Molecular Dynamics Study of One-Base Deletion Duplexes

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

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 Preetleen Kathuria, Prebhleen Singh, Purshotam Sharma, Richard A. Manderville, and Stacey D Wetmore J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.9b06489 • Publication Date (Web): 16 Jul 2019 Downloaded from pubs.acs.org on July 31, 2019

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The Journal of Physical Chemistry

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 Preetleen Kathuria,a Prebhleen Singh,a Purshotam Sharma,*a Richard A. Mandervilleb and Stacey D. Wetmore*c aComputational

Biochemistry Laboratory, Department of Chemistry and Centre for

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

of Chemistry and Toxicology, University of Guelph, Guelph, Ontario,

Canada N1G 2W1. cDepartment of Chemistry and Biochemistry, University of Lethbridge, Lethbridge, Alberta, Canada T1K 3M4.

Abstract 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 towards understanding the prevalence of OTA-induced one-base deletion mutations, the present work uses molecular dynamics (MD) simulations to analyze the conformational preferences of onebase 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 reveals 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-

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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. Nevertheless, 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.


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Introduction Ochratoxin A (OTA) is a potent mycotoxin produced as a secondary metabolite by certain species of aspergillus and penicillium,1 and has been classified as a possible (group 2B) human carcinogen by the International Agency for Research on Cancer.2 Human exposure to OTA occurs through consumption of OTA-contaminated food products,3,4,5 and has been linked to a number of toxicological effects, including nephrotoxicity6,7 and testicular carcinogenesis.8 The mechanism of OTA action has been controversial, since two divergent pathways have been proposed in the literature. Specifically, the first (direct genotoxicity) mechanism suggests the formation of OTA DNA adducts (addition products),9 whereas the second (indirect) pathway links OTA action to events such as oxidative DNA damage, mitosis disruption and chromosomal instability.9-11 However, recent studies have revealed that indirect pathways do not completely rationalize the observed OTA mediated toxicity,12-13 and support the formation of DNA adducts as one of the major pathways for OTA action.9,12,14,15 This has, in turn, revived research on OTADNA guanine adducts,16-19 which have been previously characterized using NMR and mass spectrometry.20,21 These lesions include the most prevalent22,23 C-linked C8-OT-G adduct (Figure 1a),20 the Olinked C8-OTA-G adduct21 and the N,N-linked N1,N2-OTHQ-G adduct.19 In the context of the genotoxicity of OTA, a recent analysis of the mutational spectrum of the mutation reporter (red/gam (Spi−)) gene in OTA-administered cells revealed a complicated mutational profile and indicated that the frequencies of all mutations increase upon OTA exposure.14 More importantly, despite a relatively small increase in frequency compared to large deletions and base substitutions, one-base deletions comprise the largest percentage (73%) of the total mutations that occur upon 3 ACS Paragon Plus Environment


OTA exposure.14 Further experiments have revealed that the specific mutational frequency of one-base deletion mutations undergoes a 2.7-fold increase in G/C runs of p53-deficient gpt Delta mice compared to the control upon treatment with OTA for four weeks.15 Furthermore, despite the prevalence of large deletion mutations in OTA-treated rats,14 one-base deletions in repetitive sequences are prevalent along with insertions and base substitutions in Spi mutants from the kidneys of p53-deficient gpt delta mice exposed to OTA.14 Together, this data highlights the important contribution of one-base deletion mutations in OTA-mediated genotoxicity. Since the C8-OT-G adduct is the major OTA DNA damage product, site-specific incorporation of this adduct into one-base deletion sites in DNA is expected to provide currently missing structural insights into how OTA induces one-base deletion mutations. However, despite the availability of structural information for one-base deletion duplexes containing other C8-G adducts,24-26 no structural data is available for any OTA lesion. Previous experimental studies27,28 on C8-G adducts formed by aromatic amines (AAs) have proposed a plausible mechanism for the induction of one-base deletions. Specifically, it has been suggested that the polymerase stalls at the site of DNA adduction during replication. This delay allows slippage of the damaged base to form a slipped mutagenic intermediate (SMI, Figure S1), which is composed of a one-base bulge containing the adducted nucleotide. Further studies have suggested that the conformation of the lesion in the SMI and the ability of the lesion to form discrete (hydrogen-bonding and stacking) interactions with the flanking DNA environment play crucial roles in the stabilization of one-base deletion mutations.24,29


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Structural studies on DNA duplexes containing C8-G AA adducts formed by aminofluorene

(AF),26

acetylaminofluorene

(AAF)24

and

2-amino-1-methyl-6-

phenylimidazo-[4,5-b]-pyridine (PhIP)25 within a one-base deletion site depict two possible conformations. Specifically, the B-DNA type (B) conformation observed for AAFG maintains the anti ( = 180  90, see Figure 1a for the definition of ) orientation of all nucleotides and stacks the damaged guanine in the helix. However, the stacked (S) conformation observed for AF-G,26 AAF-G24 and PhIP-G25 flips the damaged guanine into the syn orientation ( = 0  90) and intercalates the bulky moiety within the helix. These studies further suggest that the S conformation better stabilizes a one-base bulge compared to the B conformation due to stacking of the C8-moiety within the helix, and thereby facilitates the induction of one-base deletion mutations.24 Furthermore, although DNA sequence is known to alter the relative population of the B and S conformers of C8dG AA adducts,24 the effects of sequence on the propensity of C8-adducts to yield onebase deletion mutations are not fully understood. Although previous studies on AA adducts have provided significant information about the induction of one-base deletion mutations by C8-G adducts, OT-G differs from these lesions in terms of the carcinogenbase linker (C8C linker in OT-G vs. C8NC linker in the AA adducts), size of the bulky moiety attached to the damaged G (Figures 1a and S2), and the ability of the bulky moiety to exist in different ionization states depending upon the local pH (neutral for AA lesions and neutral, carboxylic ionized (COO– ), phenolic ionized (ArO–), and dianionic (both COO– and ArO–) for OT-G, Figure 1a).30, 31 Thus, the structural properties and conformational preferences of OTA-induced one-base deletion duplexes need to be studied with different OT-G ionization states in different



sequence contexts in order to provide a structural explanation for the induction of such mutations in OTA exposed cells. To accomplish this goal, the present work uses computational methods to analyze the structural characteristics of DNA duplexes containing OT-G paired opposite a one-base deletion site at three positions in the NarI sequence context (5−G1G2CG3CC),32 which is an established hotspot for deletion mutations in prokaryotes, associated with AA adducts33 and has been used to study other C8-dG adducts.34-37 The effects of altering discrete lesion-site interactions by changing the adduct conformation, sequence context and lesion ionization state have been characterized. The implications of our findings for the repair propensity and mutational outcomes of the lesion are analyzed. In conjunction with our previous studies on OT-G adducted full-length DNA duplexes,38,39 OT-G mismatched DNA duplexes,40 and duplexes containing a two-base deletion site opposite the lesion,41 the present work contributes to the development of a structural explanation for the experimentally-observed tissue specific expression and complex mutagenic profile associated with OTA.

Methodology Starting Structures To model OT-G within a one-base deletion duplex, a 12-mer DNA strand containing the NarI sequence (5−G1G2CG3CC) was paired against an 11-mer strand. The initial structure for the B duplex conformation (Figure 2, left) was derived from a crystal structure of anti-1,N2-ethenodeoxyguanosine paired opposite a one-base deletion site (PDB ID:2KTP) in the G3 sequence context.42 This approach is similar to the previous studies on damaged deletion duplexes, where the crystal structure of a deletion duplex containing a different lesion is modified to study deletion mutation induced by a different adduct.24,25 6 ACS Paragon Plus Environment

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Starting structures with the adduct at G1 or G2 were obtained by fixing the adduct position (i.e., the sixth base from the 5′ end of the 12-mer) and altering the 12-base sequence of DNA accordingly (Figure 1b). In addition, any terminal A:T pairs were replaced with stronger G:C pairs to reduce the possibility of artificial unravelling at the oligonucleotide ends during the simulations. Starting structures for the S conformers (Figure 2, right) were obtained by changing the glycosidic orientation of OT-G to a syn conformation. All starting structures were built based on the lowest energy orientation of the OT moiety with respect to damaged G isolated in our previous quantum chemical studies on OT-G (i.e., θ = (N9– C8–C10–C11) ~ 0, Figure 1a).38 Simulation Protocol Each DNA system was neutralized using 21 sodium ions and solvated in a truncated octahedral box of ~4500 TIP3P43 water molecules such that all DNA atoms are at least 8 Å from the edge of the box. The natural nucleotides were simulated with the default deoxyribonucleotide parameters using the tleap module of AMBER12,44 whereas sodium ions were simulated using the Joung and Cheatham ion parameters.45 Partial atomic charges and other force field parameters for the four ionization states of OT-G were taken from our previous studies.38,39,41 For the initial minimization step, water molecules around DNA were minimized using 500 steps of steepest descent minimization and 500 steps of conjugate gradient minimization, with a 500 kcal mol−1 Å−2 restraint on the DNA strand. In the subsequent minimization, the restraints on DNA were removed and the whole system was minimized using 1000 steps of steepest decent, followed by 1500 steps of conjugate gradient minimization. The system was then heated from 0 K to 300 K using the Langevin



temperature scheme46 by carrying out a 20 ps simulation in an NVT ensemble. Finally, a 40 ns production run was carried out on each DNA strand using the PMEMD module of AMBER 1244 with an NPT ensemble, and the Berendsen barostat47 and Langevin temperature scheme.46 In total, 24 unique duplexes were investigated representing OTG in 4 ionization states (neutral, COO–, ArO– or dianionic), each of which was simulated in 2 (B and S) conformations at 3 positions (G1, G2 or G3) in one-base deletion duplexes. For each system, no significant fluctuations were observed in the backbone rmsd calculated with respect to the first frame of the production simulation (Table S1). Furthermore, the standard deviation in the rmsd over the last 10 ns of the simulation was less than 1 Å, which is smaller than that for the total 40 ns simulation (2.3 Å, Table S1). Therefore, in accordance with previous studies of natural48 and damaged48,49 DNA oligonucleotides, detailed structural analysis was carried out on the last 10 ns of the production simulation to ensure convergence of key DNA structural parameters, and the reliability of our conclusions. To verify convergence of key structural parameters, the 40 ns simulations with the adduct at G3 were extended to 100 ns as representative examples. The lesion-site structural parameters do not deviate significantly between 40 and 100 ns (Tables S2-S4), which confirms that the structural features of these duplexes are sufficiently converged at 40 ns. To analyze the statistical relevance of the derived results, two additional 40 ns MD production simulations were carried out on each of the COO and ArO OT-G ionization states in the G3 sequence context using different initial velocities. All simulations yield similar lesion-site structural parameters, with the largest deviations in the angular (i.e., , θ, twist, roll and tilt) and translational (i.e., minor groove width, shift, slide and rise)


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parameters being < 30 and < 1.5 Å, respectively (Tables S5-S8). Although a more robust sampling including larger numbers of replicas and longer timeframe simulations may have further improved the conformational sampling, the current study is expected to provide at least qualitative estimates of the structural differences between different possible conformations of 1-base deletion duplexes containing different OT-G ionization states. Furthermore, the choice of the simulation protocol for the present application stems from our desire to compare the structural features of OTA-adducted 1-base deletion duplexes with the corresponding 2-base deletion duplexes previously determined using the same protocol.41 For analysis, each simulation was clustered with respect to the locations of OT-G atoms comprising the  and θ dihedral angles to obtain a representative structure. The binding energy of the two DNA single strands was calculated as the interaction energy between the non-terminal bases of one strand with the other (hydrogen-bond occupancies and associated base-step parameters for the non-terminal base pairs are provided in Tables S9-S10), while neglecting the terminal bases which fray during oligonucleotide simulations.50-52 These energies are used to assess the relative stability of competing (B and S) conformations of the DNA oligonucleotides. To analyze the distortions that may correlate with the repair propensity of the lesion, the lesion-site interaction energies are calculated as a sum of the stacking energy of the OT-G adduct (calculated as the interaction energy of the OT-G adduct with the flanking base pairs using only the atoms of the nucleobase moiety of the DNA nucleotide), as well as the hydrogenbonding strength (calculated as the interaction energies between the opposing bases using only the atoms of the nucleobase moiety of the DNA nucleotide) of the base-pairs



flanking the lesion. Furthermore, the helix binding energies are calculated as the sum of interaction energies between the two strands of the deletion duplex. All these energy calculations were carried out using the ‘lie’ command of the cpptraj module of AMBER 12.45 Hydrogen-bonding occupancies were determined using a cut-off value of 3.4 Å for the donor–acceptor distance and a donor–hydrogen–acceptor angle of 120°. Additionally, the ‘nastruct’ command was used to calculate the lesion-site pseudostep parameters using a base-step consisting of the 5 and 3 base pairs with respect to the OT-G adduct.39,53-56 The minor groove width was calculated as the distance between the P atoms of the 7th and 20th residues minus the sum of the van der Waals radii of the P atoms (5.8 Å).57,58

Results and Discussion General lesion-site structural features of OT-G adducted one-base deletion duplexes. In the B conformation, the  dihedral angle ((O4′–C1′–N9–C4), Figure 1a) of the OT-G nucleotide ranges between 211 and 231 (Table 1), which depicts an anti ( = 180  90, Figure 1a) adduct orientation. This conformation solvent exposes the complete OTmoiety in the DNA major groove and stacks the damaged G inside the helix (Figures 3 and S3-S4). In the S conformation,  ranges between 37 and 83 (Table 1), which is characteristic of a syn ( = 0  90) orientation. This conformation stacks the isocoumarin group of the OT-moiety within the helix, and exposes the amide-linked phenylalaninic moiety into the DNA minor groove (Figures 3 and S3-S4). Irrespective of the (B or S) conformation adopted, the θ dihedral angle ((N9–C8– C10–C11), Figure 1a) equals 0  20, which reflects planarity at the C8-linkage connecting 10 ACS Paragon Plus Environment

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the OT-moiety to the damaged G (Table 1). This θ conformation corresponds to that observed in fully-paired DNA duplexes,39 but is up to ~100 less twisted than that of the isolated OT-G nucleotide.38 The base pairs flanking the lesion remain intact (> 90% occupancies of the Watson-Crick (WC) hydrogen-bonds, Table S11), suggesting that OTG does not hydrogen bond with the flanking bases and minimally perturbs the neighboring DNA environment. However, changes in the OT-G ionization state and sequence context alter the lesion-site structure which, in turn, affects the relative conformational stabilities of the OT-G adducted one-base deletion duplexes (vide infra). Relative conformational stabilities of the OT-G adducted one-base deletion duplexes. Analysis of the helix binding energies reveals that the relative stabilities of the B and S conformations of one-base deletion duplexes containing OT-G depend on the sequence context (Figure 4, Table S12). Specifically, at G1 and G2, the B conformers are more strongly bound (by 11 – 67 kcal mol–1) compared to the S conformers. The substantial difference in the stability of the two conformations indicates that the B conformer will be exclusively preferred for all four adduct ionization states. In contrast, at G3, the S conformer is (~ 16 kcal mol–1) more stable than the B conformer for dianionic OT-G, which indicates an exclusive preference for the S conformer. Furthermore, the S conformations containing COO– and neutral OT-G are slightly more stable than than the B conformers (by 6 – 7 kcal mol–1). Although

the order of the stability of the B and S conformers

reverses for ArO– OT-G at G3, the two conformers remain within 6 kcal mol–1. This suggests conformational heterogeneity for one-base deletion duplexes containing the neutral, COO– and ArO–ionization states at G3.



Overall, although the B conformation is generally more stable for one-base deletion duplexes containing OT-G, the S conformer is comparably stable to the B conformer (for neutral, ArO– and COO) or significantly more stable (dianionic) than the B conformer in a particular sequence context (G3). This suggests that the cellular environment of the lesion, including the surrounding pH and sequence context, can alter the conformational preferences of OT-G adducted one-base deletion duplexes. Lesion-site stability and structural features of one-base deletion duplexes as a function of OT-G ionization state and sequence context. For the B conformers, dianionic OT-G exhibits the weakest lesion-site interaction energy among all ionization states at G1 and G3 (Table S13). Specifically, the lesion-site interaction energy for dianionic OT-G is less than for other ionization states by up to 24 kcal mol–1 at G1 and up to 12 kcal mol–1 at G3. In contrast, at G2, ArO– OT-G exhibits the smallest lesion-site stabilization energy (by up to 11 kcal mol–1 compared to other ionization states). However, the change in lesion-site structural parameters with respect to the corresponding natural DNA (i.e., full-length DNA duplex with no deletion) is greatest for COO OT-G at G1 (i.e., a 33.8 lesion-site untwisting and a 21.8 change in tilt, 2.9 Å change in slide and 2.6 Å change in minor groove width). The next most distorted structure occurs for COO OT-G at G2 (i.e., up to 35.2 untwisting and 13.9 change in tilt, 2.7 Å change in slide and 0.7 Å change in minor groove width, Table S14). In contrast, at G3, dianionic OT-G leads to the greatest distortion compared to other ionization states (i.e., change in minor groove widening of ~2.5 Å, change in roll of 10.1 and DNA untwisting of 27.3, Table S14).


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In contrast to the B conformation, the S conformation is only energetically accessible for the G3 sequence context irrespective of the OT-G ionization state. At G3, the lesion-site interaction energy is greatest for neutral OT-G (~ 96 kcal mol–1), followed by COO OT-G (~ 85 kcal mol–1), and smallest for dianionic and ArO– OT-G (~ 70 kcal mol–1). In terms of the lesion-site structural parameters, maximum distortion is induced by ArO– OT-G (up to an ~ 2.2 Å, 2.3 Å, 1.9 Å, and 1.4 Å change in minor groove width, shift, slide and rise, respectively, and ~ 33 untwisting) followed by neutral (up to ~ 1.7 Å minor groove widening, up to ~ 11.4 change in tilt and ~ 28.5 change in roll, and ~ 32.6 untwisting) and COO OT-G (up to ~ 1 Å minor groove widening, up to ~ 9.6 change in tilt and ~ 11.8 change in roll and ~ 26.7 untwisting). Furthermore, comparison of the accessible S conformers with the corresponding B conformers with OT-G at G3 reveals smaller lesion-site distortions for the B conformers for the neutral, COO and ArO– states (up to ~ 1.4 Å minor groove widening, ~ 1.0 Å change in rise, up to ~ 14 change in tilt, and ~ 18.5 untwisting). In contrast, a larger change in the minor groove width in the B conformation (2.5 Å) compared to the S conformation (1.8 Å) is observed for dianionic OT-G at G3. Overall, for the B conformers, dianionic OT-G results in the greatest distortion and lesion-site destabilization among all ionization states at G3. However, COO OT-G leads to the most distortion at G1 and G2. For the accessible S conformers, the structural parameters exhibit more distortion relative to natural duplexes than the corresponding B conformers of one-base deletion duplexes irrespective of the OT-G ionization state. Comparison to C8-dG aromatic amine adducts at the G3 position in the NarI sequence. 13 ACS Paragon Plus Environment


As stated in the Introduction, previous studies have structurally analyzed one-base deletion duplexes containing the AF-G,26 AAF-G24 and PhIP-G25 AA adducts. Specifically, solution-state NMR studies of one-base deletion duplexes containing these AA adducts within the NarI G3 sequence context reveal a strong preference for the S conformer,26 although a small proportion of the B conformation is also formed for AAF-G.24 Similar to the AA adducts, our results reveal that OT-G adducted one-base deletion duplexes largely prefer the S conformation at the G3 position for the biologically predominant COO and dianionic OT-G states, as well as the neutral lesion. Furthermore, despite greater stability of the B conformer, the S conformer is likely also accessible (within 6 kcal mol–1) for ArO OT-G. This indicates that the conformational preferences of the one-base deletion duplexes may be more strongly dictated by the sequence context than the adduct chemical composition. Structural comparison of the B conformations of AAF-G and OT-G-containing duplexes at G3 reveals that both adducts have a similar stacking pattern of the damaged G with the intrastrand bases and a similar (extrahelical) location of the carcinogenic moiety. Additionally, the hydrogen bonding within the flanking base pairs remains intact for both types of adducted duplexes. This suggests that despite differences in the chemical composition of the C8-moiety, both lesions cause similar minimal perturbations to the helix in the B conformation. In contrast, the structures of the S conformer of OT-G adducted one-base deletion duplexes differ remarkably from those containing AF-G,26 AAF-G,24 and PhIP-G.25 For example, due to a shorter and less flexible CC linker, the damaged guanine is not completely displaced out of the helix, but instead stacks with the intrastrand flanking bases. The OT moiety also stacks with the interstrand flanking bases.


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However, the longer and more flexible CNC linker of the AA adducts24-26 renders the damaged guanine extrahelical in order to stack the C8 moiety with the flanking base pairs. Overall, although a change in the linker size and flexibility alters discrete interactions between the adduct and the flanking DNA environment, the conformational preferences of one-base deletion duplexes largely remain similar, regardless of the chemical composition of the C8 moiety, likely being dictated by the position of the damage.

Biological Implications Implications for nucleotide excision repair (NER). (a) Effect of conformation, adduct ionization state and sequence context on the repair propensity of the OT-G lesion. Bulky DNA adducts are generally repaired by the NER pathway in cells,59 which involves removal of a lesion-containing 24 – 32 nucleotide segment of DNA,59 followed by DNA restoration through ligation of newly synthesized DNA to the existing strand.60,61 The NER pathway is initiated through lesion recognition by specialized factors such as XPC-Rad23B in eukaryotes, which constantly scan the genome to recognize DNA structural perturbations and subsequently recruit downstream factors to complete the repair process.62 Previous experimental and computational studies have revealed that the repair propensities of bulky DNA adducts correlate with a number of factors, including the extent of lesion-site thermodynamic destabilization,63,64 decreased stacking,25,65 and increased helical distortions,63,64 dynamics66-68 and global structural changes, such as helix bending.69 Specifically, a crystal structure of the yeast orthologue of XPC-Rad23B (Rad4) bound to damaged DNA revealed that lesion recognition is initiated by insertion of a -hairpin of the recognition factor from the major groove side of DNA and simultaneous flipping of the base opposing the lesion.70 More 15 ACS Paragon Plus Environment


recent studies have suggested that lesion-site structural perturbations facilitate lesion recognition by increasing the residence time of XPC-Rad23 at the lesion site and reducing the DNA opening time required to flip the opposing base.71,72 Furthermore, single molecule imaging studies reveal that a conformational rearrangement is triggered within the protein upon encountering DNA damage, which in turn facilitates lesion recognition.73 Overall, these studies indicate that structural changes induced by the lesion play a decisive role in lesion recognition for NER. Our analysis reveals that the B conformation is largely accessible and generally preferred for OT-G containing one-base deletion duplexes. At G1 and G2, greater distortions and intermediate lesion-site stabilization observed for the B conformer of COO OT-G compared to other OT-G ionization states may aid damage recognition and initiate NER when the adduct is in the COO state. In contrast, within the G3 sequence context, the B conformers of all OT-G ionization states contain similar lesion-site distortion and exhibit similar stacking stabilization. This indicates that the B conformer of OT-G at G3 will have a similar repair propensity irrespective of the adduct ionization state. Nevertheless, for one-base deletion duplexes with OT-G at G3, the S conformation is also energetically accessible. Among the accessible S conformers, ArO OT-G at G3 exhibits the weakest lesion-site stabilization and the maximum distortion, which indicates that this form of the lesion will likely be repair prone in the S conformation. However, although the lesion-site stabilization of dianionic OT-G at G3 is similar to ArO OT-G, small distortion may render dianionic OT-G repair resistant, leading to a persistent S conformer for dianionic OT-G at G3. Nevertheless, the accessible S conformers are more distorted


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compared to the corresponding B-conformers, which points towards a greater repair propensity of the S over the B conformers. Overall, the differential repair propensity of the adduct for different conformations, sequence contexts and ionization states indicates that the cellular processing of OT-G is highly dependent on physiological factors such as pH and the identity of the bases flanking the lesion. This correlates with the experimentally-observed tissue specific mutagenicity in OTA exposed cells.14 (b) Structural explanation for the experimentally-observed greater propensity of one-base deletions compared to two-base deletions upon OTA exposure. A previous experimental study revealed that one-base deletions occur more prominently than two-base deletions upon OTA exposure.14 Comparison of the relative stabilization of OTA-containing onebase deletion duplexes considered in the present work and two-base deletion duplexes previously considered41 reveals that the lesion-site interaction energies are typically greater (by up to 39 kcal mol–1, Table S13) for the one-base than the two-base41 deletion duplexes. This difference primarily arises for two reasons. First, the closer proximity of the lesion to the 5′flanking base pair due to the absence of a flanking bulged base in the one-base deletion duplexes results in larger stacking energies compared to the two-base deletion duplexes. Second, the hydrogen-bonding interactions in the 5′base pair flanking the lesion are either completely or partially disrupted in the two-base deletion duplexes for most ionization states and sequence contexts,42 but remain intact in the one-base deletion duplexes (Table S11). Weaker lesion-site interactions and greater distortions, including disrupted hydrogen bonds in the 5′base pair resulting from the presence of the bulge, suggest that the two-base deletion duplexes may be more readily identified by



NER recognition factors compared to one-base deletion duplexes. This correlates with the greater prevalence of one-base deletions compared to two-base deletions within OTA exposed cells.14 Implication for mutagenesis. As mentioned in the Introduction, it is widely believed that deletion mutations result from adduct-induced inhibition of DNA replication, which allows for the formation of a SMI that contains a bulge at the lesion-site. Structurally distinct adducts interact differently with polymerases, as well the flanking DNA environment, which in turn affects the stability of the SMI and hence the propensity of deletion mutations. In this context, previous studies on C8-G adducts have emphasized that the SMI is better stabilized by the syn (stacked) lesion orientation, which stacks the bulky moiety into the helix.29,35 However, as proposed for two-base deletion duplexes,36 the bulge formed with the B conformation for one-base deletion duplexes is more flexible and can realign, potentially leading to mismatch formation (Figure 5).36 Our analysis reveals that irrespective of the adduct ionization state, the B conformer is largely accessible and generally preferred for OT-G adducted one-base deletion duplexes. This indicates that the OT-G adducted SMI containing a one-base bulge will be unstable and susceptible to realignment, which will lead to mismatch formation reported in context of OTA40,14 (Figure 5, right). However, the accessibility of the S conformer in certain sequence contexts (e.g., G3) and the significant preference of the S conformation for select adduct ionization states (e.g., neutral, COO and dianionic) indicates the propensity for induction of one-base deletion mutations in select environments (Figure 5, right). Thus, our data points to a dependence of the replication


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outcome on sequence context and adduct ionization state. Coupled with the predicted differential NER repair propensities for different sequence contexts and ionization states, our structural data directly correlates with the observed occurrence of tissue-specific mutations upon OTA exposure.14 Nevertheless, tissue specific metabolic activation and adduct formation may also contribute to the observed tissue specific action of the OT-G adducts.

Conclusion The present structural analysis of OT-G containing one-base deletion duplexes reveals two stable (B- and S-type) conformers as previously identified for several N-linked adducts. However, the adduct ionization state and sequence context are found to alter the conformational preference, as well as the discrete lesion-site interactions and lesionsite distortions. This suggests that the biological outcome (repair and replication) of the lesion will depend on the physiological environment, which correlates with the tissuespecific mutagenicity observed in the context of OTA exposure.14 Comparison of the lesion-site interaction energies for the OTA-adducted one-base deletion duplexes with the corresponding two-base deletion duplexes suggests that the SMI containing a onebase deletion is better stabilized and less distorted compared to that containing a twobase deletion. This observation explains the smaller increase in two-base deletions compared to one-base deletions in cells upon OTA exposure.14,15 However, since there is a current lack of biochemical data regarding the repair and replication of the OT-G adduct in different physiological environments, additional experiments are required to confirm the hypothesis put forward in the present work. Associated Content



Supporting Information Available Proposed one-base slippage mechanism for deletion mutations arising from heterocyclic aromatic amine C8-dG adducts; N-linked C8-dG adducts previously studied in one-base deletion duplexes; lesion site structures of DNA one–base deletion duplexes containing OT-G at G1 and G2; comparison of the backbone rmsd over the last 10 ns and 40 ns of the production simulations; comparison of average and standard deviation for the  and θ dihedral angles of the adduct over 40 ns and 100 ns production simulations for each one base deletion duplex containing the lesion at G3; comparison of the average pseudostep parameters and minor groove width for each DNA deletion duplex over 40 ns and 100 ns production simulations when the lesion is at G3; comparison of hydrogenbond strengths and total stacking energies for one-base deletion duplexes containing the lesion at G3 calculated over 40 ns and 100 ns production simulations; average  and θ dihedral angles for the adduct in one-base deletion duplexes containing the lesion at G3 over three 40 ns MD simulations; average of pseudostep parameters for each one-base deletion duplexes containing the lesion at G3 over three 40 ns MD simulations; average minor groove width for each one-base deletion containing the lesion at G3 over three 40 ns MD simulations; comparison of hydrogen-bond strengths and total stacking energies for one-base deletion duplexes containing the lesion at G3 over three MD simulations; base step parameters of the non-terminal base pairs for the COO OT-G containing onebase deletion duplexes over the last 10 ns of the MD production simulation; hydrogenbond occupancies of the non-terminal base pairs for the COO OT-G, including the flanking bases, for the last 10 ns of the 40 ns production MD simulation for each onebase deletion duplexes; hydrogen-bond occupancies at the lesion site, including the 20 ACS Paragon Plus Environment

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flanking bases, for the last 10 ns of 40 ns production simulation for each one-base deletion duplex; average lesion-site energies calculated as the sum of the hydrogen-bonding strengths of the flanking-base pair and the interaction energy of the OT-G adduct with the flanking base pairs within each two-base and one-base deletion duplex; change in the magnitude of the pseudostep parameters and the minor groove width for one-base deletion duplexes with respect to the natural fully paired DNA duplex. Author Information Corresponding Authors E-mail: [email protected] (SDW), [email protected] (PS) Telephone: (403) 329-2323 (SDW), (91)9855855239 (PS) Funding SDW thanks the Canada Foundation for Innovation (CFI) [22770], the Natural Sciences and Engineering Research Council (NSERC) of Canada [2016-045687], and the Board of Governor Research Chair Program at the University of Lethbridge for supporting this research; RAM thanks the Natural Sciences and Engineering Research Council (NSERC) of Canada; PS thanks the Department of Science and Technology (DST), and University Grants Commission (UGC), New Delhi, for financial support through the DST INSPIRE (IFA14-CH162) and the UGC FRP (F.4-5(176-FRP/2015(BSR)) programs, respectively. Conflict of Interest The authors declare no competing financial interest. Acknowledgements



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Fig. 1 (a) Chemical structure of the OT-G adduct indicating the constituent structural moieties and atomic numbering required to define important dihedral angles ( and θ). Wavy bonds represent the 3′ and 5′ sites where the adduct is covalently linked within a DNA strand. OT-G was considered in the neutral (non-ionized; R1 = OH, R2 = OH), carboxylic group ionized (COO–; R1 = O–, R2 = OH), phenolic group ionized (ArO–; R1 = OH, R2 = O–) and dianionic (R1 = R2 = O–) forms. b) The oligonucleotide sequence used to simulate the deletion duplex containing OT-G in the G1, G2 or G3 sequence context, with the lesion position (G*) highlighted in purple.


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Fig. 2 Representative structures highlighting the lesion site for the B (left) and S (right) conformations of the OT-G adducted one-base deletion duplex. Examples are provided for the COO ionization state of OT-G at G3. The OT moiety is shown in red, the damaged G in blue and the base pairs flanking the lesion in green.



Fig. 3 Lesion site representative structures of the adducted DNA deletion duplex containing the neutral, COO–, ArO–, or dianionic form of OT-G incorporated at the G3 position. The OT moiety is shown in red, the damaged G moiety in blue, and the base pairs flanking the lesion in green.


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Fig. 4 Variation in the helix binding energy (kcal mol–1) for the B (left) and S (right) conformers of one-base deletion duplexes containing OT-G in different ionization states (color) at the G1, G2 or G3 position. The helix binding energy was calculated as the sum of interaction energies between the two strands of deletion duplex.



Fig. 5 Proposed mechanism depicting the fate of the SMI formed by C8-G adducts.26, 74


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Table 1 Average and standard deviation (in parentheses) for the  and θ dihedral angles (deg.) in the adduct from MD simulations.a Sequence

Conformer

G1

B

Ionization State



θ

Neutral 220.2 (7.3) 9.5 (6.0) 214.2 (10.2) 14.9 (7.1) COO 220.7 (7.8) 9.3 (6.3) ArO Dianionic 211.6 (8.6) 18.6 (6.3) S Neutral 66.8 (8.7) 19.9 (6.2)  68.9 (8.5) 19.8 (6.2) COO  39.5 (7.7) 346.6 (6.8) ArO Dianionic 36.9 (7.4) 346.4 (6.0) 2 G B Neutral 222.5 (8.1) 8.8 (6.4) 230.8 (9.6) 4.3 (6.5) COO  225.5 (9.2) 4.9 (6.5) ArO Dianionic 226.1 (9.3) 5.9 (6.6) S Neutral 72.1 (8.3) 19.1 (6.0) 74.7 (8.3) 14.8 (6.5) COO 42.8 (7.5) 340.8 (6.1) ArO Dianionic 73.5 (8.4) 20.0 (6.0) G3 B Neutral 220.6 (8.0) 10.2 (6.6)  222.0 (8.7) 7.5 (6.8) COO 219.3 (7.8) 9.4 (6.4) ArO Dianionic 210.7 (8.9) 19.0 (6.4) S Neutral 73.8 (8.4) 13.0 (6.5) 67.8 (22.2) 5.2 (13.3) COO 82.6 (9.4) 17.4 (6.0) ArO Dianionic 82.6 (9.4) 17.4 (6.0) aMeasured over the last 10 ns of the 40 ns production simulation for each DNA deletion duplex.



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Molecular Dynamics Study of One-Base Deletion Duplexes

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