Replication of the Aristolochic Acid I Adenine Adduct (ALI-N6-A) by a Model Translesion Synthesis DNA Polymerase: Structural Insights on the Induction of Transversion Mutations from Molecular Dynamics Simulations.

Exposure to aristolochic acid I and II (AAI and AAII) has been implicated in aristolochic acid nephropathy and urothelial carcinoma. The toxicological effects of AAs are attributed to their ability to form aristolacatam (AL)-purine DNA adducts. Among these lesions, the AL-adenine (ALI-N6-A and ALII-N6-A) adducts cause the "signature" A → T transversion mutations associated with AA genotoxicity. To provide the currently missing structural basis for the induction of these signature mutations, the present work uses classical all-atom molecular dynamics simulations to examine different (i.e., preinsertion, insertion, and postextension) stages of replication past the most abundant AA adduct (ALI-N6-A) by a representative lesion-bypass DNA polymerase (Dpo4). Our analysis reveals that, before dNTP incorporation (i.e., preinsertion step), ALI-N6-A adopts a nearly planar conformation at the N6-linkage and the ALI moiety intercalates within the DNA helix. Since this conformation occupies the dNTP binding site, the same planar lesion conformation results in a significant distortion of the polymerase active site at the insertion step and therefore replication will likely not be successful. However, if ALI-N6-A undergoes a small conformational change to introduce non-planarity at the N6-linkage during the insertion step, minimal distortion occurs in the Dpo4 active site upon incorporation of dATP. This insertion and subsequent extension would initially lead to A:A mismatches and then result in A → T transversion mutations during the second round of replication. In contrast, if a large conformation flip of the ALI moiety occurs at the insertion step to reorient the bulky moiety from an intercalated position into the major groove, dTTP (non-mutagenic) incorporation will be favored. Molecular dynamics (MD) simulations on postextension complexes reveal that damaged DNA will likely further rearrange during later replication steps to acquire a base-displaced intercalated conformation that is similar to that previously reported for (unbound) ALI-N6-A adducted DNA, with the exception of slight non-planarity at the lesion site. Overall, our results provide a structural explanation for both the successful non-mutagenic lesion bypass and the preferential misincorporation of dATP opposite ALI-N6-A and thereby rationalize the previously reported induction of A → T signature transversion mutations associated with AAs. This work should thereby inspire future biochemical experiments and modeling studies on the replication of this important class of DNA lesions by related human translesion synthesis polymerases.

Can modified DNA base pairs with chalcogen bonding expand the genetic alphabet? A combined quantum chemical and molecular dynamics simulation study.

A comprehensive (DFT and MD) computational study is presented with the goal to design and analyze model chalcogen-bonded modified nucleobase pairs that replace one (i.e., AXY:T, G:CXY, GXY:C) or two (GXY:CX'Y', X/X' = S, Se and Y/Y' = F, Cl, Br) Watson-Crick (WC) hydrogen bonds of the canonical A:T or G:C pair with chalcogen bond(s). DFT calculations on 18 base pair combinations that replace one WC hydrogen bond with a chalcogen bond reveal that the bases favorably interact in the gas phase (binding strengths up to -140 kJ mol-1) and water (up to -85 kJ mol-1). Although the remaining hydrogen bond(s) exhibits similar characteristics to those in the canonical base pairs, the structural features of the (Y-XO) chalcogen bond(s) change significantly with the identity of X and Y. The 36 doubly-substituted (GXY:CX'Y') base pairs have structural deviations from canonical G:C similar to those of the singly-substituted modifications (G:CXY or GXY:C). Furthermore, despite the replacement of two strong hydrogen bonds with chalcogen bonds, some GXY:CX'Y' pairs possess comparable binding energies (up to -132 kJ mol-1 in the gas phase and up to -92 kJ mol-1 in water) to the most stable G:CXY or GXY:C pairs, as well as canonical G:C. More importantly, G:C-modified pairs containing X = Se (high polarizability) and Y = F (high electronegativity) are the most stable, with comparable or slightly larger (by up to 13 kJ mol-1) binding energies than G:C. Further characterization of the chalcogen bonding in all modified base pairs (AIM, NBO and NCI analyses) reveals that the differences in the binding energies of modified base pairs are mainly dictated by the differences in the strengths of their chalcogen bonds. Finally, MD simulations on DNA oligonucleotides containing the most stable chalcogen-bonded base pair from each of the four classifications (AXY:T, G:CXY, GXY:C and GXY:CX'Y') reveal that the singly-modified G:C pairs best retain the local helical structure and pairing stability to a greater extent than the modified A:T pair. Overall, our study identifies two (G:CSeF and GSeF:C) promising pairs that retain chalcogen bonding in DNA and should be synthesized and further explored in terms of their potential to expand the genetic alphabet.

Molecular Dynamics Simulations of Mismatched DNA Duplexes Associated with the Major C8-Linked 2'-Deoxyguanosine Adduct of the Food Mutagen Ochratoxin A: Influence of Opposing Base, Adduct Ionization State, and Sequence on the Structure of Damaged DNA.

Exposure to ochratoxin A (OTA) is associated with chronic renal diseases and carcinogenesis. The deleterious effects of OTA have been linked to its covalent binding at the C8 position of guanine (G) to form a DNA adduct (OT-G), which causes various mutations. To contribute toward understanding the complex mutagenic profile of OTA, the present work uses a robust computational approach to characterize postreplication DNA structures containing OT-G mismatched with canonical nucleobases. Our MD simulations provide insight into the effects of the opposing base, adduct ionization state, and flanking base on duplex structural features for the competing (major groove (B-type), wedge (W), and stacked (S)) conformers. For the B-type duplexes, our data suggest that significantly more stable lesion-site hydrogen bonding may lead to preferential insertion of an opposing cytosine (C) if the OT moiety is directed toward the major groove at the replication fork. Although the W conformation is consistently predicted to be less stable than the B conformer, a G mismatch is likely the most stable and least distorted replication outcome when the bulky moiety is directed into the DNA minor groove. These findings directly correlate with the limited contribution of substitution mutations to the overall mutagenic profile of OTA and suggest that the dominant mutations are G → C transversions. In contrast, stable S conformers that are known precursors to small (one- or two-base) deletion mutations are found when the lesion is opposite cytosine, adenine, or thymine, which directly correlates with the large number of deletion mutations previously reported for animals exposed to OTA. Nevertheless, the predicted sequence and ionization-dependent distortion of the S conformer points toward the dependence of the repair propensity on the cellular environment, which rationalizes the reported tissue specific OTA-induced toxicity.

Molecular Modeling of the Major DNA Adduct Formed from Food Mutagen Ochratoxin A in NarI Two-Base Deletion Duplexes: Impact of Sequence Context and Adduct Ionization on Conformational Preference and Mutagenicity.

Exposure to ochratoxin A (OTA), a possible human carcinogen, leads to many different DNA mutations. As a first step toward understanding the structural basis of OTA-induced mutagenicity, the present work uses a robust computational approach and a slipped mutagenic intermediate model previously studied for C8-dG aromatic amine adducts to analyze the conformational features of postreplication two-base deletion DNA duplexes containing OT-dG, the major OTA lesion at the C8 position of guanine. Specifically, a total of 960 ns of molecular dynamics simulations (excluding trial simulations) were carried out on four OT-dG ionization states in three sequence contexts within oligomers containing the NarI recognition sequence, a known hotspot for deletion mutations induced by related adducts formed from known carcinogens. Our results indicate that the structural properties and relative stability of the competing "major groove" and "stacked" conformations of OTA adducted two-base deletion duplexes depend on both the OTA ionization state and the sequence context, mainly due to conformation-dependent deviations in discrete local (hydrogen-bonding and stacking) interactions at the lesion site, as well as DNA bending. When the structural characteristics of the OT-dG adducted two-base deletion duplexes are compared to those associated with previously studied C8-dG adducts, a greater understanding of the effects of the nucleobase-carcinogen linkage, and size of the carcinogenic moiety on the conformational preferences of damaged DNA is obtained. Most importantly, our work predicts key structural features for OT-dG-adducted deletion DNA duplexes, which in turn allow us to develop hypotheses regarding OT-dG replication outcomes. Thus, our computational results are valuable for the design and interpretation of future biochemical studies on the potentially carcinogenic OT-dG lesion.

How do hydrophobic nucleobases differ from natural DNA nucleobases? Comparison of structural features and duplex properties from QM calculations and MD simulations.

Computational (DFT and MD simulation) methods are employed to systematically characterize the structural and energetic properties of five hydrophobic nucleobases (FEMO, MMO2, NaM, 5SICS and TPT3) that constitute four unnatural base pairs (FEMO:5SICS, MMO2:5SICS, NaM:5SICS and TPT3:NaM). These hydrophobic bases have been recently shown to be replicated when present between natural bases in DNA duplexes, with the highest replication fidelity and efficiency occuring for the TPT3:NaM pair. Our QM calculations suggest that the preferred (anti) glycosidic orientations of nucleosides containing hydrophobic bases are similar to the natural DNA nucleosides despite differences in their chemical structures. However, due to the inability to form interbase hydrogen bonds, hydrophobic base pairs intrinsically prefer nonplanar, distorted geometries, many of which are stabilized through π-π stacking interactions. Furthermore, the intrinsic stacking potential between a hydrophobic and a natural base is similar to that between two natural bases, indicating that the strength of stacking interactions in DNA duplexes containing hydrophobic bases is likely comparable to natural DNA. However, in contrast to the isolated base-pair geometries, our MD simulations suggest that the hydrophobic base pairs adopt variable geometries within DNA, which range from stacked (5SICS:FEMO) to nearly planar (5SICS:NaM and SICS:MMO2) to planar (TPT3:NaM). As a result, the duplex structural features at the site of modification depend on the identity of the hydrophobic base pair, where the TPT3:NaM pair causes the least structural changes compared to natural DNA. Overall, the structural insight obtained from our calculations on DNA containing hydrophobic base pairs explains the experimentally-observed higher fidelity and efficiency during replication of TPT3:NaM compared to other hydrophobic nucleobase pairs. By providing valuable structural information that explains the intrinsic and duplex properties of this class of unnatural nucleobases, the present work may aid the future design of improved hydrophobic analogues.

Adenine versus guanine DNA adducts of aristolochic acids: role of the carcinogen-purine linkage in the differential global genomic repair propensity.

Computational modeling is employed to provide a plausible structural explanation for the experimentally-observed differential global genome repair (GGR) propensity of the ALII-N(2)-dG and ALII-N(6)-dA DNA adducts of aristolochic acid II. Our modeling studies suggest that an intrinsic twist at the carcinogen-purine linkage of ALII-N(2)-dG induces lesion site structural perturbations and conformational heterogeneity of damaged DNA. These structural characteristics correlate with the relative repair propensities of AA-adducts, where GGR recognition occurs for ALII-N(2)-dG, but is evaded for intrinsically planar ALII-N(6)-dA that minimally distorts DNA and restricts the conformational flexibility of the damaged duplex. The present analysis on the ALII adduct model systems will inspire future experimental studies on these adducts, and thereby may extend the list of structural factors that directly correlate with the propensity for GGR recognition.

Conformational preferences of DNA following damage by aristolochic acids: Structural and energetic insights into the different mutagenic potential of the ALI and ALII-N(6)-dA adducts.

Aristolochic acids (AAI and AAII), produced by the Aristolochiaceae family of plants, are classified as group I (human) carcinogens by the International Agency for Research on Cancer. These acids are metabolized in cells to yield aristolactams (ALI and ALII, respectively), which further form bulky adducts with the purine nucleobases. Specifically, the adenine lesions are more persistent in cells and have been associated with chronic renal diseases and related carcinogenesis. To understand the structural basis of the nephrotoxicity induced by AAs, the ALI-N(6)-dA and ALII-N(6)-dA lesions are systematically studied using computational methods. Density functional theory calculations indicate that the aristolactam moiety intrinsically prefers a planar conformation with respect to adenine. Nucleoside and nucleotide models suggest that the anti and syn orientations about the glycosidic bond are isoenergetic for both adducts. Molecular dynamics simulations and free energy calculations reveal that the anti base-displaced intercalated conformation is the most stable conformer for both types of AL-N(6)-dA adducted DNA, which agrees with previous experimental work on the ALII-N(6)-dA adduct and thereby validates our approach. Interestingly, this conformer differs from the dominant conformations adopted by other N6-linked adenine lesions, including those derived from polycyclic aromatic hydrocarbons. Furthermore, the second most stable syn base-displaced intercalated conformation lies closer in energy to the anti base-displaced intercalated conformation for ALI-N(6)-dA compared to ALII-N(6)-dA. This indicates that a mixture of conformations may be detectable for ALI-N(6)-dA in DNA. If this enhanced conformational flexibility of double-stranded DNA persists when bound to a lesion-bypass polymerase, this provides a possible structural explanation for the previously observed greater nephrotoxic potential for the ALI versus ALII-N(6)-dA adduct. In addition, the structural characteristics of the preferred conformations of adducted DNA explain the resistance of these adducts to repair and thereby add to our current understanding of the toxicity of AAs within living cells.

Molecular Dynamics Study of One-Base Deletion Duplexes Containing the Major DNA Adduct Formed by Ochratoxin A: Effects of Sequence Context and Adduct Ionization State on Lesion Site Structure and Mutagenicity.

Ochratoxin A (OTA) is a ubiquitous food toxin associated with chronic nephropathy in humans and renal carcinogenicity in rodents. The mutational spectra of cells exposed to OTA reveal that one-base deletions comprise the largest percentage (73%) of the total mutations that occur upon OTA exposure. To contribute toward understanding the prevalence of OTA-induced one-base deletion mutations, the present work uses molecular dynamics (MD) simulations to analyze the conformational preferences of one-base deletion duplexes containing OT-G, the major OTA adduct (addition product) at the C8-site of guanine. Specifically, the influence of OT-G in four possible ionization states and three sequence contexts (G1, G2 and G3 in the NarI (5'-G1G2CG3CC-3'), a prokaryotic mutational hotspot sequence) on the structure of the adducted DNA is investigated. Our data reveal that the damaged helices are stable in two (B-type (B) and stacked (S)) conformations that are structurally similar to those adopted by common N-linked C8-guanine lesions. However, the adduct ionization state and sequence context affect the degree of helical distortion and the B/S conformational heterogeneity, which will impact the lesion repair and replication outcomes. This finding correlates with the experimentally reported tissue-specific mutagenicity of OTA exposure. Furthermore, regardless of the adduct conformation, ionization state, or sequence context, more stable lesion-site interactions and lack of disruption of the flanking base pairs in the one-base deletion duplexes compared to the corresponding two-base deletion helices rationalize the greater abundance of OTA induced one-base deletions. Overall, our work provides valuable structural insights that help explain the experimentally observed mutagenicity associated with OTA.

Effect of base sequence context on the conformational heterogeneity of aristolactam-I adducted DNA: structural and energetic insights into sequence-dependent repair and mutagenicity.

Aristolochic acids (AAs) are nephrotoxic and potentially carcinogenic plant mutagens that form bulky DNA adducts at the exocyclic amino groups of the purines. The present work utilizes classical molecular dynamics simulations and free energy calculations to investigate the role of lesion site sequence context in dictating the conformational outcomes of DNA containing ALI-N6-dA, the most persistent and mutagenic adduct arising from the AAs. Our calculations reveal that the anti base-displaced intercalated conformer is the lowest energy conformer of damaged DNA in all sequence contexts considered (CXC, CXG, GXC and GXG). However, the experimentally-observed greater mutagenicity of the adduct in the CXG sequence context does not correlate with the relative thermodynamic stability of the adduct in different sequences. Instead, AL-N6-dA adducted DNA is least distorted in the CXG sequence context, which points toward a possible differential repair propensity of the lesion in different sequences. Nevertheless, the structural deviations between adducted DNA with different lesion site sequences are small, and therefore other factors (such as interactions between the adducted DNA and lesion-bypass polymerases during replication) are likely more important for dictating the observed sequence-dependent mutagenicity of ALI-N6-dA.