Anatomy of noncovalent interactions between the nucleobases or ribose and π-containing amino acids in RNA-protein complexes.

A set of >300 nonredundant high-resolution RNA-protein complexes were rigorously searched for π-contacts between an amino acid side chain (W, H, F, Y, R, E and D) and an RNA nucleobase (denoted π-π interaction) or ribose moiety (denoted sugar-π). The resulting dataset of >1500 RNA-protein π-contacts were visually inspected and classified based on the interaction type, and amino acids and RNA components involved. More than 80% of structures searched contained at least one RNA-protein π-interaction, with π-π contacts making up 59% of the identified interactions. RNA-protein π-π and sugar-π contacts exhibit a range in the RNA and protein components involved, relative monomer orientations and quantum mechanically predicted binding energies. Interestingly, π-π and sugar-π interactions occur more frequently with RNA (4.8 contacts/structure) than DNA (2.6). Moreover, the maximum stability is greater for RNA-protein contacts than DNA-protein interactions. In addition to highlighting distinct differences between RNA and DNA-protein binding, this work has generated the largest dataset of RNA-protein π-interactions to date, thereby underscoring that RNA-protein π-contacts are ubiquitous in nature, and key to the stability and function of RNA-protein complexes.

Effects of a Second Local DNA Damage Event on the Toxicity of the Human Carcinogen 4-Aminobiphenyl: A Molecular Dynamics Study of a Damaged DNA Structure.

Exposure of humans to carcinogenic aromatic amines (AAs) occurs daily. AAs are bioactivated in cells into products that attack DNA, primarily leading to N-linked C8-dG adducts. Previous work on DNA containing a single AA-derived adduct (monoadducted DNA) has shown a structure-function relationship between the damaged DNA conformation and cellular outcomes. However, relatively little is known about the conformation and biological outcomes of DNA containing two bulky adducts (diadducted DNA) in close proximity. To fill this current void in the literature, the present work uses quintuplet 0.5 µs MD simulations to understand the structural impact of DNA exposure to the potent bladder carcinogen 4-aminobiphenyl (ABP), which is found in cigarette smoke and select dyes, and results in the widely studied N-linked ABPdG adduct. Specifically, 18 unique DNA duplexes were investigated that contain one or two ABPdG adducts in the anti and/or syn glycosidic orientation(s) in all combinations of three G positions in the NarI mutation hotspot for AAs (5'-G1G2CG3CC). Monoadducted DNA displays sequence-dependent conformational heterogeneity, with the G1 site having the greatest anti preference, and highlights the range of helical structures associated with the syn lesion orientation [i.e., stacked (S), intercalated (I), and wedge (W) conformations]. Diadducted DNA results in interesting lesion separation effects on the conformational heterogeneity, including a greater anti preference for neighboring adducts (G1G2) and a greater syn preference for next-nearest neighbor damaged sites (G2G3) compared to monoadducted DNA. As a result, an increase in the number of ABPdG adducts changes the conformational heterogeneity of ABP-exposed DNA depending on the relative positions of the lesions and thereby could result in increased or decreased toxicity upon human exposure to elevated levels of ABP.

Impact of DNA Adduct Size, Number, and Relative Position on the Toxicity of Aromatic Amines: A Molecular Dynamics Case Study of ANdG- and APdG-Containing DNA Duplexes.

Human exposure to aromatic amines (AAs) can result in carcinogenic DNA adducts. To complement previous work geared toward understanding the mutagenicity of AA-derived adducts, which has almost exclusively studied (monoadducted) DNA containing a single lesion, the present work provides the first in-depth comparison of the structure of monoadducted and diadducted DNA duplexes. Specifically, molecular dynamics (MD) simulations were initially performed on DNA containing the nonmutagenic single-ringed N-(deoxyguanosin-8-yl)-aniline (ANdG) or the mutagenic four-ringed N-(deoxyguanosin-8-yl)-1-aminopyrene (APdG) lesion at G1, G2, or G3 in the AA deletion hotspot (5'-G1G2CG3CC) in the anti or syn glycosidic orientation (B/S duplex conformation). Subsequently, diadducted strands were assessed that span each combination of damaged sites (G1G2 (nearest neighbors), G2G3 (next-nearest neighbors), and G1G3 (two intervening nucleotides)) and anti/syn lesion glycosidic orientations. Despite other N-linked C8-dG adducts exhibiting sequence dependence conformational heterogeneity, a single ANdG or APdG lesion induces helical conformational homogeneity that is exclusively controlled by aryl moiety size. However, the preferred damaged DNA conformation can change upon the addition of a second adduct depending on lesion separation, with neighboring lesions stabilizing a nonmutagenic conformation and next-nearest damaged sites stabilizing a promutagenic conformation regardless of adduct size. As a result, diadducted DNA is found to adopt conformations that are unfavored for the corresponding monoadducted system, pointing to differential replication and repair outcomes for diadducted DNA compared to those for monoadducted DNA. Thus, although the toxicity of monoadducted DNA is most significantly dictated by lesion size, the toxicity can increase or decrease upon a second damaging event depending on lesion size and relative position. Overall, our work adds the number of lesions and their spatial separation to the growing list of factors that determine the structure and biological outcomes of adducted DNA.

Structure of an Unusual Tetracyclic Deoxyguanosine Adduct: Implications for Frameshift Mutagenicity of ortho-Cyano Nitroanilines.

Nitroaromatic compounds represent a major class of industrial chemicals that are also found in nature. Polycyclic derivatives are regarded as potent mutagens and carcinogens following bioactivation to produce nitrenium electrophiles that covalently modify DNA to afford N-linked C8-2'-deoxyguanosine (C8-dG) lesions that can induce frameshift mutations, especially in CpG repeat sequences. In contrast, their monocyclic counterparts typically exhibit weak mutagenicity or a lack thereof, despite also undergoing bioactivation to afford N-linked C8-dG adducts. Recently, it has been reported that cyano substitution can greatly increase the mutagenicity of nitroaniline derivatives that are components of azo dyes. The basis of this "cyano effect" may be rooted in the formation of a novel polycyclic adduct arising from initial formation of the N-linked C8-dG adduct followed by a cyclization process involving N7 of dG and the ortho-CN group of the attached C8-aryl moiety to generate a quinazolinimine ring as part of a fused tetracyclic C8,N7-dG adduct structure. The present work structurally characterizes this novel cyclic adduct using a combination of optical spectroscopies, NMR analysis, density functional theory (DFT) calculations, and molecular dynamics (MD) simulations. Our data indicate that this highly fluorescent cyclic adduct adopts the promutagenic syn conformation and can stabilize the slipped mutagenic intermediate (SMI) within the CpG repeat of the NarI sequence, which is a hotspot for frameshift mutagenesis mediated by polycyclic N-linked C8-dG adducts. In contrast, the open para-CN (4-aminobenzontrile-derived) N-linked C8-dG adduct is less likely to disrupt the canonical B-form. Together, our results provide a rationale for the potent mutagenicity of cyano-substituted nitroaniline derivatives recently reported in frameshift-sensitive tester strains.

Conformational Preference and Fluorescence Response of a C-Linked C8-Biphenyl-Guanine Lesion in the NarI Mutational Hotspot: Evidence for Enhanced Syn Adduct Formation.

Aromatic chemical carcinogens can undergo enzymatic transformations to produce a range of electrophilic species that attach covalently to the C8-site of 2'-deoxyguanosine (dG) to afford C8-dG adducts. The most studied C8-dG adducts are formed from arylamines and contain a N-linkage separating the dG from the C8-aryl moiety. Other carcinogenic species result in direct aryl ring attachment to the dG moiety, resulting in C-linked adducts. The resulting C-linked adducts have reduced conformational flexibility compared to the corresponding N-linked C8-dG adducts, which can alter their orientation in the DNA duplex. Described herein are structural studies of a fluorescent C-linked 4-fluorobiphenyl-dG (FBP-dG) that has been incorporated into the reiterated G3-postion of the 12-mer NarI sequence and those containing other 5'-flanking nucleobases. FBP-dG displays a strong preference for adopting a syn conformation in the fully paired NarI duplex to produce an intercalated structure that exhibits stacking interactions between the C-linked biphenyl and the flanking bases. FBP-dG is also shown to significantly stabilize the slippage mutagenic intermediate (SMI) duplex containing the lesion and 5'-flanking base within a 2-base bulge. FBP-dG exhibits fluorescence sensitivity to SMI duplex formation that can readily distinguish it from the fully paired duplex. Molecular dynamics simulations and optical spectroscopy for the NarI oligonucleotides containing the C-linked FBP-dG predict increased rigidity of the biphenyl in the syn conformation. The greater propensity to generate the promutagenic syn conformation for the C-linked FBP-dG adduct compared to the N-linked 4-aminobiphenyl-dG adduct (ABP-dG) suggests greater mutagenicity for the C-linked analogue. These results highlight the effect of the adduct linkage type on the conformational properties of adducted DNA. The turn-on emission response of FBP-dG in the SMI duplex may be a powerful tool for monitoring SMI formation in the NarI sequence upon synthesis with DNA polymerases.

Effect of Size and Shape of Nitrogen-Containing Aromatics on Conformational Preferences of DNA Containing Damaged Guanine.

The present work investigates the effects of the size and shape of the nitrogen-containing aromatic (NCA) skeleton on the structure of DNA damaged through adduct formation at C8 of 2'-deoxyguanosine (dG), a common DNA lesion associated with chemical carcinogenesis. Specifically, density functional theory (DFT) calculations (B3LYP-D3) and molecular dynamics (MD) simulations (AMBER) are performed on seven model adducts with systematic expansion of the NCA moiety. DFT calculations reveal that the NCA moiety shape affects the structure at the nucleobase-carcinogen linkage. Approximately 4.5 µs of MD simulations on damaged oligonucleotides adopting three established conformational themes (namely, B, W, and S) illustrate that the structure and lesion-site stabilization strongly depend on the NCA moiety shape and size, which provides insight into the repair propensity of C8-dG adducted DNA. Our results add bulky moiety shape to the growing list of previously established effects on the conformational and repair outcomes of damaged DNA (i.e., size, ionization state, substitution, linker type, and DNA sequence). Furthermore, this work illustrates the utility of a systematic set of model DNA lesions for understanding the structure-activity relationship for DNA damaged by carcinogens of different sizes and shapes, which should be used in future studies of the cellular processing of damaged DNA.

Effect of Guanine Adduct Size, Shape, and Linker Type on the Conformation of Adducted DNA: A DFT and Molecular Dynamics Study.

DNA is damaged through various exogenous sources (e.g., automobile exhaust, tobacco smoke, and processed foods), which can yield diverse C8-dG bulky aryl adducts. Adducts are known to induce structural changes to DNA that can lead to various biological outcomes, ranging from cell death to diseases such as cancer. Unfortunately, the relationship between the chemical composition of the damaged product, the adducted DNA structure, and the biological consequences is not well understood, which limits the development of disease detection and prevention strategies. The present study uses density functional theory (DFT) calculations and quintuplicate 1 µs molecular dynamics (MD) simulations to characterize the structure of DNA containing 21 model C8-dG adducts that systematically differ in size (phenyl to pyrenyl), shape (α (2,3), ß (3,4) fusion, or ring substitution), and nucleobase-aryl group linkage (N, O, and C-linked). DFT calculations reveal that the inherent structural features of the G nucleobase adducts are impacted by linker type and bulky moiety shape, but not size, with the conformational flexibility reducing with α-ring fusion and linker composition as N > O > C. These structural properties are maintained in nucleoside models, which also reveal an increased propensity for anti-to-syn rotation about the glycosidic bond with N < O < C linker type. Although these diverse chemical features do not influence the global structure of adducted DNA, the adducts differentially impact the conformation local to the adducted site, including the relative populations of structures with the bulky moiety in the major groove (B conformer) and intercalated (stacked) into the helix (S conformer). Specifically, while the smallest phenyl adducts favor the B conformation and the largest pyrenyl-derived adducts stabilize the S conformation, the B/S ratio decreases with an increase in ring size and N > O > C linker composition. The shape and size (length) of the adduct can further finetune the B/S ratio, with ß-fused naphthyl or α-fused phenanthryl N-linked adducts and O or C-linked adducts containing ring substitution increasing the prevalence of the S adducted DNA conformation. Overall, this work uncovers the significant effect of bulky moiety size and linker type, as well as the lesser impact of aryl group shape, on adducted DNA structure, which suggests differential replication and repair outcomes, and thereby represents an important step toward rationalizing connections between the structure and biological consequences of diverse DNA adducts.

Landscape of π-π and sugar-π contacts in DNA-protein interactions.

There were 1765 contacts identified between DNA nucleobases or deoxyribose and cyclic (W, H, F, Y) or acyclic (R, E, D) amino acids in 672 X-ray structures of DNA-protein complexes. In this first study to compare π-interactions between the cyclic and acyclic amino acids, visual inspection was used to categorize amino acid interactions as nucleobase π-π (according to biological edge) or deoxyribose sugar-π (according to sugar edge). Overall, 54% of contacts are nucleobase π-π interactions, which involve all amino acids, but are more common for Y, F, and R, and involve all DNA nucleobases with similar frequencies. Among binding arrangements, cyclic amino acids prefer more planar (stacked) π-systems than the acyclic counterparts. Although sugar-π interactions were only previously identified with the cyclic amino acids and were found to be less common (38%) than nucleobase-cyclic amino acid contacts, sugar-π interactions are more common than nucleobase π-π contacts for the acyclic series (61% of contacts). Similar to DNA-protein π-π interactions, sugar-π contacts most frequently involve Y and R, although all amino acids adopt many binding orientations relative to deoxyribose. These DNA-protein π-interactions stabilize biological systems, by up to approximately -40 kJ mol(-1) for neutral nucleobase or sugar-amino acid interactions, but up to approximately -95 kJ mol(-1) for positively or negatively charged contacts. The high frequency and strength, despite variation in structure and composition, of these π-interactions point to an important function in biological systems.