Urea Mimics Nucleobases by Preserving the Helical Integrity of B-DNA

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Urea Mimics Nucleobases by Preserving the Helical Integrity of BDNA Duplexes via Hydrogen Bonding and Stacking Interactions Gorle Suresh, Siladitya Padhi, Indrajit Patil, and U. Deva Priyakumar Biochemistry, Just Accepted Manuscript • DOI: 10.1021/acs.biochem.6b00309 • Publication Date (Web): 22 Sep 2016 Downloaded from http://pubs.acs.org on September 27, 2016

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Biochemistry

Urea Mimics Nucleobases by Preserving the Helical Integrity of B-DNA Duplexes via Hydrogen Bonding and Stacking Interactions

Gorle Suresh, Siladitya Padhi, Indrajit Patil and U. Deva Priyakumar*

Center for Computational Natural Sciences and Bioinformatics, International Institute of Information Technology, Hyderabad 500032, India

*Corresponding Author Email: [email protected] Phone: +91-40-6653 1161 FAX: +91-40-6653 1413

1 ACS Paragon Plus Environment

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Abstract Urea lesions are formed in DNA due to free radical damage of thymine base, and their occurrence in DNA blocks DNA polymerases, which has deleterious consequences. Recently, it has been shown that urea is capable of forming hydrogen bonding and stacking interactions with nucleobases, which are responsible for the unfolding of RNA in aqueous urea. Base pairing and stacking are inherent properties of nucleobases; since urea is able to form both, this study attempts to investigate if urea can mimic nucleobases in the context of the nucleic acid structures by examining the effect of introducing urea lesions complementary to the four different nucleobases on the overall helical integrity of B-DNA duplexes and their thermodynamic stabilities using molecular dynamics (MD) simulations. The MD simulations resulted in stable duplexes without significant changes in the global B-DNA conformation. The urea lesions occupy intrahelical positions by forming hydrogen bonds with nitrogenous nucleobases, in agreement with experimental results. Furthermore, these urea lesions form hydrogen bonding and stacking interactions with other nucleobases of the same and partner strands, analogous to nucleobases in typical B-DNA duplexes. Direct hydrogen bond interactions are observed for the urea-purine pairs within DNA duplexes, whereas two different modes of pairing, namely direct hydrogen bonds and water-mediated hydrogen bonds, are observed for the urea-pyrimidine pairs. The latter explains the complexities involved in interpreting the experimental NMR data reported previously. Binding free energy calculations were further performed to confirm the thermodynamic stability of the urea incorporated DNA duplexes with respect to pure duplexes. This study suggests that urea mimics nucleobases by pairing opposite to all the four nucleobases, and maintains the overall structure of the B-DNA duplexes. Keywords: DNA damage; urea lesions; molecular dynamics simulations; stacking interactions; binding free energies; water bridges; thermodynamic stability


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Introduction Many exogenous and endogenous agents are capable of damaging cellular DNA, thereby modifying the properties of DNA or triggering mutations; this phenomenon is referred to as DNA damage. Such modifications have been shown to be responsible for mutagenesis, carcinogenesis and cell lethality.1 DNA damage includes base and furanose sugar modifications, abasic site formation, single and double strand breaks, and cross linkages,1-3 and these factors play a central role in the etiology of cancer, neurological disorders and aging related diseases.4-8 One of the most common DNA damages is the formation of thymine glycol (Tg) from thymine upon oxidative addition. Thymine glycol undergoes further ring fragmentation to form N-(2-deoxy-β-D-erythropentofuranosyl)formamide and N-(2-deoxy-β-D-erythro-pentofuranosyl)urea.9-13 These fragmented products are considered as intermediate structures between nucleobases and abasic sites. These urea lesions were reported as being non-instructive14,15 and are known to be mutagenic similar to abasic sites.16 Previous studies have shown that urea lesions are capable of forming hydrogen bonds with nucleobases after losing part of their original coding information.9,10 Urea lesions are poorly bypassed by DNA polymerase and they block DNA polymerase in in vitro.15,17 These urea lesions are recognized by enzymes like endonuclease III, which are known to remove the lesions in Escherichia coli.18 If these modifications are not repaired, the polymerase can potentially incorporate any base opposite to it, which results in a mutation. It has been observed that the presence of urea and Tg affects the cleavage rate of RNA-DNA hybrids by ribonuclease H, and it preferentially redirects the cleavage site lying next to the mismatch base pair.19 Previous studies have also shown that the damaged site increases the flexibility of duplex DNA that increases protein recognition of DNA during DNA repair mechanisms.20 NMR studies have been performed on the B-DNA duplexes containing urea or formamide lesion as a bulge or complementary to the nucleobases.21-29 NMR studies of DNA with urea lesion opposite to the thymine base have indicated that the urea nucleotide adopts cis and trans


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conformations around the uridic bond, and these two isomers were present in the ratio of 2:3 in solution.23 It was also observed that urea-thymine base pair has two hydrogen bonds similar to regular AT base pair.23 Furthermore, the mismatch base pair occupied an intrahelical position and was held by hydrogen bonds with other nucleobases. NMR studies have also reported the structures of B-DNA helix with urea lesion as a bulge opposite to the abasic site, and explained their role in frameshift mutagenesis.26 This study also suggested that a kink is produced in the double helix due to the formation of hydrogen bonds with the nucleobases in the same strand.26 If the polymerase does not include a nucleobase opposite to the urea lesion during replication, a frameshift mutagenesis is caused. Such a structure has been reported in previous studies,27 but these studies were unable to characterize the individual structures of DNA duplexes with urea lesion in cis and trans isomers due to resonance overlap. Unlike formamide, the urea lesion occupies intrahelical and extrahelical positions because of the capability of urea lesion to form unusual hydrogen bonds. Similarly, apurinic and apyrimidinic abasic sites in DNA duplexes occupy both intrahelical and extrahelical conformations as observed in several experimental studies.30-35 Recently we have investigated the nonbonded interactions that are responsible for the unfolding of RNA in aqueous urea.36 It has been shown that urea is capable of forming hydrogen bonding interactions and more interestingly stacking interactions with nucleobases, which is responsible for stabilizing the bases in their extrahelical conformation, hence favoring the unfolded states, which is otherwise not possible in presence of only water.36-38 Hydrogen bonding and stacking interactions between nucleobases are two important phenomena that are responsible for the stability of the duplex structures of DNA. Since urea is capable of forming both, the natural question is how well urea can mimic the nucleobases within the nucleic acid structures. Given that urea is a thymine damaged product and that it is present in DNA structures, this question assumes higher significance in terms of understanding the structures, dynamics, and thermodynamic stabilities of nucleic acid duplexes containing urea lesions.


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Although NMR experiments provide valuable information about the structures of urea and formamide incorporated B-DNA duplexes, the complete characterization of DNA duplexes with urea lesions has been difficult because of the resonance overlapping of the signals.23,26 Molecular dynamics (MD) simulations are, in general, very useful in studying the structure, dynamics and thermodynamic stability of damaged and chemically modified nucleic acid duplexes.39-42 In the present study, MD simulations have been performed on the B-DNA duplexes with urea lesion opposite to four nucleobases adenine (A), thymine (T), guanine (G), and cytosine (C) in WatsonCrick (WC), Hoogsteen, and sugar edged interactions. Analyses of the MD trajectories suggest that the urea lesions prefer to form WC-like hydrogen bond interactions with the nucleobases, especially purines and can potentially mimic the nucleobases in B-DNA duplexes.

Computational Methods Initial models for urea incorporated DNA duplexes: A previous study investigated all possible hydrogen bond assisted interactions of urea with all the DNA nucleobases (A, G, C and T).36 All the urea-nucleobase binary complexes from this study were considered for generating the initial configurations of the urea incorporated DNA duplexes. Based on the structures of typical DNA duplex structures, it is known that the distance between nitrogen atoms (N9 for purines and N1 for pyrimidines) connected to the furanose sugars of the nucleobases from the two complementary strands is approximately 9 Å. Based on this, a cutoff distance of 9 ± 2 Å between either of the N atom of urea and N9 of purines or N1 of pyrimidines was used to select urea-nucleobase pairs that are suitable to fit within the B-DNA duplex (Figure 1a-g). This selection criterion resulted in eight different structures of urea-nucleobase pairs with WC, Hoogsteen and sugar edge-like interactions. The 10-mer DNA sequence d(AGCGATCACG) from the available NMR experimental study on urea-thymine base pair23 was considered as the template sequence for modeling all the pure and urea incorporated B-DNA duplexes (Figure 1h).


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The thymine-urea pair present in this structure was replaced by each of the urea-nucleobase pairs resulting in eight duplex structures containing the urea lesion (Figure 1a-g). Corresponding DNA duplexes with the typical GC, CG, AT and TA base pairs were also considered for comparison. All the urea modifications were done by using the SYBYL program (Tripos Inc).

Molecular dynamics simulations: CHARMM biomolecular simulation program43 was used to prepare the initial systems of urea incorporated B-DNA duplexes, to setup the MD simulations, to equilibrate the systems and to perform analyses of the MD trajectories. All the production simulations were performed by employing the NAMD biomolecular simulation program.44 CHARMM36 all-atom parameters42,45-47 were used for B-DNA duplex and urea. The topology and parameters corresponding to urea nucleotide were derived from the CHARMM Generalized Force Field (CGenFF) by combining urea, nucleotide and amide parameters.48 A 500-step minimization was performed on each duplex using the steepest descent (SD) method by applying harmonic restraints on the hydrogen bonds present between urea and its complementary base in all the B-DNA duplexes. Another 500-step SD minimization was performed on these systems with harmonic restraints on the heavy atoms. Then these systems were overlaid into an orthorhombic water box whose dimensions were selected by extending 10 Å beyond the duplex dimensions. The TIP3P water model49 was used for water. The negative charge of the phosphate groups of DNA duplexes were neutralized by adding sodium ions, and then the systems were minimized by performing 500 steps each of SD and adopted basis Newton Raphson (ABNR) minimizations while harmonically restraining the DNA heavy atoms. A 100 ps MD simulation was performed on each of these systems in NVT ensemble under the same conditions used above. The final coordinates obtained after the equilibration were then used for running production simulations in NPT ensemble. SHAKE algorithm50 was used to constrain the covalent bonds involving hydrogen atoms and CRYSTAL module51 in CHARMM was used to


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represent periodic boundary conditions during equilibration. Particle mesh Ewald (PME) summation method52,53 was used to treat the long range electrostatic interactions. Lennard-Jones (LJ) potential was truncated at 14 Å by using a force switch function.54 An integration time step of 2 fs was used and the coordinates were saved every 5 ps for further analysis. All the urea incorporated DNA duplexes were simulated for 25 ns without any restraints for further equilibration, which were considered as the second stage of equilibration. Further, 100 ns MD simulations were performed on the duplexes in which the urea lesions occupy intrahelical positions. The stable conformations obtained at the end of the 25 ns MD simulations were used to run these simulations (see later). The temperature and pressure were maintained using the Nosé-Hoover thermostat55 and Langevin piston56 respectively during the simulations. Four additional simulations of duration 100 ns each were performed on pure DNA duplexes corresponding to AT, GC, TA and CG base pairs in place of urea-nucleobase for comparison (Figure 1h). Visual Molecular Dynamics (VMD) program57 was used for viewing the structures and rendering the images shown in the manuscript. Hydration numbers for the urea-nucleobase pair were calculated by counting the number of water molecules that have their oxygen atom present within 3.5 Å of N or O atoms of the urea-nucleobase. The INTER command in CHARMM program was used to calculate the interaction and base stacking interactions of urea base pairs in the duplex by applying infinite nonbond cut-offs and these interactions contain both electrostatic and van der Waals contributions. The averages and standard errors of mean reported in the current manuscript were obtained by averaging the data over 5 ns blocks during the 100 ns of the simulations.

Free energy calculations: The molecular mechanics-generalized Born with surface area (MM-GBSA) method was used to calculate the binding free energies corresponding to formation of duplex from individual strands which uses molecular mechanical energy, solvation energy and entropy of the system.39,40


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= + − where represents the free energy of the system, represents the molecular mechanical energy which is the sum of all the energy terms in the force field equation (bonds, angles, dihedrals, torsion, electrostatic and van der Waals terms), is the solvation free energy which is the sum of electrostatic ( ) and non-polar ( ) solvation free energies, is the absolute temperature and is the molecular mechanical entropy of the system. The electrostatic and nonpolar solvation free energies were calculated by using the generalized Born molecular volume (GBMV) method and = ∗ , where = 0.0072 kcal mol-1Å-2 respectively. The

represents the solvent accessible surface area calculated by using a small probe with radius 1.4 Å. The binding free energy can be calculated as the difference in the free energies of the duplex, and the sum of the free energies of the individual components. ∆ = − − where , , and represent the free energies of the duplex and the individual strands present in the duplex calculated using the above equation. The free energies corresponding to individual strands were calculated from the individual strand trajectories obtained from the same simulations as that of the duplex. Previous studies39,40,58-60 have shown that such an approach is able to reproduce trends that are observed in simulations of isolated strands. The entropy term was computed using the quasiharmonic approximation61,62. The free energy difference between the pure DNA duplex and the urea substituted DNA duplex must be estimated in order to compare their relative thermodynamic stabilities. Within approximations considered here, the current approach is expected to be adequate toward this end.

Results and Discussion The structural deviations of urea incorporated duplexes were assessed by calculating root mean square deviations (rmsd) with respect to their initial conformation. The rmsd values


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corresponding to all the eight urea incorporated systems were calculated with respect to their starting conformation (Figure S1). The deviations were considerably small for all the duplexes within 25 ns simulation time. Interestingly, the non-WC urea-nucleobase pairs either transformed into WC conformations or simply moved out of the duplex within few nanoseconds of the simulation time, indicating their instability. Elimination of the structures based on the stability and helical position of the urea nucleotides decreased the number of structures under study from 8 to 5. These five structures are A2b, G1b, G1c, C2b and T2c, on which a new set of simulations for 100 ns each were performed. The current study describes the results of these five urea incorporated duplexes obtained from the 100 ns MD simulations. The urea-thymine base pair in T2c exhibits conformations and hydrogen bond interactions that are similar to those observed in the NMR structure23. Furthermore, the time series of hydrogen bond distances between pyrimidine and urea indicate that several events of switching between two modes of interactions, discussed later, have been observed (Figure S2). These observations suggest the reliability of the explanations made using the MD trajectories obtained in the present simulations.

Structure and dynamics of urea incorporated B-DNA duplexes: The structural closeness of the damaged DNA duplexes was examined by calculating the rmsd with respect to their initial conformation that resembles a typical B-form DNA. As shown in Figure 2, the rmsd values indicate that the structural deviations are small compared to their respective starting structure, indicating their similarity with an ideal B-form DNA. Visual inspection of MD snapshots indicates that the urea lesions are placed well inside the duplex for all the urea incorporated B-DNA during the course of these simulations. Furthermore, the rmsd values were calculated with respect to typical B-DNA conformation, and the small values observed indicate that the presence of urea does not affect their overall structure significantly. This suggests that the BDNA duplexes preserve their global characteristics after the incorporation of urea lesions opposite


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to all the four nucleobases. High deviations were observed for short time within these simulations, especially for T2c and C2b, which are attributed to local structural transitions that are discussed later. The simulations study resulted in stable duplexes that are similar to the structures observed in earlier NMR spectroscopy studies on DNA duplex with urea complementary to thymine base.23

Hydrogen bonding interactions between urea and nucleobases: Previous studies have shown that urea has strong capability to form hydrogen bonds with the nucleobases.36-38 There are two potential hydrogen bonding sites on the urea molecule to pair with the nitrogenous bases of the complementary strand. These are oxygen atom on the carbonyl group of urea and nitrogen atom of the amino group which is not attached to the deoxyribose sugar of the double helix. In the beginning, all the urea-nucleobase pairs have two hydrogen bonds between urea and nucleobases (Figure 1a-g). Stability of these hydrogen bonds was monitored by calculating the hydrogen bond distances corresponding to the donor/acceptor atoms involved in hydrogen bonding between urea and nucleobases during the simulations. Figure 3 shows the probability distributions of such hydrogen bond distances for all the urea-nucleobases pairs present in the urea incorporated duplexes. These distributions show interesting trends that differentiate the urea-purine and ureapyrimidine pairs. In the duplexes containing urea-purine pairs, a strong peak at 3.5 Å suggests that at least one stable hydrogen bond is observed during the simulations in spite of deviations in the second hydrogen bond (Figure 3). The probability distributions of G1c indicate the GUA-urea pair to be alternating between two-hydrogen bonded state and a bifurcated hydrogen bonded state. In case of urea-pyrimidine pairs, the peak at 3.5 Å corresponds to the direct hydrogen bond interactions between urea and nucleobase, whereas the peak at distance 5.5 Å corresponds to water mediated hydrogen bond formation between urea and nucleobase. This suggests that, while urea interacts with the purine bases through direct hydrogen bonding interactions, urea-pyrimidine pairs alternate between direct hydrogen bonding and water mediated interactions within the duplex. The


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nature of such water mediated interactions between the pyrimidine nucleobase and urea are discussed later. The base pairs other than the mismatched urea and its neighbors are reasonably stable with WC base pairing (Figure S3) indicating that inclusion of urea lesion in the B-DNA duplexes does not disturb their structural integrity. Furthermore, the perturbations observed in base pair step parameters corresponding to base pair steps away from the mismatched base pair are minimal, supporting earlier observations (Figure S4-S8).

Energetics of the hydrogen bonding and stacking interactions between urea and nucleobases: Base pairing and stacking interactions among the nucleobases are important for the stability of nucleic acid duplexes. The strength of these interactions in the presence of the urea lesion were calculated and compared to the regular DNA duplexes. The mean interaction energies of urea with its complementary WC base are shown in Figure 4 and Table S1. Unlike nucleobases, urea does not have a π-surface and can form strong interactions with the nucleobases within DNA duplex. A reduction in interaction energy values was observed when substituting one of the nucleobase with urea in typical DNA duplexes. These interaction energy values are in the range from ~ -5 to -15 kcal mol-1, suggesting that urea strongly interacts with all the nucleobases. Although the interactions are less favorable compared to those of the regular A-T and G-C base pairs (~ -14 and -24 kcal mol-1 for the canonical AT and GC base pairs) of a typical B-DNA duplex, they are significant and strong enough to stabilize these duplexes. The reduction in interactions are high when replacing purine bases compared to pyrimidine bases in the purine-pyrimidine base pairs in DNA duplex. The effect of the presence of urea on the strength of neighboring base pairs was also evaluated (Figure 4 and Table S1), which indicate no significant change in the interaction energies due to the inclusion of urea lesion. Stacking interactions of urea moiety with the nitrogenous bases present above and below in the same strand and complementary strand were also calculated, and are shown in Table 1. The energy values indicate that the urea moiety present in DNA duplexes forms significant stacking


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interactions with the neighboring bases. However, similar to the hydrogen bonding interactions, the magnitudes of stacking interaction energies of urea moiety are less favorable than that observed in the control simulations. However, these interactions are sufficient to keep the urea lesions inside the double helix in a manner characteristic of regular bases. Such strong stacking interactions between urea and nucleobases were also observed in previous quantum chemical and MD simulation studies in the context of RNA unfolding in presence of aqueous urea.36-38 The incorporation of urea lesion leads to small deviations in the stacking interaction energies of nucleobases complementary to urea (Table 1). The urea substitution does not affect stacking interactions among the base pair steps that are away from the urea-nucleobase pair (Figure S4-S8). Therefore, the energy values from hydrogen bond interactions and stacking interactions though less favorable than in canonical DNA duplexes suggest that urea can potentially replace nucleobases in DNA duplexes without distorting their overall structures. The effect of incorporating a urea lesion on the overall stability of the duplex structures is discussed later.

In order to understand the effect of incorporation of urea lesion on the thermodynamic stability of DNA duplexes, binding free energies corresponding to duplex formation were calculated using the MM-GBSA method (Table 2). The relative binding free energy values for the substitutions are shown with respect to their corresponding pure DNA duplexes. These values indicate that, in general, the urea substituted duplexes are either less stable or have comparable stability with respect to the corresponding pure duplex. However, the duplex that has urea complementary to cytosine is more stable than the corresponding pure duplex. Future studies can possibly include DNA duplexes with homo AT and GC base pairs in which all the pyrimidine bases are replaced by urea. This can provide further support for the pyrimidine-mimicking nature of urea.


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Smaller size of urea compared to the purine base leads to interesting hydrogen bond dynamics: All canonical base pairs in nucleic acids are formed between a purine and a pyrimidine nucleobases. In the duplexes considered here, one of the nucleobases is replaced by the urea moiety. While the size of the urea moiety and the positions of the hydrogen bonding groups are comparable to those of the pyrimidine base, there exists a huge mismatch in terms of the size when it comes to replacing a purine base by urea. As discussed above, at least one conventional hydrogen bond is formed between the pairs involving urea and GUA/ADE. In case of urea-CYT/THY pairs, the sugar moieties have to move towards each other to form hydrogen bonds. The distance between the N9 of purine and N1 of pyrimidine corresponding to a typical base pair is found to be approximately 9 Å. The distance between the N atom of urea covalently bonded to the sugar moiety and the paired base (N9 for purine and N1 for pyrimidine) were calculated and the distributions are given in Figure 5a. The distributions involving urea-GUA/ADE pairs sample the distance close to 9 Å with minor deviation for G1a. However, for both the urea-THY and urea-CYT pairs, two distinct states centered at approximately 7.5 and 9.5 Å are sampled with no significant probability in the intermediate distances. Careful analysis of the trajectories indicates that they exist in dynamic equilibrium between two states corresponding to these distances. Time series plot of the distances indicate that the systems alternate between these two states in the ns timescale. Analysis of the structures reveal that the state corresponding to a distance close to 7.5 is when there is direct hydrogen bond between the nucleobase and urea, whereas the other state corresponds to the state where the two moieties interact via bridging water molecules (Figure 6). Such dynamics can make detection of the imino protons of this pair difficult/impossible. Notably, previous NMR studies had difficulties in studying nucleic acid structures with urea-nucleobase pairs possibly because of this effect. This phenomenon also is expected to alter the solvation dynamics of the nucleic acid significantly.26,28 To understand this further, the number of water molecules present around the urea:nucleobase pair were calculated.


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Table 3 shows the average numbers of water molecules around the urea-nucleobase pairs, and these values indicate that more numbers of water molecules are present around the urea-pyrimidine base pairs compared to urea-purine base pairs. This is because of the difference in mode of interaction of urea lesion with purine and pyrimidine bases. As discussed above, urea lesion forms direct hydrogen bonds with purine bases while for urea-pyrimidine base pairs, water mediated hydrogen bonds are also observed in addition to direct hydrogen bonds and an equilibrium between these two states (Figure 6). No such water-mediated hydrogen bond formations are found when the urea lesion forms hydrogen bonds with purine nucleobases. This further indicates that water molecules play a significant role in stabilizing the urea-pyrimidine base pairs in DNA double helix.

Local and global conformational properties of B-DNA duplexes after the incorporation of the urea nucleotide: Conformational properties of the DNA duplexes and changes in their conformation after the incorporation of urea lesion were examined by calculating the pseudorotation angles and characteristic backbone dihedral angles such as α (O3’-P-O5’-C5’), β (P-O5’-C5’-C4’), γ (O5’-C5’C4’-C3’), ε (C4’-C3’-O3’-P), and ζ (C3’-O3’-P-O5’). The probability distributions of pseudorotation angles indicate that the conformation of the furanose sugars of urea nucleotide depends on its complementary base (Figure 7). The deoxyribose sugars generally sample populations majorly in C2'-endo and minor populations in O4'-endo regions.58,63 The furanose sugars corresponding to urea lesions opposite to guanine and cytosine predominantly sample in C2'endo regions whereas furanose sugars corresponding to urea lesion complementary to adenine and thymine nucleobases sample conformations in C2'-endo and C3'-endo regions. The changes in pseudorotation profiles of furanose sugars of urea lesion are restricted to local changes and do not alter the global conformation of B-DNA duplexes (Figure S9a and b). Similar conformations have been observed in NMR studies for the furanose sugars corresponding to the modified sites in B-


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DNA duplexes with urea or formamide lesions.26,28 Sampling of the population in O4’-endo region increases for the furanose sugars of nucleobases that are opposite to urea lesion. To understand the conformation of the urea and complementary nucleotides, glyosidic dihedral angles corresponding to sugar-base or sugar-urea bond were calculated. Probability distributions shown in Figure 8a indicate that all the urea lesions exhibit anti conformation except for guanine in G1a conformation, in which urea exhibits both syn and anti-conformations. As shown in Figure 8b, the probability distributions indicate that the complementary purine bases sample conformations that are sampled by the bases in the regular B-DNA duplex. However, the pyrimidine bases sample regions corresponding to regions sampled by bases in regular B-form and A-form duplexes. This might be associated with the change in pseudorotation profile of the furanose sugar that requires rearrangement in the orientation of sugar-base glycosidic dihedral angle. The urea incorporation does not induce considerable changes in the backbone conformation as revealed by the probability distributions of the characteristic backbone dihedral angles corresponding to the urea nucleotide and its neighboring nucleotides (Figure S10-S11). This suggests that incorporation of urea lesion does not alter the backbone conformation of the attached DNA strand significantly. The incorporation of the urea lesion preserves the B-form characteristics of the DNA duplexes, which is in agreement with previous studies.23

Conclusions Atomistic molecular dynamics simulations were performed on a number of B-DNA duplexes with the urea lesion complementary to all the four nucleobases (A, G, C and T). The nature of hydrogen bonding between urea and all the four nucleobases were considered from earlier quantum mechanical studies, and this bonding included WC, Hoogsteen and sugar edged-type interactions. Several structural and energetic calculations were performed on the resultant trajectories, and the results suggest that the urea incorporation preserves most of the global


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characteristics of typical B-DNA duplexes despite small variations being seen around the mismatched site for urea-pyrimidine base pairs. It was observed that within a DNA duplex, the urea-nucleobase pairs with WC hydrogen bonding interactions were stable, whereas other interacting conformations were either unstable or were transformed into WC conformations during the course of the simulations. Urea lesions can form stable hydrogen bonds with nucleobases and interact strongly with them which is evident from the hydrogen bond distances and base pair interaction energy calculations. This is in good agreement with the previous results obtained from NMR and quantum mechanical studies. The base pair interaction energies of urea-purine base pairs are comparable to the values of pyrimidine-purine base pairs that are present in canonical B-DNA duplex. The interaction energy values corresponding to urea-pyrimidine base pairs, however, were small compared to typical base pair values. Nevertheless, all the urea-nucleobase pairs were quite stable and the urea lesions stayed inside the helix during the course of the simulations. Urea lesions can also form strong stacking interactions with the neighboring nucleobases of the same strand and in the complementary strand. The incorporated urea lesions experience an environment similar to regular bases that are present in the same and partner strands. These urea-nucleobase pairs do not alter the geometries of the neighboring base pairs. The formation of hydrogen bonds directly between the urea lesion and the purine bases stabilizes the base pairs, whereas water mediated hydrogen bonds were also observed in case of urea-pyrimidine base pairs in addition to direct hydrogen bonds. These observations suggest that the stability of mismatched base pairs depends on the size of the complementary base paired with the urea lesions. Free energies corresponding to the duplex formation were further calculated to understand the thermodynamic stability of DNA duplexes after the incorporation of urea lesion. All these calculations support the fact that the urea lesion can potentially mimic nucleobases in typical B-DNA duplexes without affecting the helical integrity significantly. In vitro studies have shown that DNA polymerase stops at one nucleotide upstream of a urea


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lesion.15 The present study suggests that the mismatched urea-nucleobase pairs are quite stable and can potentially replace base pairs in typical B-DNA duplexes. It is also observed that there is no equilibrium between intrahelical and extrahelical conformations as observed for abasic sites and bulges. There have been a few studies that attempt to understand urea-nucleobase pairs, especially T and G, in the context of B-DNA duplex, and these show that urea-thymine base pair is quite stable.23 Due to the capability of forming strong hydrogen bonds with all four nucleobases, DNA polymerase can incorporate any base complementary to urea lesion unlike formamide lesion, which is specific to guanine. This leads to the existence of several possibilities to mutate the DNA sequence. Another interesting observation is that urea can interact with guanine base in multiple ways that are stable throughout the simulations. This suggests that DNA polymerase can specifically incorporate guanine opposite to urea. This is not a general observation reported in experiments, although formamide lesions have been shown to be specific to guanine.29 Another noteworthy observation is that decreased stacking interaction energies could be one reason why DNA polymerase stops one nucleotide upstream of the urea lesion. Replacement of the existing nucleobase with the urea lesion results in the decrease of stacking interactions among the nucleobases around the mismatched site, which could affect the activity of DNA polymerase.

Financial Support This work is supported by Department of Atomic Energy (DAE)-Board of Research in Nuclear Sciences (BRNS), India with grant number: 37(2)/14/05/2015/BRNS/20046, and Indian National Science Academy under the research support for young scientist medal awardees scheme.

Supporting Information available Figures of rmsd of initial model systems, time series of pyrimidine-urea pairs, probability distributions of hydrogen bond distances of base pairs, base pair step parameters, pseudorotation


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angles, dihedral angles around phosphodiester bonds and sugar-nucleobase glycosidic dihedral angles. Table for urea-nucleobase interaction energy. This material is available free of charge via the Internet at http://pubs.acs.org.

Abbreviations MD, molecular dynamics; RMSD, root mean square deviations; Tg, thymine glycol; WC, WatsonCrick; MM-GBSA, molecular mechanics-generalized Born with surface area; SASA, solvent accessible surface area

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of inflammation. Arch. Biochem.Biophys. 423, 12-22. 9. Evans, J., Maccabee, M., Hatahet, Z., Courcelle, J., Bockrath, R., Ide, H. and Wallace, S.S. (1993) Thymine ring saturation and fragmentation products: lesion bypass, misinsertion and implications for mutagenesis. Mutat. Res. 299, 147-156. 10. Maccabee, M., Evans, J.S., Glackin, M.P., Hatahet, Z. and Wallace, S.S. (1994) Pyrimidine ring fragmentation products. Effects of lesion structure and sequence context on mutagenesis. J. Mol. Biol. 236, 514-30. 11. Wallace, S.S. and Ide, H. (1990) Structure/function relationships involved in the biological consequences of pyrimidine ring saturation and fragmentation products. Ionizing radiation damage to DNA: molecular aspects, Wiley-Liss Inc, pp. 1–15 12. Teoule, R., Bert, C., and Bonicel, A. (1977) Thymine fragment damage retained in the DNA polynucleotide chain after gamma irradiation in aerated solutions. Radiat. Res. 72, 190-200. 13. Katcher, H.L. and Wallace, S.S. (1983) Characterization of the Escherichia coli X-ray endonuclease, endonuclease III. Biochemistry 22, 4071-4081. 14. Wallace, S.S. (1987) The biological consequences of oxidized DNA bases. Br. J. Cancer 55, suppIVIII, 118-128. 15. Ide, H., Kow, Y.W. and Wallace, S.S. (1985) Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro. Nucleic Acids Res. 13, 8035-8052. 16. Schaaper, R.M., Kunkel, T.A. and Loeb, L.A. (1983) Infidelity of DNA synthesis associated with bypass of apurinic sites. Proc. Nail. Acad. Sci. USA 80, 487-491. 17. Streisinger, G., Okada, Y., Emrich, J., Newton, J., Tsugeta, A., Terzaghi, E. and Inouye, M. (1966) Frameshift mutations and the genetic code. Cold Spring Harbor Symp. Quant. Biol. 31, 77-84. 18. Wallace, S.S. (1994) DNA damages processed by base excision repair: biological consequences. Int. J. Radiat. Biol. 66, 579-589.


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28. Maufrais, C., Fazakerley, G.V., Cadet, J. and Boulard, Y. (2003) Structural study of DNA duplex containing an N-(2-deoxy-β-D-erythro-pentofuranosyl)formamide frameshift by NMR and restrained molecular dynamics. Nucleic Acids Res. 31, 5930-5940. 29. Maufrais, C., Fazakerley, G.V., Cadet, J. and Boulard, Y. (2000) Solution structure by NMR and molecular dynamics of a duplex containing a guanine opposite a N-(2-deoxy-b-Derythro-pentofuranosyl)formamide lesion. Biochemistry 39, 5614-5621. 30. Cuniasse, Ph., Fazakerley, G.V., Guschlbauer, W., Kaplan, B.E. and Sowers, L.C. (1990) The abasic site as a challenge to DNA polymerase: A nuclear magnetic resonance study of G, C and T opposite a model abasic site. J. Mol. Biol. 213, 303-314. 31. Joshua-Tor, L., Frolow, F., Appella, E., Hope, H., Rabinovich, D. and Sussman, J.L. (1992) Three-dimensional structures of bulge-containing DNA fragments. J. Mol. Biol. 225, 397431. 32. Roll, C., Ketterle, C., Fazakerley, G.V. and Boulard, Y. (1999) Solution structures of a duplex containing an adenine opposite a gap (absence of one nucleotide). An NMR study and molecular dynamic simulations with explicit water molecules. Eur. J. Biochem. 264, 120-131. 33. Woodson, S.A. and Crothers, D.M. (1988) Structural model for an oligonucleotide containing a bulged guanosine by NMR and energy minimization. Biochemistry 27, 31303141. 34. Morden, K.M. and Maskos, K. (1993) NMR studies of an extrahelical cytosine in an A.T rich region of a deoxyribodecanucleotide. Biopolymers 33, 27-36. 35. Van den Hoogen, Y.T., van Beuzekom, A.A.V., van den Elst, H., van den Marel, G.A., van Boom,

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Figures and Tables

Figure 1: Model systems of urea-nucleobase pairs and DNA duplexes. (a-g) Representation of the possible hydrogen bonding between urea and all the four nucleobases (A,G,T and C), and (h) the DNA sequence used to model the pure and mismatched B-DNA duplexes. This sequence is taken from an earlier NMR spectroscopic study on 10-mer DNA duplex with urea-thymine mismatch pair.21 The terms used for representing the urea incorporated DNA duplexes and throughout the manuscript are also mentioned.


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Figure 2: Time series of root mean square deviations (in Å) of the urea incorporated B-DNA duplexes. These values are calculated with respect to their initial conformation used to run the simulations.


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Figure 3: Probability distributions of distances (in Å) corresponding to the hydrogen bonds between urea and all the four nucleobases that are present in urea incorporated B-DNA duplexes. The hydrogen bonding between urea and nucleobase are shown in a separate panel on the right hand side. The colour scheme used to represent probability distribution lines is same as the colour scheme of hydrogen bonds between the urea-nucleobase pairs.


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Biochemistry

Figure 4: Interaction energies of the urea-nucleobase mismatched pairs and their neighbor AT and GC base pairs of all the urea incorporated B-DNA duplexes (blue). The interaction energies corresponding to base pairs in pure DNA are also included for comparison (red). All the interaction energies are in kcal mol-1.


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Figure 5: Probability distribution of the distances (in Å) between connecting nitrogen atoms of nucleobase or urea with furanose sugar for (a) urea-nucleobase mismatched pairs and (b) base pairs next to the mismatched base pair (CG:red and AT:black) present in all the urea incorporated B-DNA duplexes.


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Biochemistry

Figure 6: Two modes of interactions between urea and pyrimidine bases. Depiction of direct hydrogen bonds and water mediated hydrogen bonds of urea with its complementary bases, thymine (a:T2c) and cytosine (b:C2b), during the course of the molecular dynamics simulations.


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Figure 7: Probability distributions of pseudorotation angles of furanose sugars of urea nucleotides (Ur) and their respective complementary nucleotides (cU) in all the urea incorporated B-DNA duplexes. The probabilities corresponding to Ur and cU are shown in blue and green respectively, while the probability corresponding to pure DNA duplexes is shown in purple.


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Biochemistry

Figure 8: Probability distributions of sugar-base glycosidic angle (in degree) for urea nucleotides (a) and respective complementary nucleobases (b) of all the urea incorporated B-DNA duplexes.


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Figure 9: Probability distributions of dihedral angle (in degree) of uridic bond of urea in urea nucleotides in all the urea incorporated B-DNA duplexes. Structures corresponding to cis (A2b) and trans (T2c) conformation of urea along with its complementary nucleobase are also shown.


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Biochemistry

Table 1: Stacking interaction energies of urea and its complementary nucleobases with the nucleobases of the same and their complementary strands. All the energies are in kcal mol-1.

Duplex

Urea

Complementary

Adenine B-DNA A2b

-19.0 ± 0.0

-19.5 ± 0.0

-5.6 ± 0.0

-19.4 ± 0.0

Guanine B-DNA

-18.5 ± 0.3

-20.8 ± 0.0

G1a

-5.6 ± 0.0

-21.6 ± 0.1

G1c

-7.3 ± 0.0

-20.4 ± 0.0

Thymine B-DNA T2c

-12.0 ± 0.0

-9.6 ± 0.0

-6.4 ± 0.1

-9.9 ± 0.1

Cytosine B-DNA C2b

-13.9 ± 0.0

-8.9 ± 0.1

-5.2 ± 0.1

-10.8 ± 0.2


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Table 2: Absolute and relative binding free energies (∆G and ∆∆G, respectively, in kcal mol-1) of urea incorporated duplexes and pure DNA duplexes considered in the present study. Duplex

∆G

∆∆G

(a) Adenine Pure DNA -112.5 A2b

0.00

-114.5

-2.0

Pure DNA -128.0

0.00

(b) Guanine

G1a

-114.7

13.3

G1c

-110.5

17.5

(c) Thymine Pure DNA -108.9 T2c

-106.1

0.00 2.8

(d) Cytosine Pure DNA C2b

-83.9

0.00

-105.7 -21.8


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Table 3: Number of water molecules present around the mismatched base pair (urea and complementary base) for all the urea incorporated B-DNA duplexes considered in the present study. Duplex

Hydration Number

A2b

10.57 ± 0.04

G1a

11.72 ± 0.04

G1c

10.49 ± 0.02

T2c

13.43 ± 0.04

C2b

13.23 ± 0.05


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Table of contents (TOC) image


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Urea Mimics Nucleobases by Preserving the Helical Integrity of B-DNA

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