Structural grounds for the 2-aminopurine mutagenicity: a novel insight into the old problem of the replication errors

Abstract

Within the framework of the classical Watson–Crick tautomeric hypothesis it has been reliably established by quantum-chemical calculations that amino–imino tautomerization of the 2-aminopurine (2AP), implemented in any way present in the literature, is not the root cause of its mutagenicity. It has been shown for the first time that the mutagenic pressure of the 2AP is exerted on DNA by the generation of the T* mutagenic tautomers (marked by an asterisk) within the 2AP·T(WC) pair with Watson–Crick (WC) geometry at its transformation into the wobble (w) pair, 2AP·T*(w), according to the reaction pathway 2AP·T(WC) → 2AP·T*(w). This mutagenic process proceeds with considerably greater probability than in the case of the Watson–Crick A·T(WC) DNA base pair tautomerization – A·T(WC) → A·T*(w).

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Introduction

Nowadays, the problem of a better understanding and clarification of the various mechanisms of action of mutagens, which are the derivatives of the nucleotide bases, has become increasingly important.^1–5 This is due, primarily, to the need for human protection against possibly mutagenic chemicals or environmental agents,^6,7 or due to the possibility of the application of these compounds in medical practice, in particular for antivirus and anticancer therapy,^8–12 which is of high importance for clinical research and drug discovery.^13–17

2-Aminopurine (2AP) is extensively used as a fluorescent nucleotide analog to monitor local conformational changes in nucleic acids in experiments with single-molecule approaches.^18–21 Moreover, 2AP is known as a classical mutagen, of which the mechanisms of action have been intensively studied both experimentally^22–35 and theoretically,^35–43 starting from the pioneering work⁴⁴ that has already become classical.

Despite the fact that 2AP enzymatically incorporates into the DNA double helix in only trace concentrations,²⁴ it causes a powerful mutagenic effect, inducing point mutations, the lion's share of which make up the A·T → G·C transitions in comparison with the G·C → A·T transitions.^24,37

It was experimentally registered that by its coding properties 2AP resembles adenine (A) and guanine (G) DNA bases; moreover, the first property is manifested more strongly.⁴⁵ Thus, within the framework of the Watson–Crick tautomeric hypothesis, the 2AP mutagenicity is usually explained by its incorrect binding with the imino tautomer of the cytosine (C) into the pair,^26–32,39 which architecture is close to the characteristic dimensions of the canonical Watson–Crick (WC) A·T(WC) and G·C(WC) DNA base pairs. In this case, usually the 2AP·C*(WC) pair is preferred over the 2AP*·C(WC) pair, which has a much higher energy than the first pair^46,47 (here and below mutagenic tautomers are marked with an asterisk). However, the intimate mechanism of the origin of the C* tautomers has not been established to date. Literature analysis^22–38 indicates that this is due to the fact that until this time the problem of the 2AP mutagenicity has not been considered with the involvement of all the approaches and model representations present in the literature.

We have comprehensively and thoroughly addressed the solution of this extremely important and at the same time very difficult biological problem by analyzing the structural mechanisms of the 2AP mutagenic pressure on DNA.

First of all, taking into account that in recent years the classical Watson–Crick tautomeric hypothesis⁴⁸ significantly expanded its evidentiary capabilities and confirmed its performance,^49–55 we chose this theoretical platform as a basis.^{42,43,56–60} Secondly, within the framework of this hypothesis we have analyzed in detail all the literature-presented physico-chemical mechanisms of the origin of rare tautomers of the classical DNA bases, such as 2AP, that are opposite to the basic, canonical tautomeric forms, with changed coding properties.

The most widespread and at the same time the most simple mechanism of the DNA bases tautomerization is intramolecular proton transfer within the isolated molecule (see overview⁶¹ and refs. therein).

But above all, a deservedly honored place is occupied in the literature by a classical and also, historically, the first mechanism of DNA base tautomerization proposed by Löwdin^62–67 that connects the emergence of mutagenic tautomers of the nucleobases with tautomerization of their Watson–Crick DNA base pairs by counter proton transfer along adjacent antiparallel intermolecular H-bonds.

We have also analyzed the possibility of nucleotide base tautomerization by a single water molecule (see work⁶¹ and refs. therein). Such processes commonly occur faster in comparison with the rate of the DNA replication in living cells and appear quite realistic, especially in those cases where, in virtue of certain circumstances, the replisome becomes permeable to water.⁴³ At the same time, it should be noted, in fairness, that currently there are no experimental or theoretical data on the probability of the penetration of water molecules into the area of DNA replication and their binding with DNA bases in a suitable configuration for the mutagenic tautomerization.

Finally, we also took into account novel ideas according the tautomeric bistability of the Watson–Crick DNA base pairs, as realized by the intrapair movement of protons, which is accompanied by a significant change in the geometry of the pair – from Watson–Crick to the wobble (w) (shifted) and vice versa.^{42,43,58–60,68–70} This approach is an alternative to Löwdin's mechanism and has proven its productivity at explaining the mechanisms of the origin of all 12 spontaneous point mutations,⁴³ including both transitions and transversions, from the unified physico-chemical positions. The first experimental confirmation for these mechanisms have already appeared.^54,55

By implementing this approach, we were the first to establish that amino-imino tautomerization of 2AP in any of the known literature ways is not the reason for its mutagenic pressure on DNA. We have also shown that the facilitated transition of the thymine (T) base into its mutagenic tautomeric form T* obligatory for the 2AP·T(WC) → 2AP·T*(w) WC → w tautomeric conversion underlies the mutagenic action of 2AP. In fact, 2AP induces the T → T* mutagenic tautomerization within the 2AP·T(WC) pair with significantly greater probability than takes place in the case of the A·T(WC) DNA base pair. Thus, 2AP of necessity produces A·T → G·C transitions, and also some transversions.

Computational details

Density functional theory geometry and vibrational frequency calculations

All calculations of the geometries and harmonic vibrational frequencies of the considered base mispairs and transition states of their interconversion were performed using the Gaussian'09 package⁷¹ at the DFT B3LYP/6-311++G(d,p) level of theory,^72–74 which has been applied for analogous systems and verified to give accurate geometrical structures, normal mode frequencies, barrier heights and characteristics of intermolecular H-bonds.^75–77 A scaling factor that is equal to 0.9668 has been applied in the present work for the correction of the harmonic frequencies of all the studied base pairs.^78,79 We have confirmed the minima and transition states (TSs), located by means of the Synchronous Transit-guided Quasi-Newton method,⁸⁰ on the potential energy landscape by the absence or presence, respectively, of the imaginary frequency in the vibrational spectra of the complexes. We applied standard TS theory⁸¹ to estimate the activation barriers of the tautomerization reaction.

Surrounding effects

In order to examine the surrounding effect on the considered tautomerization processes, we reoptimized geometries of the complexes at the B3LYP/6-311++G(d,p) level of theory using the commonly applied Conductor-Like Polarizable Continuum Model (CPCM),^82–84 choosing the continuum with a dielectric constant of ε = 4 typical for the hydrophobic interiors of proteins⁸⁵ and for the hydrophobic interfaces of specific protein–nucleic acid interactions.^86–89

Intrinsic reaction coordinate calculations

Reaction pathways were monitored by following the intrinsic reaction coordinates in the forward and reverse directions from each TS using a Hessian-based predictor-corrector integration algorithm⁹⁰ with tight convergence criteria. These calculations eventually ensure that the proper reaction pathway, connecting the expected reactants and products on each side of the TS, has been found.

Single point energy calculations

In order to consider electronic correlation effects as accurately as possible, we followed geometry optimizations with single point energy calculations using the MP2 level of theory⁹¹ and 6-311++G(2df,pd) Pople's basis set of valence triple-ζ quality^92,93 and aug-cc-pVDZ Dunning's cc-type basis set,⁹⁴ augmented with polarization and/or diffuse functions.

The Gibbs free energy G for all structures was obtained in the following way:

G = E_el + E_corr, (1)

where E_el is the electronic energy, while E_corr is the thermal correction.

Evaluation of the interaction energies

Electronic interaction energies E_int were calculated at the MP2/6-311++G(2df,pd) level of theory in vacuum as the difference between the total energy of the base pair and the energies of the isolated monomers. Gibbs free energy of interaction were obtained using a similar equation. In each case the interaction energy was corrected for the basis set superposition error (BSSE)^95,96 through the counterpoise procedure.^97,98

Estimation of the kinetic parameters

The time τ_99.9% necessary to reach 99.9% of the equilibrium concentration of the reactant and product in the system of reversible first-order forward (k_f) and reverse (k_r) reactions can be estimated by the formula:⁸¹

The lifetime τ of the formed mismatches was calculated using the formula 1/k_r, where the values of the reverse k_r and forward k_f rate constants for the tautomerization reactions were obtained as:⁸¹

where quantum tunneling effects are accounted for by Wigner's tunneling correction,⁹⁹ which has been successfully used for the DPT reactions:^{56–60,100,101}where k_B is Boltzmann's constant, h is Planck's constant, ΔΔG_f,r is the Gibbs free energy of activation for the tautomerization reaction in the forward (f) and reverse (r) directions, and ν_i is the magnitude of the imaginary frequency associated with the vibrational mode at the TSs.

QTAIM analysis

Bader's quantum theory of atoms in molecules (QTAIM) was applied to analyze the electron density distribution ^102–107. The topology of the electron density was analyzed using program package AIMAll ¹⁰⁸ with all the default options. The presence of a bond critical point (BCP), namely the so-called (3, −1) BCP, and a bond path between hydrogen donor and acceptor, as well as the positive value of the Laplacian at this BCP (Δρ > 0), were considered as criteria for the H-bond formation ^102–107. Wave functions were obtained at the level of theory used for geometry optimization.

Calculation of the energies of the intermolecular specific contacts

The energies of the weak CH⋯O/N and NH⋯C H-bonds, and CH⋯HN dihydrogen bond, and attractive N⋯N van der Waals contacts in the base mispairs and TSs of their interconversion were calculated by the empirical Espinosa–Molins–Lecomte (EML) formula based on the electron density distribution at the (3, −1) BCPs of the specific contacts:^109–113

E = 0.5 × V(r), (5)

where V(r) is the value of a local potential energy at the (3, −1) BCP.

The energies of all the other conventional AH⋯B H-bonds were evaluated by the empirical Iogansen's formula:¹¹⁴

where Δν is the magnitude of the frequency shift of the stretching mode of the AH H-bonded group involved in the AH⋯B H-bond relative to the unbound group. A partial deuteration was applied to minimize the effect of vibrational resonances.¹¹⁵

The atomic numbering scheme for the DNA bases is conventional.¹¹⁶

Substantiation of the computational model

Similarly to our recent studies on a related topic,^117,119 we considered the simplest physico-chemical model of the base mispairs in the base-pair recognition pocket of the high-fidelity DNA-polymerase, namely the H-bonded pairs of nucleotide bases in the continuum with ε = 1/ε = 4. In this case, we have relied on the results obtained in the work¹¹⁸ in which the adequacy of this model was convincingly proved, at least for the study of the tautomerization of the H-bonded pairs of nucleotide bases, where insignificance of the influence of the stacking and the sugar-phosphate backbone on the tautomerization process has been demonstrated. Thereby, their impact can be neglected in the first approximation. In addition, the applied model can help to distinguish the lowest structural level at which the tautomerization effects can be observed, and to estimate the changes at the sequential complications of the model.

In this study we have chosen the simplest level of the base pairs that adequately reflects the processes occurring in real systems^54,55 without deprivation of the structurally functional properties of the bases in the composition of DNA. In this case, the value of the effective dielectric constant ε (1 < ε < 4) characteristic for the anhydrous molecular crystals satisfactorily models the substantially hydrophobic recognition pocket of the DNA-polymerase machinery as a part of the replisome.^85–89

Results and their discussion

In this work we have carefully analyzed, without any exception, all the mechanisms of the generation of rare tautomers, including mutagenic ones that are represented in the literature.

Initially, we examined the intramolecular amino–imino tautomerization of the 2AP molecule and compared it with similar tautomerization for the A DNA base. It was found that this process cannot be responsible for the 2AP mutagenicity, since the population of its tautomerized state 2AP* is almost five orders of magnitude lower than the population of the A* mutagenic tautomer (Fig. 1a and b, Tables 1–3, S1 and S2†).

Fig. 1 Reaction pathways of the biologically important tautomerizations and conformational transitions of the investigated structures containing canonical DNA bases and 2-aminopurine (2AP) (B3LYP/6-311++G(d,p) level of theory, ε = 1). Relative Gibbs free energies ΔG (kcal mol⁻¹), populations P of the presented structures and imaginary frequencies ν_i (cm⁻¹) at the TSs of their interconversions are presented below them in brackets (MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of theory in the continuum with ε = 1/ε = 4 at T = 298.15 K). Dotted lines indicate AH⋯B H-bonds and continuous lines indicate loosened A–H–B covalent bridges (their lengths in the continuum with ε = 1 are presented in angstroms; for more detailed physico-chemical characteristics of the H-bonds, see Table S1†); carbon atoms are in light-blue, nitrogen in dark-blue, hydrogen in grey and oxygen in red; numeration of the atoms is standard.

Table 1 Energetic and kinetic characteristics of the biologically important tautomerizations and conformational transitions of investigated structures obtained at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of theory in the continuum with ε = 1/ε = 4 (see also Fig. 1 and Tables S1–S4)

Tautomerization	ε^a	ΔG^b	ΔE^c	ΔΔGTS^d	ΔΔETS^e	ΔΔG^f	ΔΔE^g	τ99.9%^h	τ^i	P^j
a The dielectric constant.b The Gibbs free energy of the product relative to the reactant of the tautomerization reaction (T = 298.15 K), kcal mol^−1.c The electronic energy of the product relative to the reactant of the tautomerization reaction, kcal mol^−1.d The Gibbs free energy barrier for the forward reaction of tautomerization, kcal mol^−1.e The electronic energy barrier for the forward reaction of tautomerization, kcal mol^−1.f The Gibbs free energy barrier for the reverse reaction of tautomerization, kcal mol^−1.g The electronic energy barrier for the reverse reaction of tautomerization, kcal mol^−1.h The time necessary to reach 99.9% of the equilibrium concentration between the reactant and the product of the tautomerization reaction, s.i The lifetime of the product of the tautomerization reaction, s.j Populations of the final tautomerized structures.
2AP ↔ 2AP*	1	21.69	21.63	47.07	50.55	25.38	28.92	1.18 × 10^6	1.70 × 10^5	1.2 × 10^−16
	4	19.26	19.24	47.59	51.27	28.33	32.03	1.64 × 10^8	2.37 × 10^7	7.5 × 10^−15
A ↔ A*	1	13.60	11.82	46.16	48.05	32.56	36.23	2.13 × 10^11	3.08 × 10^10	1.1 × 10^−10
	4	11.49	10.45	46.39	49.22	34.90	38.77	1.07 × 10^13	1.54 × 10^12	3.7 × 10^−9
2AP·H2O ↔ 2AP*·H2O	1	17.41	16.55	22.05	24.06	4.63	7.51	1.23 × 10^−9	1.79 × 10^−10	1.5 × 10^−13
	4	16.78	15.84	21.61	22.91	4.83	7.07	2.24 × 10^−9	3.24 × 10^−10	4.9 × 10^−13
A·H2O ↔ A*·H2O	1	9.77	8.96	18.40	20.60	8.63	11.64	1.03 × 10^−6	1.50 × 10^−7	4.6 × 10^−8
	4	9.74	8.65	18.51	19.61	8.77	10.96	2.11 × 10^−6	3.05 × 10^−7	7.2 × 10^−8
2AP·T(WC) ↔ 2AP·T*(w)	1	8.62	8.33	18.66	17.53	10.04	9.21	2.53 × 10^−5	3.66 × 10^−6	4.8 × 10^−7
	4	7.33	7.79	15.48	14.72	8.15	6.93	1.04 × 10^−6	1.50 × 10^−7	4.2 × 10^−6
2AP·T*(w) ↔ 2AP*·T(w)	1	7.83	7.50	7.02	9.14	−0.81	1.64	1.46 × 10^−13	2.11 × 10^−14	8.6 × 10^−13
	4	7.90	6.98	7.10	8.64	−0.80	1.66	1.33 × 10^−13	1.93 × 10^−14	6.7 × 10^−12
A·T(WC) ↔ A·T*(w)	1	13.08	14.84	20.28	20.41	7.20	5.57	2.09 × 10^−7	3.03 × 10^−8	2.5 × 10^−10
	4	10.69	12.47	16.03	16.42	5.34	3.94	9.20 × 10^−9	1.33 × 10^−9	6.1 × 10^−9
	1	17.10	17.91	22.47	21.97	5.36	4.06	9.52 × 10^−9	1.38 × 10^−9	2.9 × 10^−13
	4	13.95	15.09	17.95	17.88	4.00	2.79	9.43 × 10^−10	1.36 × 10^−10	5.8 × 10^−11
	1	10.91	11.25	16.72	16.02	5.81	4.77	2.02 × 10^−8	2.92 × 10^−9	9.9 × 10^−9
	4	10.19	10.48	14.41	13.56	4.22	3.07	1.36 × 10^−9	1.97 × 10^−10	3.3 × 10^−8
A*·2AP(WC) ↔ A*·2APsyn	1	−0.83	−0.59	6.54	8.55	7.37	9.14	5.59 × 10^−8	4.07 × 10^−8	—
	4	−0.49	−0.07	5.13	7.45	5.62	7.52	4.49 × 10^−9	2.15 × 10^−9	—
A*·A(WC) ↔ A*·Asyn(TF)	1	0.56	1.23	8.09	8.09	7.53	6.85	2.66 × 10^−7	5.33 × 10^−8	—
	4	0.44	0.38	5.68	7.80	5.25	7.42	5.32 × 10^−9	1.14 × 10^−9	—
G*·2AP(w) ↔ G·2AP(WC)	1	1.33	1.07	18.04	16.58	16.70	15.51	1.77	0.28	—
	4	−1.20	−1.09	12.30	11.61	13.50	12.71	1.03 × 10^−3	1.28 × 10^−3	—
G·2AP(WC) ↔ G·2APsyn	1	0.60	1.51	8.23	10.31	7.63	8.80	3.22 × 10^−7	6.35 × 10^−8	—
	4	1.64	1.33	7.26	9.49	5.62	8.16	1.39 × 10^−8	2.14 × 10^−9	—
G*·A(w) ↔ G·A(WC)	1	−4.30	−6.73	12.33	9.97	16.64	16.70	1.22 × 10^−3	0.25	—
	4	−4.87	−6.90	8.42	6.08	13.29	12.98	1.66 × 10^−6	9.03 × 10^−4	—
G·A(WC) ↔ G·Asyn	1	0.76	0.58	8.39	8.89	7.64	8.31	3.47 × 10^−7	6.42 × 10^−8	—
	4	0.30	−0.02	7.41	7.99	7.11	8.01	1.14 × 10^−7	2.65 × 10^−8	—

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Table 2 Interbase interaction energies (in kcal mol⁻¹) for the investigated nucleobase pairs/complexes and TSs of their interconversions obtained at the MP2/6-311++G(3df,2pd)//B3LYP/6-311++G(d,p) level of theory (ε = 1)

Pair/complex	−ΔEint^a		∑EHB^b		∑EHB/|ΔEint|, %		−ΔGint^c
	X		X		X		X
	A	2AP	A	2AP	A	2AP	A	2AP
a The BSSE-corrected electronic interaction energy.b The total energy of the intermolecular H-bonds (see Table S1).c The BSSE-corrected Gibbs free energy of interaction (T = 298.15 K).
X·H2O	9.60	8.97	9.35	8.76	97.5	97.6	−3.06	−1.94
TSX·H2O↔X*·H2O	182.68	—	26.38	—	14.4	—	182.04	—
X*·H2O	12.72	14.13	11.37	12.47	89.4	88.3	1.07	2.23
X·T(WC)	14.92	14.28	12.97	12.26	86.9	85.9	1.43	2.88
	121.09	122.78	18.83	12.87	15.6	10.5	108.85	108.58
X·T*(w)	13.44	20.95	10.83	15.25	80.6	72.8	1.61	9.18
	123.44	120.41	13.17	14.67	10.7	12.2	110.37	107.85
X^+·T^−(w)	131.37	—	23.31	—	17.7	—	119.92	—
	24.75	15.38	17.62	12.11	71.2	78.8	11.20	4.39
X*·T(w)	5.18	20.96	2.74	15.62	52.9	74.6	−7.01	8.84
A*·X(WC)	17.89	9.82	13.89	7.99	77.6	81.4	4.32	−1.29
TSA*·X(TF)↔A*·Xsyn	9.88	2.45	7.92	1.52	80.2	62.0	−3.74	−6.65
A*·Xsyn	16.73	10.47	12.52	7.16	74.8	68.4	3.83	−0.40
G*·X(w)	12.41	15.80	9.83	13.74	79.2	87.0	0.90	4.25
	117.62	114.69	14.63	11.34	12.4	9.9	105.01	101.11
G·X(WC)	17.54	12.04	12.87	9.01	73.4	74.8	3.57	0.15
TSG·X(WC)↔G·Xsyn	8.97	3.07	6.63	2.13	73.9	69.5	−4.55	−6.75
G·Xsyn	17.00	11.15	11.20	7.37	65.9	66.1	2.80	0.16
C*·X(WC)	15.73	14.25	14.44	12.27	91.8	86.1	2.27	3.05

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Table 3 Selected structural and energetic characteristics of the incorrect purine·purine and purine·pyrimidine base pairs by the participation of canonical DNA bases and 2AP obtained at the MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) level of theory (ε = 1)

Base mispair	R(HN1/N9-HN1/N9)^a, Å	α1^b, °	α2^c, °	ΔEdef^d, kcal mol^−1
				To A·T(WC)	To G·C(WC)
a The distance between the glycosidic protons at the N1/N9 atoms in the purine·purine and purine·pyrimidine base pairs, respectively.b Glycosidic angle for the base situated on the left within the base pair.c Glycosidic angle for the base situated on the right within the base pair.d The deformation energy necessary to apply for the mismatch to acquire the sizes of the A·T(WC) and G·C(WC) Watson–Crick DNA base pairs.
2AP·C*(WC)	10.260	49.6	59.2	0.21	0.35
A·C*(WC)^43,56,58,59	9.996	55.3	58.2	0.10	0.29
2AP·T(WC)	8.215	49.6	53.0	0.27	0.43
A*·2APsyn	10.699	50.2	31.7	2.59	4.50
A*·Asyn(TF)^41	10.322	53.9	41.2	2.18	2.72
G·2APsyn	10.745	47.3	29.1	3.06	4.88
G·Asyn^101	10.399	51.6	38.5	3.00	3.61
A·T(WC)^42,43	10.130	54.3	54.8	0.00	0.25
G·C(WC)^42,43	10.209	52.9	55.3	0.11	0.00

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Then we aimed to analyze the tautomeric properties of the 2AP·T(WC) pair with a geometry close to Watson–Crick, which is stabilized by the participation of three intermolecular C6H⋯O4, N3H⋯N1 and N2H⋯O2 H-bonds (Fig. 1c, Table S1†). It is interesting to note that the A·T(WC) and 2AP·T(WC) H-bonded pairs possess similar energetic characteristics^40–43 (Tables 2, 3 and S1†). This means that for the explanation of the weak enzymatic integration of the 2AP into the composition of the 2AP·T(WC) pair in DNA arguments other than energetical should be considered.³⁶

Thus, we have not detected on the hypersurface of the potential energy of the 2AP·T(WC) pair the local minimum corresponding to the tautomerized Löwdin's mechanism pair (Fig. 1c). This result does not depend either on the applied level of the QM theory (B3LYP/6-311++G(d,p), MP2/6-311++G(d,p)), or on the environment of the pair (ε = 1, ε = 4). This means that the 2AP·T(WC) pair does not tautomerize via the Löwdin's mechanism for reasons of the principal character, and other mechanisms of the generation of the rare tautomers should be considered.

At the mutagenic tautomerization of the canonical DNA bases (X = A, G, T, C) by the water molecule the probability of the origin of the mutagenic tautomers of the DNA bases is determined by the population of the tautomerized X*·H₂O complexes in comparison with the starting X·H₂O H-bonded structures.⁴³ It is evident from the numerical data presented in Fig. 1d, e and Tables 1, 2, S1 and S2† that the population of the tautomerized 2AP*·H₂O complex is by orders (∼5!) less than the population of the similar complex A*·H₂O because of the participation of the canonical A DNA base. Thus, this mechanism also cannot provide a more effective tautomerization of 2AP in comparison with the canonical A DNA base. This effect has a simple explanation – isolated 2AP tautomerizes worse, since it has a higher energy of tautomerization in comparison with the free A base⁴⁶ (Fig. 1a, b, Tables 1 and S1†).

We have also explored the 2AP tautomerization within the 2AP·T(WC) pair by a newly proposed mechanism^{42,43,58–60,68–70} that is an alternative to Löwdin's scheme. As a result of a thorough analysis of all the possible ways of the WC → w tautomeric transformations of the 2AP·T(WC) base pair, it was found that formally 2AP·T(WC) → 2AP*·T(w) tautomerization occurs via two transition states, and TS_{2AP·T*(w)↔2AP*·T(w)}, and an intermediate – the long-lived 2AP·T*(w) mispair (Fig. 1f, Tables 1, 2, S1, and S3†). The final 2AP*·T(w) complex, which stabilizes by two H-bonds, N1H⋯O4 and N3H⋯N2, is a dynamically unstable structure, since under the barrier ΔΔE = 1.64 kcal mol⁻¹ of its reverse tautomerization, 2AP*·T(w) → 2AP·T*(w), it could not be arranged at a zero vibrational level, which frequency becomes imaginary in the transition state of tautomerization. Moreover, the barrier ΔΔG = −0.81 kcal mol⁻¹ is negative (Table 1): this means that the final stage of the 2AP·T*(w) → 2AP*·T(w) transformation via the TS_{2AP·T*(w)↔2AP*·T(w)} transition state does not represent a real physico-chemical process. Moreover, the 2AP*·T(w) pair has an extremely low population (Fig. 1f and Table 1) and a very small lifetime (∼2 × 10⁻¹⁴ s), which is by orders less than the time spent by the DNA-polymerase machinery on the dissociation of the base pair into the monomers (∼10⁻⁹ s).⁴³ By combining these data with those given above, we come to the final conclusion that amino–imino tautomerization of 2AP is not the root cause of its mutagenic pressure on DNA.

Among two other tautomerization processes of the 2AP·T(WC) base pair by the participation of the T* and tautomers – 2AP·T(WC) → 2AP·T*(w) (Fig. 1f, Tables 1, 2, S1 and S3†) and (Fig. 1h, Tables 1, S1 and S3†) – the first attracts special attention. It is controlled by the transition state, representing a tight ion pair 2AP⁺·T⁻, which in addition to strong electrostatic interactions are stabilized by the four N1H⋯O4, N1H⋯N3, N2H⋯N3 and N2H⋯O2 H-bonds; at this the population of the final 2AP·T*(w) complex, maintained by the two O4H⋯N1 and N2H⋯N3 H-bonds, significantly (1887 times) exceeds the population of the analogical complex A·T*(w) by the participation of the canonical A base, stabilized by two O4H⋯N1 and C2H⋯N3 H-bonds (Fig. 1g). This effect is caused by the fact that the interaction energy in the 2AP·T*(w) (ΔE_int = −20.95 and ΔG_int = −9.18 kcal mol⁻¹) pair greatly exceeds the analogical stabilization energy of the A·T*(w) (ΔE_int = −13.44 and ΔG_int = −1.61 kcal mol⁻¹) pair (Table 2).

It is logical to associate exactly with this process the mutagenic pressure of 2AP on DNA, since another tautomerized complex (Fig. 1h) has much lower (in 3.4 × 10⁴ times) occupancy than the similar complex (Fig. 1i). The opposite situation takes place for these systems – the stabilization energy of the pair (ΔE_int = −24.75 and ΔG_int = −11.20 kcal mol⁻¹) significantly exceeds the value for the pair (ΔE_int = −15.38 and ΔG_int = −4.39 kcal mol⁻¹) (Table 2).

In such a case, the microstructural mechanism of the mutagenic action of 2AP consists in the generation with the highest probability of the mutagenic tautomer T* due to the 2AP·T(WC) → 2AP·T*(w) reaction than takes place in the Watson–Crick A·T(WC) DNA base pair due to the A·T(WC) → A·T*(w) tautomerization. At this, the mutagenic effect is achieved due to the greater stability of the 2AP·T*(w) complex in comparison with the analogical A·T*(w) base pair.

Let us follow up with how point replication errors induced by 2AP are fixed in the genome. It should be noted that the 2AP·T*(w) pair meets all the necessary conditions⁴³ for its successful dissociation by the replication machinery into the monomers without alteration of their tautomeric status 2AP·T*(w) → 2AP + T*. All four canonical DNA bases belonging to the incoming nucleotide compete with the mutagenic tautomer T*.

Thus, the T* + A → T*·A(w) → T·A(WC) (Fig. 1g)^42,43 process partially “treats” the arising damage and the mispair resumes its original canonical status.

The T* + G → T*·G(WC) → T·G*(WC)^57,60 process inevitably leads to the A·T → G·C transitions. This process is described in details in the works.^43,57,60

Formation of the short Watson–Crick T*·T(WC) mispair causes transversions, when purine base is substituted by the pyrimidine.⁷⁰ The origin of another transversion of a similar type is more complicated: initially the wobble T*·C(w) pair is formed, which then tautomerizes into the short Watson–Crick-like T·C(WC) mispair.⁷⁰

Obviously, transversions arise with a significantly lower probability because, in particular, the deformation of the short Watson–Crick-like T*·T(WC) and T·C(WC) DNA base mispairs during the thermal fluctuations to the classical Watson–Crick sizes requires significant energy consumptions.^43,70

The theoretical results obtained by us are in complete accordance with experimental data, according to which 2AP produces point mutations, among which 95% are A·T → G·C transitions.¹²⁰

For the completeness of the microstructural representations of the mechanism of the 2AP mutagenic action it is necessary to analyze its interaction in the case that it represents itself as a base of the incoming nucleotide with mutagenic tautomers of the four canonical DNA bases belonging to the template and compare these processes with similar events for the canonical A base. These tautomers are formed according to known mechanisms.^42,43

2AP interacts with A* forming the long A*·2AP(WC) Watson–Crick base pair that is stabilized by the participation of the three C6H⋯N6, N1H⋯N1 and C2H⋯HN2 H-bonds (Fig. 1j, Tables 1, 2 and S1–S4†). Moreover, this pair converts into the A*·2AP_syn pair by a non-dissociative conformational mechanism via the quasi-orthogonal TS_{A*·2AP(WC)↔A*·2APsyn} transition state, maintained by the single C6H⋯N6 H-bond (Fig. 1j, Table S1†). The formed A*·2AP_syn pair is able to acquire enzymatically-competent conformation during the process of thermal fluctuations (Table 3). Nevertheless, this process does not provide a mutagenic action of 2AP, since the interaction energy of the monomers in the A*·2AP(WC) and A*·2AP_syn pairs is far less than the similar energy for the A*·A(WC)¹¹⁷ and A*·A_syn(TF)¹¹⁷ pairs by the participation of the canonical A DNA base, respectively (Fig. 1k, Table 3). Figuratively speaking, 2AP loses the competition with A for binding with the A* mutagenic tautomer.

At the same time, 2AP binds to G* more strongly than A forming the wobble G*·2AP(w) pair, which stabilizes by the participation of the two O6H⋯N1 and N2H⋯N1 H-bonds (Fig. 1l, Tables 1, 2, S1 and S4†). Moreover, the G*·2AP(w) pair is able to tautomerize into the long Watson–Crick G·2AP(WC) pair, which in its turn converts by a non-dissociative conformational mechanism through the quasi-orthogonal TS_{G·2AP(WC)↔G·2APsyn} transition state, in the stabilization of which the C6H⋯O6 and N1H⋯C6 H-bonds take part, into the G·2AP_syn, able to take an enzymatically-competent conformation during the process of thermal fluctuations (Table 3). But also in this case 2AP does not act as a mutagen since the tautomeric transformation G*·2AP(w) → G·2AP(WC) through the transition state TS_{G*·2AP(w)↔G·2AP(WC)} is much slower than a similar G*·A(w) → G·A(WC) transformation (Fig. 1m, Tables 1, 2, S1 and S4†), which completely negates the advantages of the 2AP in comparison with A for binding with G*. Indeed, the ratio of the multiplication of the probabilities⁶⁰ P(G*·2AP(w))·P(G*·2AP(w) → G·2AP(WC))/P(G*·A(w))·P(G*·A(w) → G·A(WC)) = 1.1 × 10⁻⁶ is much less than 1.

It is well known that for the effective incorporation of incorrect purine–purine pairs of nucleotide bases by DNA-polymerase into the DNA double helix, the base belonging to the incoming nucleotide must convert from an anti- to a syn-conformation. For the pairs involving canonical A and G DNA bases these transitions occur by a non-dissociative mechanism: the presence of the mobile (OH or NH₂) group as a molecular joint in position 6 of the base⁴³ plays the key role here. In the constitution of the 2AP molecule a group of this kind is absent. Nevertheless, the presence of the intermolecular C6H⋯N6 H-bond (in the A*·2AP(WC) pair (Fig. 1j))/C6H⋯O6 (in the G*·2AP(WC) pair (Fig. 1l)) allows 2AP to convert into a syn-conformation according to a non-dissociative mechanism, turning by 180 degrees as a single entity around this H-bond.

Comparison of the interaction energy of the C*·2AP(WC) and C*·A(WC)^40,56,58,59 pairs with Watson–Crick architecture, each of which is stabilized by the participation of three intermolecular H-bonds (C6H⋯N4, N3H⋯N1, N2H⋯O2 and N6H⋯N4, N3H⋯N1, C2H⋯O2, respectively) (Fig. 1n, Tables 2 and S1†), does not provide compelling reasons to believe that the first pair is incorporated into the DNA structure with a considerably greater probability than the C*·A(WC), which includes a canonical A DNA base.

And only in the case of the mutagenic tautomer T* we have established a significant excess of the affinity of this tautomer to 2AP in comparison with the A base. This indicates an additional channel of the 2AP incorporation into the structure of DNA with the formation of the 2AP·T*(w) pair, which quite quickly tautomerizes into the 2AP·T(WC) pair (Fig. 1g, Tables 1, 2, S1 and S3†), which in itself is not mutagenic.

The obtained results allow us to make some interesting physico-chemical findings.

In all the investigated complexes the total energy of the intermolecular H-bonds is less than the electronic interaction energy of the monomers. The energy of the weakest C2H⋯HN2 H-bond consists of 0.17 kcal mol⁻¹.

All the studied pairs with the participation of the 2AP adjust their geometry to the enzymatically-competent worse than similar complexes involving canonical DNA bases.

Although heterocycles of nucleotide bases are rather soft structures on the non-planar bend,^121,122 we have not detected any case of violation of their planarity.

Finally, it is important to note that the low frequency of the enzymatic incorporation of 2AP into the structure of the DNA double helix²⁴ remains an obscure phenomenon. In our opinion, the key to the solution of this problem is a substantial difference between the dynamical properties of the A·T(WC) and 2AP·T(WC) base pairs, associated with anisotropic rotational mobility¹²³ of the amino group. It is not excluded that this difference in the dynamical behavior of the pairs is critical for DNA-polymerase.

Conclusions

Incorporation of 2AP into the DNA double helix is obligatory to two channels of interaction – with thymine T and its mutagenic tautomer T*. Mutagenic pressure of 2AP on DNA boils down to the increased generation of the mutagenic T* tautomers due to the spontaneous 2AP·T(WC) → 2AP·T*(w) tautomerization. Further, this causes A·T → G·C transitions and, to a lesser extent, transversions, when a purine base is substituted with a pyrimidine base. These theoretical concepts make it possible to explain the existing experimental data in a non-contradictory manner.^22–38

Acknowledgements

We gratefully appreciate technical support and computational facilities from the joint computer cluster of SSI “Institute for Single Crystals” of the National Academy of Sciences of Ukraine (NASU) and the Institute for Scintillation Materials of the NASU incorporated into the Ukrainian National Grid. This work was partially supported by the Grant of the NASU for young scientists for 2015–2016 years and by the Scholarship of the President of Ukraine for young scientists for the years 2014–2016 given to O. O. B., as well as by the Fundación Séneca del Centro de Coordinación de la Investigación de la Región de rcMuia under Project 18946/JLI/13. O. O. B. expresses sincere gratitude to the organizing committee of the FEBS Workshop on Chromatin Proteomics (October 3–8, 2016, Crete, Greece), mainly to Prof John Strouboulis (Institute of Molecular Biology and Biotechnology Foundation of Research & Technology, Heraklion, Crete, Greece), and FEBS organisation for the Youth Travel Fund (YTF) grant for visiting. The authors sincerely thank Dr Ivan S. Voiteshenko (Institute of High Technologies, Taras Shevchenko National University of Kyiv) and Dr Fernando R. Clemente (Gaussian, Inc.) for their technical support of the work.

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Footnote

† Electronic supplementary information (ESI) available: (i) Physico-chemical parameters of the specific intermolecular contacts in the investigated DNA base pairs and transition states (TSs) of their tautomerizations and conformational transitions; (ii) energetic and kinetic characteristics of the tautomeric and conformational interconversions of the investigated monomers/complexes containing canonical DNA bases and 2-aminopurine. See DOI: 10.1039/c6ra17787e

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