stacking and hydrogen bonding on the (N1) acidity of uracil and hydrolysis of 2'-deoxyuridine.

M06-2X/6-31+G(d,p) is used to study the simultaneous effects of π-π stacking interactions with phenylalanine (modeled as benzene) and hydrogen bonding with small molecules (HF, H(2)O, and NH(3)) on the N1 acidity of uracil and the hydrolytic deglycosylation of 2'-deoxyuridine (dU) (facilitated by fully (OH(-)) or partially (HCOO(-)···H(2)O) activated water). When phenylalanine is complexed with isolated uracil, the proton affinity of all acceptor sites significantly increases (by up to 28 kJ mol(-1)), while the N1 acidity slightly decreases (by ~6 kJ mol(-1)). When small molecules are hydrogen bound to uracil, addition of the phenylalanine ring can increase or decrease the acidity of uracil depending on the number and nature (acidity) of the molecules bound. Furthermore, a strong correlation between the effects of π-π stacking on the acidity of U and the dU deglycosylation reaction energetics is found, where the hydrolysis barrier can increase or decrease depending on the nature and number of small molecules bound, the nucleophile considered (which dictates the negative charge on U in the transition state), and the polarity of the (bulk) environment. These findings emphasize that the catalytic (or anticatalytic) role of the active-site aromatic amino acid residues is highly dependent on the situation under consideration. In the case of uracil-DNA glycosylase (UNG), which catalyzes the hydrolytic excision of uracil from DNA, the type of discrete hydrogen-bonding interactions with U, the nature of the nucleophile, and the anticipated weak, nonpolar environment in the active site suggest that phenylalanine will be slightly anticatalytic in the chemical step, and therefore experimentally observed contributions to catalysis may entirely result from associated structural changes that occur prior to deglycosylation.

Effects of extending the computational model on DNA-protein T-shaped interactions: the case of adenine-histidine dimers.

The MP2/6-31G*(0.25) π-π or π(+)-π T-shaped (edge-to-face) interactions between neutral or protonated histidine and adenine were considered using computational models of varying size to determine the effects of the protein and DNA backbones on the preferred dimer structure and binding strength. The overall consequences of the backbones are reasonably subtle for the neutral adenine-histidine T-shaped dimers. Furthermore, the minor changes in the binding strengths of these dimers upon model extension arise from additional (attractive) backbone-π (bb-π) contacts and changes in the preferred π-π orientations, which is verified by the quantum theory of atoms in molecules (QTAIM). Since the binding strength of the extended dimer equals the sum of the individual backbone-π and π-π contributions, the π-π component is not appreciably affected by polarization of the ring upon inclusion of the biological backbone. In contrast, the larger effect of the backbone on the protonated histidine dimers cannot simply be predicted as the sum of changes in the π-π and bb-π components regardless of the dimer type or model. This suggests, and QTAIM qualitatively supports, that the magnitude of the π(+)-π contribution changes, which is likely due to alterations in the electrostatic landscape of the monomer rings upon inclusion of the biological backbone that largely affect T-shaped dimers. These findings differ from those previously reported for (neutral) π-π stacked and (metallic) cation-π interactions, which highlights the distinct properties of each (π-π, π(+)-π, and cation-π) classification of noncovalent interaction. Furthermore, these results emphasize the importance of considering backbone-π interactions when analyzing contacts that appear in experimental crystal structures and cautions the use of truncated models when evaluating the magnitude of the π(+)-π contribution present in large biological complexes.

Concerning the hydrolytic stability of 8-aryl-2'-deoxyguanosine nucleoside adducts: implications for abasic site formation at physiological pH.

Direct addition of aryl radical species to the C(8)-site of 2'-deoxyguanosine (dG) affords C(8)-aryl-dG adducts that are produced by carcinogenic arylhydrazines, polycyclic aromatic hydrocarbons (PAHs), and certain phenolic toxins. A common property of C(8)-arylpurine adduction is the accompaniment of abasic site formation. To determine how the C(8)-aryl moiety contributes to sugar loss, UV-vis spectroscopy has been employed to determine N(7) pK(a1) values and hydrolysis kinetics, while density functional theory (DFT) calculations have been utilized to probe the structural features and stability of the C(8)-aryl-dG adducts bearing different para and ortho substituents. In all cases, the C(8)-aryl-dG adducts adopt a syn conformation containing a strong O(5)'-H...N(3) hydrogen bond with the aryl ring twisted with respect to the nucleobase. The adducts undergo N(7)-protonation with ionization constants and calculated N(7) proton affinity (PA) values similar to those measured for dG. The hydrolysis kinetics shows that C(8)-aryl-dG nucleoside adducts are more prone than dG to acid-catalyzed hydrolysis, with those bearing para substituents having k(1) values that are ca. 90- to 200-fold larger than k(1) for dG, while the effects for the ortho adducts are only ca. 9- to 60-fold larger. Changes in the rate of hydrolysis are further explained by calculations showing that glycosidic bond cleavage in the syn orientation of both neutral and N(7)-protonated dG has a lower barrier than the anti orientation, and the bulky (phenyl) group further decreases the barrier. Despite adduct reactivity in acidic media, all adducts are relatively stable at physiological pH with t(1/2) approximately 25 days, suggesting that they are unlikely intermediates leading to abasic site formation at physiological pH. This information has allowed development of a new rationale for the tendency of abasic site formation to accompany C(8)-arylpurine adduction within duplex DNA at neutral pH.

Effects of the biological backbone on DNA-protein stacking interactions.

The pi-pi stacking (face-to-face) interactions between the five natural DNA or RNA nucleobases and the four aromatic amino acids were compared using three different types of dimers: (1) a truncated nucleoside (nucleobase) stacked with a truncated amino acid; (2) a truncated nucleoside (nucleobase) stacked with an extended amino acid; and (3) a nucleoside (extended nucleobase) stacked with a truncated amino acid. Systematic (MP2/6-31G*(0.25)) potential energy surface scans reveal important information about the effects of the deoxyribose sugar and protein backbone on the structure and binding energy between truncated nucleobase and amino acid models that are typically implemented in the literature. Most notably, electrostatic and steric interactions arising from the bulkiness of the biological backbones can change the preferred relative orientations of DNA and protein pi-systems. More importantly, the protein backbone can strengthen the stacking energy (by up to 10 kJ mol(-1)), while the deoxyribose moiety can strengthen or weaken the stacking interaction depending on the positioning of the amino acid relative to the sugar residue. These effects are likely due to additional interactions between the amino acid or nucleobase ring and the backbone in the extended monomer rather than significant changes in the properties of the biological pi-systems upon model extension. Since the present work reveals that all calculated DNA-protein stacking interactions are significant and approach the strength of other noncovalent interactions between biomolecules, both pi-pi and backbone-pi interactions must be considered when attempting to gain a complete picture of DNA-protein binding.