Molecular dynamics simulations of mismatched DNA duplexes

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Molecular dynamics simulations of mismatched DNA duplexes associated with the major C-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 8

Preetleen Kathuria, Purshotam Sharma, Richard A. Manderville, and Stacey D Wetmore Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.8b00064 • Publication Date (Web): 20 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018

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Chemical Research in Toxicology

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 Preetleen Kathuria,1 Purshotam Sharma,1 Richard A. Manderville2 and Stacey D. Wetmore3* 1Computational

Biochemistry Laboratory, Department of Chemistry and Centre for Advanced

Studies in Chemistry, Panjab University, Chandigarh, India 160014.

2Departments

of

Chemistry and Toxicology, University of Guelph, Guelph, Ontario, Canada N1G 2W1. 3Department

of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta,

Canada T1K 3M4. *E-mail: [email protected]. Telephone: (403) 329-2323. Fax: (403) 329-2057

TOC Graphic:

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ABSTRACT 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 C8 position of guanine (G) to form DNA adduct (OT-G), which causes various mutations. To contribute towards understanding the complex mutagenic profile of OTA, the present work uses a robust computational approach to characterize post-replication 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 suggests 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. While 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 deletions mutations previously reported for animals exposed to OTA. Nevertheless, the predicted sequence and ionization dependent distortion of the S


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conformer points towards the dependence of the repair propensity on the cellular environment, which rationalizes the reported tissue specific OTA-induced toxicity. INTRODUCTION Ochratoxin A (OTA) is a mycotoxin mainly produced by some species of Aspergillus1 and Penicillium,2 which commonly infect agricultural produce. OTA is a wellknown human nephrotoxin3 and is the most potent rodent renal carcinogen studied by the National Cancer Institute/National Toxicological Program.4 As a result, OTA has been classified as a group 2B (possible human) carcinogen by the International Agency for Research on Cancer.5 Due to considerable and unavoidable human exposure,6 understanding the impact of OTA on human health is critical for devising ways to manage the associated risks. Although a disagreement exists in the literature regarding the mechanism of action (MoA) of OTA-mediated toxicity and carcinogenicity, recent experiments in rodents favor a MoA based on direct genotoxicity,7-10 which involves OTA bio-activation to generate electrophilic species that form covalent adducts (addition products) at specific DNA sites.7 A number of studies have attempted to elucidate and characterize the chemical structure of DNA adducts arising from OTA exposure.11-14 This literature suggests that OTA forms adducts specific to guanine,14 and mainly affords a C8-bonded carbon-linked adduct (denoted OT-G, Figure 1A).13 In addition, a recent analysis of the mutational spectrum of DNA extracted from OTA exposed cells revealed an increase in deletion, insertion and substitution mutations in the presence of OTA compared to control cells.9 Molecular dynamics (MD) simulations on damaged DNA is a useful approach for gaining structural insights into the role of adduct formation in the associated mutagenicity. 3 ACS Paragon Plus Environment


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Specifically, MD simulations permit the study of DNA structural dynamics, which can be further used to explain experimental observations.15-17 Therefore, as a first step towards understanding the impact of OTA-exposure on DNA structure, we previously employed MD simulations to characterize the conformational preferences of DNA containing the OT-G adduct.18,

19

The adduct was paired against complementary cytosine in a DNA oligomer

containing the NarI recognition sequence (5′−G1G2CG3CC), which is the known hotspot for mutations induced by other carcinogenic C8-G adducts.20 Our analysis revealed that OTAdamaged DNA likely adopts a mixture of three distinct (i.e., the major groove (B), wedge (W) and base-displaced intercalated (S)) conformations (Figure 1B–D) regardless of the adduct ionization state and the lesion-site sequence context. Each of these conformations differ in the location of the OT moiety in the helix, and therefore likely interact with critical cellular machinery, including polymerases and repair enzymes, in unique ways. Although previous studies provide structural insights into the OT-G lesion in matched duplex DNA, analysis of post-replication structures can significantly contribute towards currently missing structural explanations for the complicated mutational profile of the lesion, including the preferred replication outcomes and the propensity of the adduct to be targeted by repair pathways. Unfortunately, however, no definitive information is available for the relative importance of different point mutations in OTA carcinogenesis, and post-replication structures of DNA duplexes containing OT-G mismatched against the canonical nucleobases have not been studied to date. Furthermore, in contrast to previously studied C8-G lesions,21-26 the bulky moiety of OT-G is larger and inherently more flexible. Furthermore, OT-G contains phenolic and carboxylic groups, and therefore can exist in different ionization states (carboxylic ionized (COO−), phenolic ionized (ArO−) and 4 ACS Paragon Plus Environment

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dianionic form (both carboxylic and phenolic ionized)) depending on the local pH.27, 28 It is not immediately clear how the bulky moiety chemical composition and ionization state will influence the structure of mismatched DNA duplexes or the relative occurrence of various OT-G mispairs. In an attempt to provide the molecular level clues required to further understand the mutational profile of OTA, the present work uses MD simulations to analyze the structure of DNA duplexes containing OT-G substitution mutations in varying sequence contexts. These newly characterized conformers of OT-G mismatched duplexes are compared to the corresponding duplexes containing OT-G correctly paired against complementary C.19 In addition to revealing the effect of the identity of the pairing base, sequence and OT-G ionization state on the structural features of OTA adducted DNA, postprocessing free energy calculations are used to predict the preferred DNA conformation and lesion-site base pairing. Our subsequent analysis of possible replication and repair outcomes associated with OT-G adducted DNA provides a viable explanation for the observed tissue-specific selective mutagenicity of OTA,8, 9 and highlights a novel theme of the dependence of the replication and repair propensity of adducted DNA on the cellular pH, which should be further probed and validated with future experimental biochemical studies. In addition, our results broaden our current understanding of how sequence context affects the structural features of adducted DNA,29-31 and specifically emphasize sequence effects on the structural features of duplexes containing an adduct mispair, which have only been investigated in one other context thus far (i.e., AF-G paired against A).24 COMPUTATIONAL DETAILS



MD

simulations

were

carried

out

on

a

12-mer

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DNA

oligonucleotide

(5′−CTCG1G2CG3CCATC) containing the NarI recognition sequence (underlined).20 The OT-G adduct was reiteratively placed in each of four ionization states at the G1, G2 or G3 position, and paired opposite A, T or G. Initial conformations were considered in accordance with the three (i.e., B, W and S) conformational themes previously reported for the C8-bonded aromatic amine adducts,31, 32 as well as OT-G paired opposite C.19 The natural and modified DNA nucleotides were described using the parmbsc0 modification33 of parm99,34 and additional parameters and partial charges for the OT-G nucleotide were taken from our previous studies.18,

19, 35

To isolate different conformations of the adduct in DNA and

maintain hydrogen bonding in the terminal base pairs, we performed a number of trial simulations. In the cases where a consistent conformation was not obtained, simulations were repeated with new starting structures, built from either the representative structure of a different OT-G ionization state or by adjusting the original starting structure. Following extensive exploratory trial simulations, a 40 ns explicit solvent MD production simulation was performed on each of the resulting 108 conformers of OT-G mismatched DNA duplexes. In synchrony with previous MD studies on natural36 and damaged36-38 DNA oligonucleotides, all simulation frames between 30 and 40 ns were used for comprehensive structural and free energy analyses. This was done to ensure the convergence of key DNA structural parameters and obtain the most reliable conclusions. Full details of the calculations, including the choice of initial structures, force field, MD protocol, and structural and energetic analyses are provided in the Supporting Information. RESULTS AND DISCUSSION Structural features of the B conformation of OT-G adducted mismatched DNA. 6 ACS Paragon Plus Environment

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In the B conformation, OT-G maintains an anti glycosidic orientation (χ ~ 204 – 221° and θ ~ 10 – 22°, Tables S1–S3). As a result, the OT moiety is directed towards the DNA major groove and the Watson-Crick (WC) edge of the damaged G is available to pair with the opposing base (Figures 1B and S2–S4). Specifically, two persistent (> 90% occupancy) and one transient (~ 32 − 80% occupancy) hydrogen bonds are formed between the WC edge of the lesion and the Hoogsteen edge of A for all adduct ionization states and sequence contexts (Figures 2A, S5–S7 and Table S4). However, against G, either normal Hoogsteen (i.e., (OT-G)N1−H∙∙∙O6(G) and (OT-G)N2−H∙∙∙N7(G) interactions, ~ 28 – 98% occupancy) or bifurcated (i.e., (OT-G)N2−H∙∙∙N7(G) and (OT-G)N1−H∙∙∙N7(G) interactions, ~ 62 – 97% occupancy) hydrogen bonding occurs with the Hoogsteen edge of the opposing base depending on the adduct ionization state and sequence context (Figures 2A, S5–S7 and Table S5). Specifically, except for dianionic OT-dG, persistent Hoogsteen hydrogen bonding occurs at G1 and the bifurcated pattern occurs at G2 for all OT-G ionization states. However, at G3, the ArO– state exclusively forms bifurcated bonding, whereas the Hoogsteen hydrogen bonding is more common for all other ionization states. In contrast, against anti T, although two interbase hydrogen bonds ((OT-G)N1−H∙∙∙O2(T) and (OT-G)O6∙∙∙H−N3(T)) persist in almost all structures (> 85% occupancy), a transient (OT-G)N2−H∙∙∙O2(T) interaction (~ 22 − 42% occupancy) forms in a few sequences for select OT-G ionization states (Figures 2A, S5–S7 and Table S6). However, neutral OT-G at G1 forms a bifurcated hydrogen bond with T ((OT-G)N1−H∙∙∙O2(T) and (OT-G)N2−H∙∙∙O2(T)) that persists throughout the simulation (~ 95 − 97% occupancy for each interaction, Figure S5 and Table S6). Overall, despite variations in the hydrogen-bonding occupancies, the lesion-site hydrogen-bonding pattern is unaffected by the adduct ionization state and 7 ACS Paragon Plus Environment


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sequence for the B conformers containing an A mismatch or the previously studied Cmatch,19 and is largely maintained for the T mismatch. However, the hydrogen-bonding pattern for G mismatched duplexes exhibits a marked ionization state and sequence dependence. In terms of the stability of the lesion pair, C hydrogen bonds more strongly with OTG (by 9 – 22 kcal mol–1) compared to A, G or T irrespective of adduct ionization state or sequence context (Figures 3A and S8−S10, and Table S7). However, the relative hydrogenbond strength of the mismatched pairs depends on the ionization state and sequence. For example, the G mispair is weaker than A or T mismatches for ArO− OT-G in all three sequence contexts (by up to 8 kcal mol–1, Figure S9). Alternatively, although neutral and COO– OT-G exhibit similar hydrogen-bonding interactions with A, T or G at G1 and G3 (within ~ 3 kcal mol–1, Figures 3A and S8), the stability of the G mismatch is less than that of the A and T mismatches at G2 (by ~ 4 – 8 kcal mol–1, Figures 3A and S8). In contrast to hydrogen-bond interactions, the strength of the lesion-site stacking weakly depends on the identity of the opposing base and sequence. Specifically, for a particular ionization state and sequence context, stacking energies change by only up to ~ 4 kcal mol–1 with the identity of the opposing base (i.e., C,19 G, A or T; Figures 3B and S11−S13, and Table S8). Similarly, stacking energies are minimally altered by the sequence (by up to ~ 4 kcal mol–1, Table S8). In contrast, ionization state has a greater effect on the stacking energies, with dianionic OT-G typically exhibiting the weakest stacking in all mismatched and C matched duplexes (by up to ~ 6 kcal mol–1, Table S8),19 which arises at least in part from disruption in the helical location of the damaged G due to greater charge on OT-G. 8 ACS Paragon Plus Environment

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In synchrony with the weakest lesion-site stacking energies, dianionic OT-G generally causes the greatest helical distortions compared to unmodified DNA19 (untwisting of up to ~ 30° and change in minor groove width up to ~ 4 Å) compared to all other ionization states in OT-G mismatched duplexes (untwisting of up to ~ 11° and change in minor groove width up to ~ 2 Å, Table S9-S11). This trend largely occurs irrespective of the identity of the flanking or opposing base, including the C matched pairs.19 Furthermore, when sequence effects are taken into account, the greatest distortions for dianionic OT-G are observed when mispaired with G at G2 followed by G1 and G3 (Table S11). Overall, our analysis reveals that the higher charge on the dianionic OT-G leads to greater helical distortions for the B-conformers. Structural features of the W conformation of OT-G adducted mismatched DNA. In contrast to the B conformation, OT-G acquires a syn glycosidic orientation (χ ~ 53 – 83° and θ ~ 4 – 20°, Tables S1–S3) in the W conformation. This locates the OT moiety in the DNA minor groove and directs the Hoogsteen edge of the damaged G towards the opposing base (Figures 1C and S14–S16). Similar to C,19 when the lesion is paired against A, only one hydrogen bond typically occurs between the adduct and the opposing base (Figures 2B, S17–S19 and Tables S4 and S6). Specifically, for dianionic OT-G paired opposite A, a (OT-G)O6∙∙∙H−C2(A) interaction forms at G1 and G2, but a stronger (OTG)O6∙∙∙H−N6(A) interaction occurs at G3 (Table S4). Additionally, at G3, neutral, COO− and ArO− OT-G form a weak (OT-G)O6∙∙∙H−C2(A) interaction, while a stronger (OTG)O6∙∙∙H−N6(A) contact occurs for dianionic OT-G (Table S4). For the T mismatch, although all conformers typically form the (OT-G)O6∙∙∙H−N3(T) interaction, an additional transient (OT-G)N1−H∙∙∙O4(T) hydrogen bond occurs for the dianionic adduct at G1 and G2 and COO− 9 ACS Paragon Plus Environment


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or ArO− form at G1 (Figures 2B, S17–S19 and Table S6). In addition, ArO− or neutral OT-G at G2 does not form a hydrogen bond with the opposing T, mainly since the nonpolar Hoogsteen edge of T is oriented towards OT-G (Figure S18 and Table S6). In contrast, against G, bifurcated hydrogen bonding consistently occurs between N2 and N1 of G, and O6 of OT-G (Figures 2B, S17–S19), with at least one of these interactions sustained throughout the simulation (> 97% occupancy) and the occupancy of the other interaction varying (~ 57 – 95%, Table S5). Thus, unfavorable steric interactions between the OT moiety and the opposing G prevents the formation of Hoogsteen hydrogen-bonding pattern observed in the G:G mismatches in natural DNA, and result in the bifurcated hydrogenbonding patterns within the OT-G:G pair. Overall, although the A and T mismatched duplexes exhibit diversity in lesion-site hydrogen bonding, adduct ionization and sequence does not affect the hydrogen-bonding pattern in the W conformer of the C matched19 or G mismatched (Table S5) duplexes. Unlike the B conformers, OT-G pairs significantly more strongly with G in the W conformers (by up to 22 kcal mol–1) compared to A, T or C,19 irrespective of the adduct ionization state and sequence context (Figures 3A and S8−S10). However, the relative hydrogen-bonding strengths between OT-G and C, A, or T are ionization state and sequence dependent. For example, although the A mismatch leads to the weakest lesion-site hydrogen-bonding for COO−, ArO− or neutral OT-G irrespective of the sequence context (Figures 3A, S8 and S9), the relative strength of the lesion pair for dianionic OT-G varies with the sequence context. Specifically, although the dianionic OT-G:A pair is not stabilized by hydrogen bonding at G1 or G3, appreciable hydrogen-bonding interactions exist between the adduct and the opposing A at G2 (~ 8 kcal mol–1, Table S7). 10 ACS Paragon Plus Environment

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In contrast to the hydrogen-bond strengths, the W conformers with OT-G paired opposite A, G or C19 generally exhibit similar lesion-site stacking (up to ~ 39 kcal mol–1), which is up to ~ 8 kcal mol–1 more stable than for T mismatch duplexes (Figures 3B and S11−S13, and Table S8). Although a minimal sequence dependence of the stacking energies is observed for T or G mismatched duplexes (up to ~ 3 kcal mol–1), the lesion-site stacking in A mismatched duplexes exhibits significant sequence effects, as reported for C matched duplexes.19 For example, the stacking energy of COO− OT-G paired against A is substantially enhanced at G2 (−38.6 kcal mol–1) compared to G1 (−32.2 kcal mol–1) and G3 (−31.7 kcal mol–1, Figure 3B and Table S8). Despite the syn orientation of the damaged nucleotide, the lesion-site structural parameters are not significantly different from natural DNA (Tables S9–S11), mainly due to the extrahelical (minor groove) location of the OT moiety. Nevertheless, the minor groove width changes compared to unmodified DNA19 (by up to ~ 3 Å, Table S9–S11). Furthermore, the A mismatched strands undergo the greatest change in the minor groove width compared to unmodified DNA (up to ~ 3 Å, Table S9), followed by the strands with the adduct against C19 and T (up to ~ 2.5 Å , Table S10), or G (up to ~ 1.5 Å, Table S11). The extent of helical distortion is largely independent of the sequence context for T and G mismatched duplexes, while a slight sequence dependence is observed for A mismatches. For example, dianionic OT-G opposite A at G2 or G3 (~ 2 Å) leads to a greater change in the minor groove width than at G1 (up to ~ 1.5 Å). Overall, the extent of the helical distortions for the W conformer exhibits a greater dependence on the mismatched base compared to the B conformer. Structural features of the S conformation of OT-G adducted mismatched DNA. 11 ACS Paragon Plus Environment


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Similar to the W conformation, the damaged base maintains a syn orientation (χ ~ 30 – 43° and θ ~ 347 – 358°, Tables S1–S3) in the S conformation. As observed for OT-G against C,19 the intrahelical portion of the bulky moiety and the extrahelical location of the opposing base completely disrupt interstrand hydrogen bonding at the lesion site irrespective of the opposing base, adduct ionization state and sequence (Figures 1D and S20–S22). However, the hydrogen bonding within the flanking base pairs remains intact (Table S12–S14). Furthermore, the lesion-site stacking energies are minimally affected by the identity of the (extrahelical) opposing base (Figures 3B and S11−S13, and Table S8). Nevertheless, there are marked ionization state and sequence dependencies on the lesionsite stacking interactions. Specifically, similar to the B and W conformers, dianionic OT-G typically exhibits the weakest lesion-site stacking among all ionization states regardless of the opposing base, although deviations from this trend occur at G1. In terms of sequence, the greatest effects on stacking are observed for the G mismatch, where the interaction energy decreases by ~ 6 kcal mol–1 upon changing the dianionic OT-G site from G1 to G2. Due to the intrahelical location of the OT moiety, more significant lesion-site distortions are observed for the S conformer (lesion-site untwisting of up to ~ 43° and change in minor groove width of up to ~ 7 Å) than the B (untwisting of up to ~ 30° and change in minor groove width of up to ~ 4 Å) and W (untwisting of up to ~ 11° and change in minor groove width of up to ~ 3 Å) conformers relative to unmodified DNA19 (Tables S9– S11). Furthermore, the extent of these distortions within the S conformers depends more strongly on the opposing base, ionization state and sequence compared to the B and W conformers. Specifically, in terms of the opposing base, the lesion site is most distorted for G mismatched duplexes (untwisting up to ~ 43° and change in minor groove width by up to 12 ACS Paragon Plus Environment

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~ 7 Å, Table S11), followed by T and A mismatch duplexes (untwisting up to ~ 29 and 39°, respectively, and change in minor groove width by up to ~ 5 Å, Tables S9 and S10), as well as the C match duplexes (untwisting up to ~ 37° and change in minor groove width by up to ~ 5 Å).19 In terms of the ionization state, dianionic OT-G causes the greatest lesion-site distortions (untwisting up to ~ 43° and change in minor groove width by up to ~ 7 Å), which largely holds true for the C matched duplexes.19 In terms of sequence, when the adduct is opposite A, G or C,19 the lesion-site untwisting is greatest at G1 (~ 39°, 43° and 37°, respectively), while the minor groove width changes the most at G2 (~ 5 Å for A and C,19 and ~ 7 Å for G, Tables S9 and S11). In contrast, for the T mismatched duplexes both the lesion-site untwisting (~ 29°) and change in minor groove width (~ 5 Å) are greatest at G2 (Table S10). Regardless, the least distortion in the S conformers typically occurs when the adduct is at G3 irrespective of the lesion ionization state and the mismatched base. Structural comparison with mismatched duplexes containing other C8-G adducts. Despite the general lack of structural data on C8-G adducted mismatch duplexes in the literature, DNA containing select mismatched N-linked (AF-G22-24 and AP-G21, Figure S1A), C-linked (Fur-G,26 Ph-G,26 CNPh-G,26 Q-G,26 BTh-G25 and Py-G25 Figure S1B) and Olinked (PCP-G39 and PhO-G,39, 40 Figure S1C) adducts at G3 of the NarI sequence or in a similar (5′-CGC) sequence context have been previously studied. Comparison of the structures adopted by DNA containing C-linked OT-G in its physiologically most relevant COO– form with those of DNA containing these other adducts reveals changes in the lesion site due to differences in the linker length and the associated flexibility (i.e., C–C versus C– O–C or C–N–C, Figure S1). Specifically, in the B conformation, the O-linked adducts form more persistent hydrogen bonds with an opposing A, and less persistent hydrogen bonds 13 ACS Paragon Plus Environment


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with an opposing T,39 compared to C-linked OT-G. Nevertheless, these differences do not significantly affect the lesion-site hydrogen bonding or duplex structure. However, the change in the linker properties have a more significant effect on the lesion pairing within W conformers. For example, although N-linked AF-G does not hydrogen-bond with the opposing A in the W conformer adopted at neutral pH,22 the C-linked OT-G adduct forms a weak (OT-G)O6∙∙∙H−C2(A) interaction. Similarly, within the S conformation with an opposing A, N-linked AP-G adopts a more distorted conformation at the C8-linkage due to its longer linker, which results in weaker lesion-site stacking compared to the planar OTG.21 In addition to the linker, the size of the C8-moiety affects the lesion-site structure of mismatched adducted duplexes. For example, for the W-conformers containing a G mismatch, model C-linked (Fur-G,26 Ph-G,26 CNPh-G,26 and Q-G26) and the O-linked (PCP-G29 and PhO-G28,

29)

adducts form a Hoogsteen hydrogen bond with the opposing G (N7 of

damaged G and N2 of opposing G and O6 of damaged guanine and N1 of the opposing G), which is similar to the (unmodified) G:G pair.41 However, in contrast, the larger OT-G adduct forms bifurcated hydrogen bonds with G (N2 and N1 of OT-G, and O6 of G). Overall, these examples emphasize the influence of the linker properties, as well as the size of the bulky moiety, on the structure of mispaired adducted DNA, which may play a role in determining replication and repair outcomes. Preferred conformations of OT-G adducted mismatched DNA. When OT-G is paired opposite A, the energetic separation between two of the three (B, W and S) duplex conformers is generally < 5 kcal mol–1, although the overall energetic separation between all three conformations can be as large as 9 – 14 kcal mol–1 (Table 14 ACS Paragon Plus Environment

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S15). Furthermore, the order of the conformational stability is OT-G ionization state and sequence dependent. For example, at G1, the B conformation is generally the most stable (by > 6 kcal mol–1) irrespective of the adduct ionization state, although the S conformation lies very close in energy for neutral OT-G (within 2.4 kcal mol–1). However, at G2, all three conformations generally lie close in energy (within 6 kcal mol–1) for neutral, COO− and ArO− OT-G, although dianionic OT-G prefers the B conformation by 6 – 11 kcal mol–1. In contrast, at G3, although the three conformations lie within 4 kcal mol–1 for the neutral and COO− states, ArO− and dianionic OT-G prefer a mixture of the B and W conformations (within 5 kcal mol–1, Table S15). Similar to A mismatched duplexes, the overall energetic separation between all three conformations is generally within 5 kcal mol–1 for T mismatches, but can be up to 8 – 14 kcal mol–1 (Table S15), and the energetic difference is ionization and sequence dependent. For example, at G2, the B conformation is the most stable irrespective of the OTG ionization state, although the W conformation lies within 0.3 kcal mol–1 for the dianionic state. However, at G1, the W and S conformations lie close in energy (within 1 kcal mol–1) for the neutral state, B and W conformations lie close (within 2 kcal mol–1) for the dianionic state, and all three states lie within 5 kcal mol–1 for the COO– state (Table S15). Compared to the A and T mismatches, adducted DNA containing OT-G mispaired with G is less conformationally heterogeneous. Furthermore, the sequence context and ionization state do not influence the conformational preference. Specifically, the W conformer is exclusively preferred by 7 – 22 kcal mol–1 for all strands considered. Interestingly, for each sequence context, the energetic separation for the adducted DNA conformers containing a C match19 is smaller than any mismatched (A, G or T) duplex (by 2 15 ACS Paragon Plus Environment


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– 8 kcal mol–1). Thus, the greatest conformational heterogeneity exists for the matched over the mismatched adducted DNA duplexes. The newly identified dependence of the conformational heterogeneity of OT-G adducted DNA on the base paired against the lesion may affect the biological fate of the adduct (i.e., replication outcomes or repair propensity). Comparison with conformational preferences of mismatched duplexes containing C8G adducts. The present work reveals that DNA containing C-linked COO– OT-G mismatched against A and T adopts a mixture of the B, W and S conformations at G3 in the NarI sequence (i.e., all conformers are within ~3 kcal mol–1). However, a previous computational study reported a B conformational preference for the O-linked PCP-G39 and PhO-G39 adducts (Figure S1) opposite A at G3, while NMR disclosed the W or S conformations for duplexes containing the N-linked AF-G22 or AP-G21 adduct (Figure S1) paired opposite A in an analogous 5′-CGC sequence context, respectively. Similarly, against T, MD simulations predict O-linked PhO-G and C-linked OT-G adducted DNA adopt a mixture of conformations (B and W for PhO-G, and B, W and S for OT-G), while O-linked PCP-G adducted DNA exclusively adopts the B conformation. This suggests that the chemical composition of the bulky moiety and the linker type at least in part dictate the conformation of A and T mismatched duplexes, and thereby play a role in the biological outcomes associated with adduct formation. In contrast, mismatched DNA duplexes containing OT-G or many other C8-G adducts paired with G, including N-linked AF-G,23 C-linked Fur-G,26 CNPh-G,26 Ph-G26, Q-G,26 and BTh-G,25 and O-linked PhO-G39,

40

and PCP-G,39 strictly prefer the W conformer. This

similarity in the conformational preference arises in part due to favourable hydrogen 16 ACS Paragon Plus Environment

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bonding between the syn C8-G nucleoside adduct and the opposing anti G. Overall, although the conformational preferences of adducted DNA containing G mismatches appear to be independent of the linker type, the structure of adducted DNA can be influenced by the chemical composition of the bulky moiety, which may have profound biological implications. Relevance to OTA mutagenesis Propensity of substitution mutations upon replication of OTA-adducted DNA: Bulky DNA lesions such as OT-G are typically replicated by translesion synthesis (TLS) polymerases that possess more open active sites than standard (replicative) polymerases and can accommodate bulky damaged sites.42 Although interactions between the bulky moiety and the polymerase active site may influence replication outcomes, including preventing extension past damaged sites, it has been reported that the conformation of a lesion in the duplex environment correlates reasonably well with the conformation at the single strand–double strand junction within the polymerase active site.43, 44 Therefore, the adducted DNA conformations characterized in the present work provide clues regarding possible replication outcomes. The extent of hydrogen bonding between the lesion in the parent strand and the incoming dNTP is an essential component for determining which dNTP will be preferentially inserted against the lesion during TLS.45-47 When the OT moiety is directed toward the major groove of DNA, our calculations reveal that the hydrogen-bonding interactions are strongest between the lesion and the opposing complementary C in the Btype duplexes (Figures 3A and S8-S10). This indicates that (complementary) dCTP will be preferentially inserted when OT moiety is directed towards the major groove, and the B 17 ACS Paragon Plus Environment


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conformation will be non-mutagenic. On the other hand, if the bulky moiety is directed toward the minor groove of the growing DNA strand at the replication junction, significantly stronger hydrogen-bonding interactions with G irrespective of the sequence context and ionization state (Figures 3B and S8−S10) suggest that the G mismatch is more likely to form over the competing mismatches or C match, which will lead to G→C transversion mutations upon subsequent replication. Overall, our results reveal that OTAinduced substitution mutations will likely only occur in the less accessible W conformation, which directly correlates with the limited contribution of substitution mutations to the overall mutagenic profile of OTA.9 Since studies on select aromatic amine adducts have revealed that small (one- or two-base) deletion mutations are stabilized upon intercalation of the bulky moiety into the helical environment, the S conformation of adducted DNA has become an accepted precursor of deletion mutations.48 The present work predicts that a stable S conformation of adducted DNA is accessible when OT-G is paired with A, C or T in most of the adduct ionization states and DNA sequence contexts. Thus, by analogy to the aromatic amine adducts, it is anticipated that OT-G can lead to deletion mutations upon replication when originally paired with A, C or T in the duplex environment. This proposal directly correlates with the large number of deletion mutations observed in laboratory animals upon OTA exposure.9 Repair of OTA-adducted mismatched DNA duplexes: The mutagenicity of bulky DNA lesions is dictated by their persistence within the cell, which is mediated by the nucleotide excision repair (NER) machinery.49 Various experimental and computational studies have emphasized that the initial recognition step of the NER pathway is facilitated by 18 ACS Paragon Plus Environment

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thermodynamic destabilization,50,

51

decreased stacking52,

53

and helical distortions50,

51

induced by the lesion. Coupled with more recent investigations into mechanism of NER recognition,54-56 these studies suggest that DNA structural changes induced by the lesion play a decisive role in lesion repair. Our free energy analysis suggests that DNA with OT-G mismatched with A and T prefer a mixture of B, W or S conformations, depending on the adduct ionization state and sequence. However, these DNA structures are distorted, exhibiting pronounced changes in the minor groove width in all three conformations and lesion-site untwisting in the S conformation, compared to unmodified DNA.19 When coupled with the insignificant dependence of the stacking interactions on the opposing base within the S conformations, this suggests that OT-G adducted DNA containing A or T mismatches may be prone to repair. On the other hand, the preferred (W) conformation of the G mismatched duplexes exhibits the smallest distortions, including the smallest change in the minor groove width, among the C match19 and all mismatched duplexes compared to unmodified DNA.19 This suggests that the W-type G mismatched duplexes may bypass repair, which will ultimately lead to G→C transversions upon replication of the mismatched strand. Our observed sequence dependence of the distortion of adducted mismatched DNA duplexes suggests that NER recognition of OT-G will likely depend on sequence. For example, the S conformer with the lesion in the G3 context exhibits the smallest distortions compared to G1 or G2 paired opposite A or T. In addition, our simulations shed light on the dependence of the NER repair propensity on the adduct ionization state. Specifically, barring few exceptions (e.g., wedge conformers containing OT-G paired opposite a mismatched base), adducted DNA with dianionic OT-G exhibits the greatest distortions and 19 ACS Paragon Plus Environment


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weakest lesion-site stacking interactions, which occurs irrespective of the identity of the opposing base and the sequence context. This suggests that duplexes containing the dianionic lesion will likely be more repair prone or, in other words, the repair propensity of OT-G will depend on the pH environment. Together our data points towards dependence of lesion persistence (and consequently mutagenicity) on the cellular environment, which correlates with available experimentally observed tissue specific carcinogenicity associated with OTA-exposure.8 Although a number of facts can be responsible for such differential expression of OTAinduced toxicity, the pH conditions in these kidney regions may result in greater stabilization of the less repair prone COO– and ArO– OT-G adducts compared to the more repair prone dianionic form that may exist in different pH environments in other tissues. Thus, our analysis provides a viable explanation for the observed tissue-specific selective mutagenicity of OTA, which points towards a novel theme of the dependence of the NER propensity on the cellular pH in the context of DNA adducts that should be experimentally investigated. ASSOCIATED CONTENT SUPPORTING INFORMATION Additional details of the computational protocol; structures of N-linked, C-linked and Olinked C8-G nucleoside adducts previously studied in mismatched DNA duplexes; representative lesion-site structures of the B conformers of DNA containing OT-G in different ionization states paired opposite A, G or T; lesion-site hydrogen bonding for OT-G in different ionization states and sequence contexts mispaired against A, T or G in the B adducted DNA conformation; hydrogen-bonding energies between OT-G

in different


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ionization states and the opposing base in the B and W adducted DNA conformations for three sequence contexts; van der Waals stacking energies between OT-G in different ionization sates and the flanking bases in each adducted DNA conformations for three sequence contexts; representative lesion-site structures of the W conformers of DNA containing OT-G in different ionization states paired opposite A, G or T; lesion-site hydrogen bonding for OT-G in different ionization states and sequence contexts position paired against A, T or G in the W adducted DNA conformation; representative lesion-site structures of the S conformers of DNA containing OT-G in different ionization states paired opposite A, G or T; backbone RMSD versus time for OT-G in all MD simulations considered in the present work; overlay of the representative structure of the lesion site obtained from the last 10 ns of MD simulations carried out using different non-bonded cut-offs; overlay of the representative structures of the lesion site obtained from 40 ns and 500 ns MD simulations; average values and standard deviations for χ and θ, and backbone rmsd for the different conformations of DNA containing OT-G in different ionization states and sequence contexts paired against A, T and G; occupancies of the hydrogen bonds in the trimers composed of the OT-G mismatch, and the 3′− and 5′−flanking base pairs for the major groove and wedge conformations of damaged DNA; hydrogen-bonding energies for different conformations of adducted DNA containing OT-G paired against C, A, T and G; van der Waals stacking energies for different conformations of adducted DNA containing OT-G paired against C, A, T and G; average values and standard deviations for pseudostep parameters and minor groove dimension for DNA containing OT-G paired against A, T and G, occupancies of the Watson-Crick hydrogen bonds of the 3′− and 5′− base pairs flanking the lesion in the stacked conformation of damaged DNA containing OT-G paired against A, T 21 ACS Paragon Plus Environment


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and G; relative MM-PBSA free energies for different conformations of DNA containing OT-G paired against A, T or G; hydrogen-bond occupancies for the base pairs at the terminal ends of DNA containing OT-G paired against A, T and G; partial charges and atom types of the OTG adduct in different ionization states; structural parameters for simulations with COO− OTG positioned at G3 and mismatched against A with 10 Å box-length and 9 Å non-bonded cutoffs; structural parameters for 500 ns simulations with COO− OT-G positioned at G2 mismatched against T. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Telephone: (403) 329-2323. Fax: (403) 329-2057. ORCID Preetleen Kathuria: 0000-0003-0007-2234 Purshotam Sharma: 0000-0002-5164-9833 Richard A. Manderville: 0000-0003-4035-8093 Stacey D. Wetmore: 0000-0002-5801-3942 ACKNOWLEDGMENT Calculations were conducted on the New Upscale Cluster for Lethbridge to Enable Innovative Chemistry (NUCLEIC), as well as additional resources provided by WestGrid and Compute/Calcul Canada. Funding Canada Foundation for Innovation [22770] and the Natural Sciences and Engineering Research Council (NSERC) [2016-04568] for SDW; NSERC [311600-2013] for RAM; Department of Science and Technology (DST) INSPIRE (IFA14-CH162), India and 22 ACS Paragon Plus Environment

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University Grants Commission (UGC) FRP (F.4-5(176-FRP/2015(BSR)), India for PS; and Council for Scientific and Industrial Research (CSIR), India for PK. Notes The authors declare no competing financial interest. ABBREVIATIONS A, adenine; AA, aromatic amines; AAF, acetylaminoflourene; AF, aminoflourene; AP, aminopyrene, BTh, benzothiophene; C, cytosine; dCTP, deoxycytidine triphosphate; dG, 2′deoxyguanosine; dNTP, deoxynucleotide triphosphate ; Fur, furan; G, guanine; MD, molecular dynamics; MM-PBSA, molecular-mechanics-Poisson-Boltzmann surface area; MoA, mechanism of action; NER, nucleotide excision repair; OTA, ochratoxin A; PCP, pentachlorophenol; Ph, phenyl; Py, pyrene; TLS, translesion synthesis; T, thymine; WC, Watson Crick; XPC, Xeroderma pigmentosum C.



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Wolfle, W. T., Washington, M. T., Kool, E. T., Spratt, T. E., Helquist, S. A., Prakash, L., and Prakash, S. (2005) Evidence for a Watson-Crick hydrogen bonding requirement in DNA synthesis by human DNA polymerase κ. Mol. Cell Biol. 25, 7137-7143. Mao, B., Cosman, M., Hingerty, B. E., Broyde, S., and Patel, D. J. (1995) Solution conformation of [AF] dG opposite a-1 deletion site in a DNA duplex: intercalation of the covalently attached aminofluorene ring into the helix with base displacement of the C8-modified syn guanine into the major groove. Biochemistry 34, 6226-6238. Costa, R. M., Chiganças, V., da Silva Galhardo, R., Carvalho, H., and Menck, C. F. (2003) The eukaryotic nucleotide excision repair pathway. Biochimie 85, 1083-1099. Geacintov, N. E., Broyde, S., Buterin, T., Naegeli, H., Wu, M., Yan, S., and Patel, D. J. (2002) Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery. Biopolymers 65, 202-210. Wu, M., Yan, S., Patel, D. J., Geacintov, N. E., and Broyde, S. (2002) Relating repair susceptibility of carcinogen-damaged DNA with structural distortion and thermodynamic stability. Nucleic Acids Res. 30, 3422-3432. Yang, W. (2006) Poor base stacking at DNA lesions may initiate recognition by many repair proteins. DNA Repair 5, 654-666. Reeves, D. A., Mu, H., Kropachev, K., Cai, Y., Ding, S., Kolbanovskiy, A., Kolbanovskiy, M., Chen, Y., Krzeminski, J., and Amin, S. (2011) Resistance of bulky DNA lesions to nucleotide excision repair can result from extensive aromatic lesion–base stacking interactions. Nucleic Acids Res., gkr537. Min, J.-H., and Pavletich, N. P. (2007) Recognition of DNA damage by the Rad4 nucleotide excision repair protein. Nature 449, 570-575. Chen, X., Velmurugu, Y., Zheng, G., Park, B., Shim, Y., Kim, Y., Liu, L., Van Houten, B., He, C., and Ansari, A. (2015) Kinetic gating mechanism of DNA damage recognition by Rad4/XPC. Nat. Commun. 6. Velmurugu, Y., Chen, X., Sevilla, P. S., Min, J.-H., and Ansari, A. (2016) Twist-open mechanism of DNA damage recognition by the Rad4/XPC nucleotide excision repair complex. Proc. Natl. Acad. Sci. 113, E2296-E2305.



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Figure 1. (A) Chemical structure of OT-G nucleoside indicating important dihedral angles (χ and θ). Wavy bonds represent the DNA strand that is covalently linked at the 3′ and 5′ sites of the adduct. The adduct can exist in four ionization states (neutral: R1 = OH, R2 = OH, carboxylic group ionized (COO−): R1 = O–, R2 = OH, phenolic group ionized (ArO–): R1 = OH, R2 = O–, and dianionic: R1 = O–, R2 = O–). (B, C and D) Possible conformational themes of adducted DNA containing OT-G paired against dN (N = A, T, G or C) depicting the trimer of pairs about the lesion. The OT moiety is shown in red, the damaged G moiety and the opposing base in green, and the base pairs flanking the lesion in blue.


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Figure 2. Lesion-site hydrogen bonding for COO− OT-G at the G3 position (taken as a representative example) mispaired against dN (N = A, G and T) in the (A) B and (B) W adducted DNA conformations. Percent occupancies of the hydrogen bonds according to the MD simulations are provided.



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Figure 3. (A) Hydrogen-bonding strengths (kcal mol–1) between COO− OT-G (taken as a representative example) and the opposing base in the B and W adducted DNA conformations for three sequence contexts. (B) van der Waals (vdW) stacking energies (kcal mol–1) of COO− OT-G (taken as a representative example) and the opposing base with the flanking base pairs (for the B and W conformers), and COO− OT-G with the flanking base pairs (for the S conformer) for four different opposing bases and three sequence contexts. Data for C match is taken from reference 19.


Molecular dynamics simulations of mismatched DNA duplexes

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