Whether the amino–imino tautomerism of 2-aminopurine is involved into its mutagenicity? Results of a thorough QM investigation

Abstract

The biologically important issue of whether the amino–imino tautomerism of 2-aminopurine (2AP) is the cause of the replication and incorporation point errors induced by this mutagen, has been debated for a long time in the literature. Exploring by quantum-mechanical methods the ability of the irregular pairs formed by the H-bonding of the imino tautomer of the 2-aminopurine (2AP*) with canonical DNA bases to acquire enzymatically-competent conformation that guarantees their successful incorporation into the structure of the DNA double helix by the high-fidelity DNA-polymerase, we came to the conclusion that 2AP tautomerism is not implicated into the origin of the replication point errors induced by this mutagen. We have authentically established that 2AP* mutagenic tautomer is able to induce only one single incorporation mistake, namely transversion, by pairing through the two N1H⋯O6 and N1H⋯N2 H-bonds with guanine (G) DNA base into the wobble (w) G·2AP*(w) pair. At this, the formed pair acquires enzymatically-competent Watson–Crick (WC) conformation by following the pathway of the tautomeric and conformational transformations G·2AP*(w) → G*·2AP(w) → G·2AP(WC) → G·2AP_syn → G·2AP_syn(WC). In this case, the long G·2AP(WC) wrong pair, stabilized by the participation of the three C6H⋯O6, N1H⋯N1 and N2H⋯N2 H-bonds, acts as a predecessor of the enzymatically-competent conformation – the G·2AP_syn(WC) mispair.

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Introduction

For the past dozen years, a highly-energetic geometric isomer of the canonical DNA base adenine (A)^1–7 – 2-aminopurine (2AP) – has continued to intrigue by its mutagenic properties both experimentalists and theorists, who work in the field of molecular and structural biology, biochemistry and biophysics.^8–21 Despite intensive investigations in this direction,^22–25 the intimate structural mechanisms underlying its mutagenicity still remain obscure. Without any exaggeration it could be said that this biologically important topic continues to be a challenge for both theory and experiment.

Indeed, it was experimentally established that 2AP induces both A·T → G·C transitions, as well as G·C → A·T transitions in the opposite direction.^10,11 But to this date it was not shown for certain, which of these two transitions are predominant. Experimental data according possible transversions induced by 2AP in DNA are absent at all in the literature.^8–16 The possible reason for experimental challenges at the study of the mechanisms of the 2AP mutagenic action is most likely that it is incorporated into DNA in trace concentrations.¹⁰

From the other side, a modest theoretical understanding of the mechanisms of the 2AP mutagenic action could be associated with the fact that until recently there was no complete, logically closed theory of spontaneous point mutagenesis. That is without clear physico-chemical conceptions about how originate spontaneous point incorporation and replication errors, it is not possible to succeed in understanding the mechanisms of the occurrence of the events of the similar content, induced by the modified nucleotide bases.

Recently, based on the theory of the spontaneous point mutagenesis, which has been originally created by Brovarets' & Hovorun^26–32 within the framework of the classical Watson–Crick tautomeric hypothesis,³³ we managed to significantly advance in understanding of the mechanisms underlying the mutagenicity of 2AP.^26,34–38 Obtained theoretical data^37,38 allow us to interpret existing experimental results.^8–16

Thus, in particular, we came to the conclusion, that amino–imino tautomerism of 2AP is not involved into the DNA replication point errors induced by this mutagen. This conclusion has been made basing on the fact that 2AP* rare tautomers are generated in the genome with the probability that by orders of magnitude lower than the probability of the A* mutagenic tautomers. Basing on this result, we have not considered the possible impact of the 2AP* imino tautomers at the emergence of the induced incorporation errors.

In this paper we decided to focus on the role of the amino–imino tautomerism of 2AP at the origin of the induced by it point errors as of incorporation, so replication. The point is, that similarly to the canonical DNA bases,^39,39 it could be assumed that 2AP as the base of the incoming nucleotide can be tautomerised by endogenous water molecules, despite the fact that experimental data on this subject are not available in the literature yet. From the other side, it could be also theoretically suggested that despite the low probability of the tautomerisation of the 2AP as a template base,^37,38 this factor could be compensated as by a greater energy of interactions with canonical DNA bases, so by the faster (in comparison with the A base) acquisition of the enzymatically-competent conformation by the wrong pairs involving 2AP* rare tautomer.

Aiming to realize these assumptions, we have analyzed the ability of all possible incorrect pairs involving 2AP* rare tautomer – 2AP*·X (X = T, A, G, C) and X·2AP* mispairs – to acquire enzymatically-competent conformation and compared it with a similar ability of the incorrect A*·X and X·A* pairs, respectively, by the participation of the A* mutagenic tautomer.

In such a case we have managed to show that amino–imino tautomerism of 2AP is not involved into the origin of the induced by this compound replication point errors in DNA. At the same time, we have shown for the first time that 2AP* as a base of the incoming nucleotide may produce one single transversion, when 2AP* binds with the G base by two N1H⋯O6 and N1H⋯N2 H-bonds and the G·2AP*(w) base pair formed in such a case transforms via the pathway of the transformations G·2AP*(w) → G*·2AP(w) → G·2AP(WC) → G·2AP_syn → G·2AP_syn(WC). In this regard, it is actual the experimental determination of the constant of the tautomeric equilibrium in aqueous solution for 2AP, K_2AP↔2AP*, in particular by using new approaches for the identification of the variables of this type.⁴¹

Computational details

Density functional theory calculations of the geometry and vibrational frequencies

All calculations of the geometries and harmonic vibrational frequencies of the considered base mispairs and transition states of their interconversion have been performed using Gaussian'09 package⁴² at the DFT B3LYP/6-311++G(d,p) level of theory,^43–45 that has been applied for analogous systems and verified to give accurate geometrical structures, normal mode frequencies, barrier heights and characteristics of intermolecular H-bonds.^46–50 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 studied base pairs.^51,52 We have confirmed the minima and transition states (TSs), located by means of Synchronous Transit-guided Quasi-Newton method,⁵³ 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 theory⁵⁴ to estimate the activation barriers of the tautomerisation reaction.

All calculation have been carried in vacuum, that adequately reflects the processes occurring in real systems without deprivation of the structurally functional properties of the bases in the composition of DNA and satisfactorily models the substantially hydrophobic recognition pocket of the DNA-polymerase machinery as a part of the replisome.^55–59

Intrinsic reaction coordinate calculations

Reaction pathways have been monitored by following intrinsic reaction coordinate in the forward and reverse directions from each TS using Hessian-based predictor–corrector integration algorithm⁶⁰ 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 MP2 level of theory⁶¹ and 6-311++G(2df,pd) Pople's basis set of valence triple-ζ quality^62,63 and aug-cc-pVDZ Dunning's cc-type basis set,⁶⁴ 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 – electronic energy, while E_corr – thermal correction.

Evaluation of the interaction energies

Electronic interaction energies E_int have been calculated at the MP2/6-311++G(2df,pd) level of theory as the difference between the total energy of the base pair and the energies of the isolated monomers. Gibbs free energy of interaction has been obtained using similar equation. In each case the interaction energy was corrected for the basis set superposition error (BSSE)^65,66 through the counterpoise procedure.^67,68

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:⁵⁴

The lifetime τ of the formed mismatches has been calculated using the formula 1/k_r, where the values of the forward k_f and reverse k_r rate constants for the tautomerisation reactions were obtained as:⁵⁴

where quantum tunneling effects are accounted by Wigner's tunneling correction,⁶⁹ that has been successfully used for the DPT reactions:^70,71where k_B – Boltzmann's constant, h – Planck's constant, ΔΔG_f,r – Gibbs free energy of activation for the tautomerisation reaction in the forward (f) and reverse (r) directions, ν_i – 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 analyse the electron density distribution.^72–77 The topology of the electron density was analysed using program package AIMAll⁷⁸ with all 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.^72–77 Wave functions were obtained at the level of theory used for geometry optimisation.

Calculation of the energies of the intermolecular specific contacts

The energies of the intermolecular specific contacts in the base mispairs and TSs of their interconversions 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:^79–83

E = 0.5V(r), (5)

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

The energies of all others conventional AH⋯B H-bonds were evaluated by the empirical Iogansen's formula:⁸⁴

where Δν – magnitude of the frequency shift of the stretching mode of the AH H-bonded group involved in the AH⋯B H-bond relatively the unbound group. The partial deuteration was applied to minimize the effect of vibrational resonances.⁸⁵

The atomic numbering scheme for the DNA bases is conventional.⁸⁶

Substantiation of the computational model

Similarly to our recent studies on the related topic,⁸⁷ we have 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 vacuum. In this case, we have relied on the results obtained in the work,⁸⁷ in which the adequacy of this model was convincingly proved, at least at the study of the tautomerisation of the H-bonded pairs of nucleotide bases, where insignificance of the influence of the stacking and the sugar-phosphate backbone on the tautomerisation process has been demonstrated.^{27–29,88,89} 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 tautomerisation effects can be observed, and to estimate the changes at the sequential complication of the model.^{27–29,88,89}

Results and their discussion

Incorporation errors

Analysis of the processes of the pairing of 2AP in its rare, imino tautomeric form 2AP* with canonical DNA bases belonging to the template represents itself significant interest. The reason for such consideration is tautomerisation of 2AP – the base of the incoming nucleotide – by the endogenous water by analogy with the canonical nucleotide bases.^39,40 Unfortunately, experimental data on the relevant constant of tautomerisation in aqueous solution K_2AP↔2AP* are not available in the literature yet.

In this context we have thoroughly studied the ability of the X·2AP* pairs (X = T, A, G, C), in which monomers interact with each other by the Watson–Crick edges, to acquire enzymatically-competent conformation and compare them with analogical properties of the X·A* pairs, involving mutagenic tautomer of the canonical A DNA base. Obtained results are presented on Fig. 1–4 and in Table 1.

Fig. 1 Reaction pathways of the biologically important tautomerisations and conformational transitions of the (a) T·2AP*(w) and (b) T·A*(w) base mispairs containing canonical DNA bases and 2-aminopurine (2AP) base in the main and rare tautomeric forms leading to incorporation and replication errors caused by the rare tautomer of the 2AP (B3LYP/6-311++G(d,p) level of theory, vacuum). Relative Gibbs free energies ΔG, electronic ΔE_int and Gibbs free ΔG_int energies of interaction, the electronic deformation energies ΔE_def(A·T)/ΔE_def(G·C) necessary to apply to the mismatch to acquire the sizes of the A·T(WC)/G·C(WC) Watson–Crick DNA base pairs (in kcal mol⁻¹), respectively, imaginary frequencies ν_i (cm⁻¹) at the TSs of the interconversions are presented below them in brackets (MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of theory in vacuum at T = 298.15 K). Dotted lines indicate AH⋯B H-bonds and continuous – loosened A–H–B covalent bridges (their lengths H⋯B are presented in angstroms); carbon atoms are in light-blue, nitrogen – in dark-blue, hydrogen – in grey and oxygen – in red. The base, belonging to the template strand of DNA, is situated on the left, while the base of the incoming nucleotide – on the right.

Fig. 2 Reaction pathways of the biologically important tautomerisations and conformational transitions of the (a) G·2AP*(w) and (b) G·A*(w) base mispairs containing canonical DNA bases and 2-aminopurine (2AP) base in the main and rare tautomeric forms leading to incorporation and replication errors caused by the rare tautomer of the 2AP. See the caption of Fig. 1 for details.

Fig. 3 Reaction pathways of the biologically important tautomerisations and conformational transitions of the (a) C·2AP*(w), (b) C·2AP*(w₁), (c) C·A*(WC), (d) A·2AP*(WC), (e) A·2AP*(w) and (f) A·A*(WC) base mispairs containing canonical DNA bases and 2-aminopurine (2AP) base in the main and rare tautomeric forms leading to incorporation and replication errors caused by the rare tautomer of the 2AP. See the caption of Fig. 1 for details.

Fig. 4 Reaction pathways of the biologically important tautomerisations and conformational transitions of the (a) 2AP*·C(w), (b) 2AP*·A(w) and (c) 2AP*·G(w) base mispairs containing canonical DNA bases and 2-aminopurine (2AP) base in the main and rare tautomeric forms leading to incorporation and replication errors caused by the rare tautomer of the 2AP. See the caption of Fig. 1 for details.

Table 1 Energetic and kinetic characteristics of the biologically important tautomerisations and conformational transitions of the investigated structures containing canonical DNA bases and 2-aminopurine base in the main or rare tautomeric forms leading to incorporation and replication errors caused by the rare tautomer of the 2AP obtained at the MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of theory in vacuum

Tautomerisation/conformational transition	νi^a	ΔG^b	ΔE^c	ΔΔGTS^d	ΔΔETS^e	ΔΔG^f	ΔΔE^g	kf^h	kr^i	τ99.9%^j	τ^k
a Imaginary frequency at the TSs of the interconversions, cm^−1.b The Gibbs free energy of the product relatively the reactant of the tautomerisation/conformational reaction (T = 298.15 K), kcal mol^−1.c The electronic energy of the product relatively the reactant of the tautomerisation/conformational reaction, kcal mol^−1.d The Gibbs free energy barrier for the forward tautomerisation/conformational reaction, kcal mol^−1.e The electronic energy barrier for the forward tautomerisation/conformational reaction, kcal mol^−1.f The Gibbs free energy barrier for the reverse tautomerisation/conformational reaction, kcal mol^−1.g The electronic energy barrier for the reverse tautomerisation/conformational reaction, kcal mol^−1.h The forward rate constant for the tautomerisation/conformational reaction, s^−1.i The reverse rate constant for the tautomerisation/conformational reaction, s^−1.j The time necessary to reach 99.9% of the equilibrium concentration between the reactant and the product of the tautomerisation reaction, s.k The lifetime of the product of the tautomerisation reaction, s.
T·2AP*(w) ↔ T*·2AP(w)	1018.0	−7.83	−7.50	−0.82	1.64	7.02	9.14	5.27 × 10^13	9.52 × 10^7	1.31 × 10^−13	1.05 × 10^−8
T*·2AP(w) ↔ T·2AP(WC)	134.2	−8.62	−8.33	10.04	9.21	18.66	17.53	2.73 × 10^5	0.13	2.53 × 10^−5	7.68
T·A*(w) ↔ T^−·A^+(WC)	835.7	2.53	3.77	6.82	6.43	4.29	2.66	1.01 × 10^8	7.21 × 10^9	9.45 × 10^−10	1.39 × 10^−10
T^−·A^+(WC) ↔ T·A(WC)	128.5	−12.43	−13.36	−0.76	0.47	11.67	13.83	2.29 × 10^13	1.74 × 10^4	3.01 × 10^−13	5.76 × 10^−5
G·2AP*(w) ↔ G*·2AP(w)	1099.7	−10.70	−9.96	−0.11	2.31	10.59	12.26	1.57 × 10^13	2.21 × 10^5	4.39 × 10^−13	4.52 × 10^−6
G*·2AP(w) ↔ G·2AP(WC)	130.1	1.33	1.07	18.04	16.58	16.70	15.51	0.76	7.18	1.77	0.28
G·2AP(WC) ↔ G·2APsyn	17.7	0.60	1.51	8.23	10.31	7.63	8.80	5.71 × 10^6	1.58 × 10^7	3.22 × 10^−7	6.35 × 10^−8
G·A*(w) ↔ G·A(WC)	126.8	−6.93	−6.73	16.98	16.32	23.92	23.05	2.21	1.83 × 10^−5	3.14	5.54 × 10^4
G·A(WC) ↔ G·Asyn	20.7	0.76	0.58	8.39	8.89	7.64	8.31	4.33 × 10^6	1.56 × 10^7	3.47 × 10^−7	6.42 × 10^−8
C·2AP*(w) ↔ C·2AP(w)	314.8	−26.99	−28.84	14.39	13.17	41.37	42.01	189.21	3.04 × 10^−18	3.65 × 10^−2	3.29 × 10^17
C·2AP(w) ↔ C*·2AP(WC)	146.9	1.85	1.42	21.95	20.30	20.11	18.88	4.99 × 10^−4	1.13 × 10^−2	5.87 × 10^2	8.88 × 10^1
C·A*(WC) ↔ C*·A(WC)	909.5	−4.52	−3.40	3.73	6.44	8.26	9.84	1.98 × 10^10	9.53 × 10^6	3.48 × 10^−10	1.05 × 10^−7
A·2AP*(w) ↔ A·2AP(w)	423.8	−24.89	−26.85	15.12	13.69	40.01	40.54	58.62	3.25 × 10^−17	0.18	3.08 × 10^16
A·2AP(w) ↔ A*·2AP(WC)	117.0	13.71	13.57	29.85	31.00	16.14	17.43	8.01 × 10^−10	9.10	0.76	0.11
A*·2AP(WC) ↔ A*·2APsyn	18.0	−0.83	−0.59	6.54	8.55	7.37	9.14	9.91 × 10^7	2.46 × 10^7	5.59 × 10^−8	4.07 × 10^−8
A·A*(WC) ↔ A*·A(WC)	497.5	0.00	0.00	6.39	9.71	6.39	9.71	1.56 × 10^8	1.56 × 10^8	2.28 × 10^−8	6.42 × 10^−9
A*·A(WC) ↔ A*·Asyn(TF)	15.8	0.56	1.23	8.09	8.09	7.53	6.85	7.25 × 10^6	1.88 × 10^7	2.66 × 10^−7	5.33 × 10^−8
2AP·A(w) ↔ 2AP*·A(WC)	121.4	23.13	23.53	37.23	36.89	14.10	13.36	3.12 × 10^−15	2.88 × 10^2	2.40 × 10^−2	3.47 × 10^−3
2AP*·A(WC) ↔ 2AP*·Asyn	25.9	−2.33	−2.59	3.32	4.62	5.65	7.21	2.28 × 10^10	4.48 × 10^8	2.97 × 10^−10	2.23 × 10^−9

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I. T·2AP*(w) ↔ T*·2AP(w) ↔ T·2AP(WC). Initially we have comprehensively studied the energy of interaction of 2AP* rare tautomer with T base (ΔE_int = −20.96; ΔG_int = −8.84 kcal mol⁻¹) that is higher, than energy of interaction of A* with T (ΔE_int = −18.10; ΔG_int = −6.06 kcal mol⁻¹) (Fig. 1a and b, Table 1).²⁶ This interaction, which is accompanied by the quite rapid (Table 1) (in comparison with the speed of the DNA replication) T·2AP*(w) → T·2AP(WC) tautomeric transformation (here and below the base, belonging to the template strand, is situated on the left), is additional to the two described by us earlier channels^37,38 of the 2AP involvement into the synthesized DNA double helix, which in itself is not mutagenic.

II. G·2AP*(w) → G*·2AP(w) → G·2AP(WC) → G·2AP_syn → G·2AP_syn(WC). Energy of the monomers interaction in the G·2AP*(w) complex (ΔE_int = −27.11; ΔG_int = −14.60 kcal mol⁻¹) (Fig. 2a, Table 1), which is stabilized by the two classical N1H⋯O6 (7.42) and N1H⋯N2 (8.47 kcal mol⁻¹) H-bonds, significantly exceeds the analogical value in the G·A*(w) mispair³¹ (ΔE_int = −15.24; ΔG_int = −2.83 kcal mol⁻¹) (Fig. 2b, Table 1). Estimation of the ratio of probabilities P_G·2AP*/P_G·A* = [P(G + 2AP* → G·2AP*(w))·P(G·2AP*(w) → G*·2AP(w))·P(G*·2AP(w) → G·2AP(WC))·P(G·2AP(WC) → G·2AP_syn)·P(G·2AP_syn → G·2AP_syn(WC))]/[P(G + A* → G·A*(w))·P(G·A*(w) → G·A(WC))·P(G·A(WC) → G·A_syn)·P(G·A_syn → G·A_syn(WC))] = 1.90 × 10⁷ obtained on the basis of the numerical data, presented in Fig. 2a and b and Table 1, points that this route of structural transformation G + 2AP* → G·2AP*(w) → G*·2AP(w) → G·2AP(WC) → G·2AP_syn → G·2AP_syn(WC) is mutagenic, generating appropriate transversions, when pyrimidine bases (in this case C) is replaced by the analogue of the purine base – 2AP. This also causes low-probable transitions and transversions, since in the next rounds of the DNA replication 2AP pairs not only with T, but also with the C and A bases, as we have shown earlier.^37,38

III. C·2AP*(w) → C·2AP(w) → C*·2AP(WC). Formation of the incorrect pairs of 2AP* with C or A bases, which are the template bases (Fig. 3a, b, d and e, Table 1), does not induce mutagenic effect. Firstly, the energy of monomers interaction in incorrect pairs C·2AP*(w) (ΔE_int = −8.25; ΔG_int = 1.52 kcal mol⁻¹) (Fig. 3a) and C·2AP*(w₁) (ΔE_int = −11.44; ΔG_int = 1.82 kcal mol⁻¹) (Fig. 3b) is significantly less than energy of monomers interaction in the C·A*(WC) mispair (ΔE_int = −23.50; ΔG_int = −10.76 kcal mol⁻¹) (Fig. 3c).^27,28

Moreover, the C·2AP*(w₁) (Fig. 3b) mispair stabilized by the C6H⋯N3 (1.94) and N1H⋯O2 (2.87 kcal mol⁻¹) H-bonds is not capable^27,28 to gain the enzymatically-competent conformation C·2AP*(WC) in the process of thermal fluctuations due to the essential steric constraints.

At the same time, other incorrect pair C·2AP*(w) able to acquire enzymatically-competent conformation by transforming into it according to the chemical pathway C + 2AP* → C·2AP*(w) → C·2AP(w) → C*·2AP(WC) → C*·2AP(WC) (Fig. 3a, Table 1). However, the estimation of the ratio of probabilities P_C·2AP*/P_C·A* = [P(C + 2AP* → C·2AP*(w))·P(C·2AP*(w) → C·2AP(w))·P(C·2AP(w) → C*·2AP(WC))·P(C*·2AP(WC) → C*·2AP(WC))]/[P(C + A* → C·A*(WC))·P(C·A*(WC) → C*·A(WC))·P(C*·A(WC) → C*·A(WC))] = 5.78 × 10⁻¹⁷ authentically indicates that this process is not mutagenic.

IV. A·2AP*(WC) → → . Formation of the A·2AP*(WC) mispair, stabilized by the participation of the three intermolecular C6H⋯HN6 (0.15), N1H⋯N1 (5.87) and C2H⋯N2 (1.94 kcal mol⁻¹) H-bonds, also does not induce mutations, since the mispair, supported by the participation of the two non-canonical^90–93 C6H⋯N1 (2.09) and C2H⋯N7 (1.11 kcal mol⁻¹) H-bonds, could not pretend on the role of the predecessor of the enzymatically-competent conformation at the transformation A·2AP*(WC) → → due to the steric clashes (Fig. 3d).

V. A·2AP*(w) → A·2AP(w) → A*·2AP(WC) → A*·2AP_syn → A*·2AP_syn(WC). Another pathway of the structural transformations A·2AP*(w) → A·2AP(w) → A*·2AP(WC) → A*·2AP_syn → A*·2AP_syn(WC) (Fig. 3e and f), that terminates with the formation of the enzymatically-competent conformation, is not mutagenic, since the ratio of probabilities P_A·2AP*/P_A·A* = [P(A + 2AP* → A·2AP*(w))·P(A·2AP*(w) → A·2AP(w))·P(A·2AP(w) → A*·2AP(WC))·P(A*·2AP(WC) → A*·2AP_syn)·P(A*·2AP_syn → A*·2AP_syn(WC))]/[P(A + A* → A·A*(WC))·P(A·A*(WC) → A*·A(WC))·P(A*·A(WC) → A*·A_syn(TF))·P(A*·A_syn(TF) → A*·A_syn(WC))] = 8.00 × 10⁻²¹ is significantly less than 1.

Here we discuss the mechanism of the 2AP* base binding with canonical DNA bases, which is the basis of its mutagenic pressure on DNA that remains imperfectly understood. The primary aim of this study was to distinguish the physico-chemical principles of the 2AP mutagenic action on DNA, causing ultimately incorporation errors in DNA.

We survey the use of 2AP as a probe of DNA–enzyme interaction and enzyme-induced distortion, focusing particularly on its use for the investigation of base flipping and the enhanced mechanistic insights that can be gained by a detailed analysis of the decay parameters, rather than merely monitoring changes in fluorescence intensity.^17–21 Finally, we reflect on the merits and shortcomings of 2AP and the prospects for its wider adoption as a fluorescence-decay-based probe.

Replication errors

In our previous paper³⁸ we came to the conclusion that amino–imino tautomerisation of the 2AP base does not make mutagenic pressure on DNA at the replication of the latest. Obtained in the current article data according the energetically-kinetic characteristics of the acquisition by the incorrect 2AP*·C(w) (Fig. 4a), 2AP*·A(w) (Fig. 4b) and 2AP*·G(w) (Fig. 4c) base mispairs additionally confirms this important conclusion.

Indeed, as it was already noted, the energy of the bases interaction in the first two mispairs is significantly smaller than a similar magnitude in the A*·C(WC) (ΔE_int = −23.50; ΔG_int = −10.76 kcal mol⁻¹) (Fig. 3c)^27,28 and A*·A(WC) mispairs (ΔE_int = −17.89; ΔG_int = −4.32 kcal mol⁻¹) (Fig. 3f) (Table 1).⁶ Moreover, the process of the incorporation of the 2AP*·C(w) mispair into the DNA is significantly slower than of the A*·C(WC) mispair, since it occurs under the kinetic control (τ_99.9% = 5.87 × 10² s) (Table 1). Eventually, the ratio of probabilities P_2AP*·C/P_A*·C = 5.78 × 10⁻¹⁷ and P_2AP*·A/P_A*·A = 8.00 × 10⁻²¹ was established to be much less than 1. From the other side, the tautomeric and conformational⁹⁴ conversion of the 2AP*·G(w) mispair (Fig. 4c), for which the energy of the monomers interaction (ΔE_int = −27.11; ΔG_int = −14.60 kcal mol⁻¹) is significantly higher than in the A*·G(w) analogical pair (ΔE_int = −15.24; ΔG_int = −2.83 kcal mol⁻¹),³¹ does not lead to the enzymatically-competent structure, since the 2AP·G_syn mispair, stabilized by the participation of the two non-canonical^90–93 C6H⋯N7 (1.18) and C8H⋯N1 (1.76 kcal mol⁻¹) H-bonds, that could pretend on this role, is not able to acquire enzymatically-competent conformation 2AP·G_syn(WC) in the process of the thermal fluctuations due to the considerable steric constraints, by moving along the route of structural transformations 2AP* + G → 2AP*·G(w) → 2AP·G*(w) → 2AP·G(WC) → 2AP·G_syn (Fig. 4c), Table 1).

Thus, the amino–imino tautomerism of 2AP has no relation to the emergence of point replication errors in DNA induced by this mutagen.

Conclusions

Thus, in this paper we have reliably shown, that amino–imino tautomerism of 2AP is not involved into the origin of the induced by this compound replication point errors in DNA. At the same time, we have proven for the first time that 2AP* as a base of the incoming nucleotide may produce only one single transversion, when 2AP* rare tautomer pairs with G base by two N1H⋯O6 and N1H⋯N2 H-bonds and formed mispair moves along the route of the structural transformations G·2AP*(w) → G*·2AP(w) → G·2AP(WC) → G·2AP_syn → G·2AP_syn(WC). In this regard, it is actual the experimental determination of the constant of the tautomeric equilibrium in aqueous solution for 2AP (K_2AP↔2AP*), in particular by using new approaches for the identification of the variables of this type.

Acknowledgements

Authors gratefully appreciate technical support and computational facilities of joint computer cluster of SSI “Institute for Single Crystals” of the National Academy of Sciences of Ukraine (NASU) and Institute for Scintillation Materials of the NASU incorporated into 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 Murcia 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 also to FEBS organisation for the Youth Travel Fund (YTF) grant for visiting. The authors sincerely thank Corresponding Member of NASU, Prof. Dmytro M. Hovorun (Institute of Molecular Biology and Genetics of the NASU) for his invaluable suggestions and comments at the fruitful manuscript discussion.

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