Molecular Principles of the Spontaneous Mutagenesis in DNA

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1 Molecular Principles of the Spontaneous Mutagenesis in DNA Ol ha Brovarets 1,2 1 Department of Molecular and Quantum Biophysics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, UKRAINE, Kyiv, Akademika Zabolotnoho street Department of Molecular Biotechnology and Bioinformatics, Institute of High Technologies, Taras Shevchenko National University of Kyiv, UKRAINE, Kyiv, Akademika Hlushkova avenue 2-h o.o.brovarets@imbg.org.ua Abstract Reported results are crucial for understanding of the microstructural mechanisms of the spontaneous transitions and transversions, since they allow us to explain, from the one side, the origin of the mutagenic tautomers at the separation of the DNA strands before its replication and, from the other side, in what way occurs the adaptation of the incorrect purine pyrimidine, purine purine and pyrimidine pyrimidine wobble pairs to the enzymatically competent sizes in the recognition pocket of the high-fidelity DNA-polymerase. Кеу words spontaneous point mutagenesis, incorporation and replication errors, tautomerisation, pairs of nucleotide bases, enzymatically-competent conformation, hydrogen bond, quantum chemistry. I. Introduction High-fidelity DNA replication is the central issues of the molecular biology [1]. At present time, it was established the important biological role of the spontaneous point mutations [2-4], arising with frequencies during DNA replication [5-8], in the functioning of the living cell. Nowadays, it is reliably known that the root cause of the origin of the spontaneous point mutations is the formation in the recognition pocket of the high-fidelity DNA-polymerase in its close state the wrong DNA base pairs (i.e. mismatches) able to adapt the conformation of the correct Watson-Crick DNA base pair (i.e. enzymatically-competent conformation) that guarantees their incorporation into the chemical structure of the synthesized DNA double helix [4]. In the literature it is currently presented two approaches according physico-chemical principles of the occurrence of the mispairs leading to spontaneous point mutations in DNA. One of them is the tautomeric hypothesis suggested by J. Watson and F. Crick [9] that consists in the spontaneous tautomeric transition of the DNA bases from the canonical to mutagenic tautomeric form leading to the formation of the adenine cytosine* (A C*)/A* C and guanine* thymine (G* T)/G T* (here and below mutagenic tautomers are marked with asterisk) Watson- Crick-like mispairs with correct enzymatically-competent conformation [10] containing mutagenic tautomers [11-13]. Despite great advances in experimental, in particular X-ray analysis [14, 15] and NMR relaxation dispersion measurements [16], and theoretical [17, 18] investigations, there are no unique approach to the physico-chemical mechanisms enabling DNA bases in the canonical tautomeric form to acquire rare or mutagenic tautomeric form before the dissociation of the Watson- Crick nucleobase pairs into the monomers by the replication machinery in order to produce mispairs resulting in further misincorporations and as a result the spontaneous point mutations at the DNA replication. It is generally accepted in the literature that mutagenic tautomers of the DNA bases can arise via the double proton transfer (DPT) along intermolecular H-bonds in the Watson-Crick [19 22] and wobble [23] base pairs, and also in the protein-dna complexes [24]. On contrary, other researchers believe that spontaneous point mutations arise due to the formation of the incorrect base pairs involving only DNA bases in the main, canonical tautomeric form so-called wobble or shifted A C and G T base pairs [25]. However, it remains unclear the mechanisms of their adaptation to the enzymaticallycompetent sizes in the very tight, slightly deformable base pair recognition pocket of the high-fidelity DNApolymerase [26, 27]. The common feature of these two approaches is the absence of the general physico-chemical conception according the nature of these mispairs causing spontaneous point mutations and the emergence of each of them is considered as a unique phenomenon. In the literature it is not represented attempts or ideas aimed at combining these approaches into the unique, internally non-contradictory conception. Nevertheless, creation of such theory is extremely actual interdisciplinary challenge called to solve urgent needs of the fundamental and applied character. Thus, without the clear understanding of the basic mechanisms of the origin of the spontaneous point mutations, it is impossible to advance in the development of the management strategy of the genome instability and in the explanation of the physico-chemical grounds of evolution [28, 29]; at the design of the highly-efficient mutagens analogs of the nucleotide bases with targeted action for different purposes, in particular, for anti-viral and anti-cancer therapy [30, 31]; for essential increase in precision of the DNA-based nanodevices of biomolecular electronics as information carriers [32, 33]; creation of synthetic macromolecular structures able to replicate with a predetermined accuracy [34] and so on. In our study we have pursued the goal to reveal at the miscrostructural level the molecular grounds of the intrinsic DNA mutability without involvement of the external agents. II. Computational methods All geometric, energetic and vibration calculations of the considered base mispairs and transition states (TSs) of their conversion have been performed by Gaussian 09 package [35] using B3LYP [36, 37] and MP2 [38] levels of quantum-mechanical (QM) theory combined with a wide variety of basis sets followed by the intrinsic reaction coordinate (IRC) calculations in the forward and reverse directions from each ТS using Hessian-based predictor-corrector integration algorithm [39]. Bader's quantum theory of Atoms in Molecules (QTAIM) was applied to analyse the electron density distribution [40]. Physico-chemical parameters have been estimated by the known formulas of the physico-chemical kinetics [41]. INTERNATIONAL YOUTH SCIENCE FORUM LITTERIS ET ARTIBUS, NOVEMBER 2016, LVIV, UKRAINE 23

А С*/С* A G* T/T G* G A syn A* G* syn A* A syn G G* syn C T/T C C C* T T* Fig. 1.

Here and below dotted lines indicate AH B H-bonds and continuos loosened A-H-B covalent bridges (their lengths are presented in Å); carbon atoms are in light-blue, nitrogen in dark-blue, hydrogen in

Obtained results and their discussion For the first time it was outlined a complete set of the 12 incorrect DNA base pairs representing a primary cause of the spontaneous point mutations and

consist solely of canonical tautomers of the DNA bases) (Fig. 1).

fluctuations (Table 1), should be experimentally observed in the closed conformation of the latter.

mutagenic tautomers of the DNA bases in the Watson-Crick [43] [45] and wobble [46] base pairs, and also in the protein- DNA complexes [47] due to the dynamical instability of the final tautomerised

dipole moment of the base pair, the distances and the angle of the intermolecular H-bonds, the electron density, the Laplacian of the electron density, ellipticity and the energy at the (3,-1) bond

along the IRC) along entire internal reaction coordinate, not only in the stationary structures such as reagent, product and transition state on the reaction pathways of the tautomeric

2 А С*/С* A G* T/T G* G A syn A* G* syn A* A syn G G* syn C T/T C C C* T T* Fig. 1. Geometrical structures of the 12 incorrect DNA base mispairs causing spontaneous point incorporation and replication errors (B3LYP/ G(d,p) level of theory, ε=1). Here and below dotted lines indicate AH B H-bonds and continuos loosened A-H-B covalent bridges (their lengths are presented in Å); carbon atoms are in light-blue, nitrogen in dark-blue, hydrogen in grey and oxygen in red III. Obtained results and their discussion For the first time it was outlined a complete set of the 12 incorrect DNA base pairs representing a primary cause of the spontaneous point mutations and determining both incorporation and replication errors: А С*/С* A, G* T/T G*, G A syn, A* G* syn, A* A syn, G G* syn, C T/T C, C* C/C C* and T* T/T T* (three of these mispairs G A syn, C T and T C consist solely of canonical tautomers of the DNA bases) (Fig. 1). Precisely these mismatches, which quite easily acquire enzymaticallycompetent conformations in the recognition pocket of the high-fidelity replication DNA-polymerase in the process of thermal fluctuations (Table 1), should be experimentally observed in the closed conformation of the latter. For the first time it was established from the physicochemical point of view, that generally accepted mechanism of the DPT along intermolecular H-bonds can t be the source of the formation of the mutagenic tautomers of the DNA bases in the Watson-Crick [43] [45] and wobble [46] base pairs, and also in the protein- DNA complexes [47] due to the dynamical instability of the final tautomerised complexes containing mutagenic tautomers: the value of the zero-point energy of the corresponding vibrational mode, which frequency becomes imaginary at the transition state, is higher than the value of the reverse barrier. We have developed original author s methodology allowing to track the evolution of all physico-chemical parameters (electronic energy, the first derivative of the electronic energy by the IRC, the dipole moment of the base pair, the distances and the angle of the intermolecular H-bonds, the electron density, the Laplacian of the electron density, ellipticity and the energy at the (3,-1) bond critical points of the intrapair H- bonds, the NBO charges of the hydrogen atoms involved in the tautomerisation, the glycosidic angles and the distance between the glycosidic hydrogens at each step along the IRC) along entire internal reaction coordinate, not only in the stationary structures such as reagent, product and transition state on the reaction pathways of the tautomeric transformations. Additionally, based on the electron-topological characteristics of the neighboring intermolecular bonds, along which protons migrate, namely the value of the electron density and its Laplacian in the corresponding critical points, for the first time we have introduced the conception of the key points (their maximum number reaches 9), which comprehensively describe the mechanism of tautomerisation. In this case 3 key points correspond to the two abovementioned local minima (the first and the last, ninth reagent and product, respectively) and transition state of the tautomerisation. Other 6 key points include 2 key points, in which migrating proton is localized midway between the electronegative atoms and are characterized by the loosened covalent bonds, and also 4 key points, in which H-bonds begin to acquire the features of the covalent bond and vice versa, that is where the Laplacian of the electron density passes through zero. TABLE 1 SELECTED STRUCTURAL AND ENERGETIC CHARACTERISTICS OF THE INCORRECT DNA BASE PAIRS, RESPONSIBLE FOR THE ORIGIN OF THE SPONTANEOUS TRANSITIONS AND TRANSVERSIONS (MP2/ G(2df,pd)//B3LYP/ G(d,p) LEVEL OF THEORy, ε=1) Mispairs Geometrical parameters R(H N1/N9 -H N1/N9) a α 1 b α 2 c ΔE def d Energetical parameters -ΔE int e ΣE HB/ ΔE int f -ΔG int g A T G C A* A syn G A syn A* G* sy G G* syn А C* A* С G* T G T* C C* C T T T* A T G C Note: a The distance between the glycosidic protons at the N1/N9 atoms, Å; b The glycosidic angle for the base situated on the left within the base pair, º; c The glycosidic angle for the base situated on the right within the base pair, º; d The electronic energy of deformation, necessary to apply to the mismatch to acquire the sizes of the A T and G C Watson-Crick DNA base pairs, kcal mol -1 ; e The electronic energy of interaction, kcal mol -1 ; f The contribution of the total energy of the intermolecular H-bonds to the electronic energy of interaction, %; g The Gibbs free energy of interaction (T= K), kcal mol -1. Selected structural and energetic characteristics of the incorrect DNA base pairs, responsible for the origin of the spontaneous transitions and transversions (MP2/ G(2df,pd)//B3LYP/ G(d,p) level of theory, ε=1) Arrangement of the extremums of the derivative of the energy by the IRC, which coincide with the second and penultimate key points, allows to separate the pathway of 24 INTERNATIONAL YOUTH SCIENCE FORUM LITTERIS ET ARTIBUS, NOVEMBER 2016, LVIV, UKRAINE

the tautomerisation reaction to the areas of reagent, transition state and product of the reaction.

TABLE 2 ENERGETIC AND KINETIC CHARACTERISTICS OF THE TAUTOMERIC TRANSFORMATIONS OF THE INCORRECT LONG, SHORT AND WATSON-CRICK-LIKE PAIRS OF NUCLEOTIDE BASES VIA THE DPT ALONG THE NEIGHBORING

07 9.58 9.63 11.46-0.44 1.88 4.83 10-14 G G* G* G 0.00 0.00 5.51 8.33 5.51 8.33 8.22 10-10 A C* A* C 3.99 3.64 8.17 10.53 4.18 6.89 1.08 10-10 G* T G T* 1.22 1.19 2.63 5.61 2.63 5.61 8.

07 12.17-1.96 1.02 4.10 10-15 A* A syn A A* syn 13.98 14.71 14.15 16.43 0.16 1.72 1.12 10-13 A* G* syn A G* syn 1.89 2.20 2.42 4.60 0.52 2.40 2.

15 K), kcal mol -1 ; b The electronic energy of the product relatively the reactant of the tautomerisation reaction, kcal mol -1 ; c The Gibbs free energy barrier for the forward reaction of

3 the tautomerisation reaction to the areas of reagent, transition state and product of the reaction. This set of these key points could be considered as "fingerprints" of the tautomerisation process via the DPT. TABLE 2 ENERGETIC AND KINETIC CHARACTERISTICS OF THE TAUTOMERIC TRANSFORMATIONS OF THE INCORRECT LONG, SHORT AND WATSON-CRICK-LIKE PAIRS OF NUCLEOTIDE BASES VIA THE DPT ALONG THE NEIGHBORING INTERMOLECULAR H-BONDS (MP2/cc-pVQZ//B3LYP/ G(d,p) LEVEL OF THEORY, ε=1) Tautomeric transition G a E b G c TS E d TS G e E f τ g A A* A* A A G A* G* G G* G* G A C* A* C G* T G T* C C* C* C C T C* T* T T* T* T G G* syn G* G* syn A* A syn A A* syn A* G* syn A G* syn Note: a The Gibbs free energy of the product relatively the reactant of the tautomerisation reaction (T= K), kcal mol -1 ; b The electronic energy of the product relatively the reactant of the tautomerisation reaction, kcal mol -1 ; c The Gibbs free energy barrier for the forward reaction of tautomerisation, kcal mol -1 ; d The electronic energy barrier for the forward reaction of tautomerisation, kcal mol -1 ; e The Gibbs free energy barrier for the reverse reaction of tautomerisation, kcal mol -1 ; f The electronic energy barrier for the reverse reaction of tautomerisation, kcal mol -1 ; g The lifetime of the product of the tautomerisation reaction, s. A A* TS A A* A* A A* A G G* TS G G* G* G G* G C C* TS C C* C* C C* C Fig. 2. Geometrical structures of the 3 stationary structures (initial, transition state and terminal) describing the progression of the tautomerisation via the DPT along the intermolecular H-bonds in some mispairs (B3LYP/ G(d,p) level of theory, ε=1) This methodology enables to make an objective conclusion about the character of tautomerisation (concerted, synchronous or asynchronous), quantitatively estimate the cooperativity of the specific intermolecular interactions (H-bonds and attractive van der Waals contacts) and trace how these interactions are grouped 13 into the patterns and how they consistently substitute each other along the IRC of tautomerisation. Withing the framework of this approach we have investigated for the first time the microstructural mechanisms of the tautomerisation of the base pairs involved into the origin of the spontaneous point mutations. It was established that for the A G A* G* [48], A C* A* C [[49]], G* T G T* [50], C T C* T* [51], G G* syn G* G* syn [52], A* A syn A A* syn [53] and A* G* syn A G* syn [54] tautomerisation processes final, tautomerised base pairs are dynamically unstable: low-frequency intermolecular vibrations can t develop during their lifetime (Fig. 2, Table 2). At the tautomerisation of the dynamically stable short Т Т* [55] and С С* [56] mispairs, as well as long А А* [57] and G G* [58] mispairs mutagenic tautomers are distributed among the monomers with equal probability, that is important for understanding of the consolidation of the point mutations in the subsequent rounds of DNA replication (Fig. 2, Table 2). It was established that short-lived, low-populated А* С and G T* mispairs are providers of the long-lived enzymatically-competent А С* [49] and G* T base pairs [50], respectively, at the origin of the replication errors in DNA. TABLE 3 ENERGETIC AND KINETIC CHARACTERISTICS OF THE TAUTOMERIC TRANSFORMATIONS OF THE CLASSICAL WATSON-CRICK (WC) OR WOBBLE (W) DNA BASE PAIRS, THAT ARE INVOLVED INTO THE PROCESSES OF THE SPONTANEOUS POINT MUTAGENESIS, VIA THE DPT ACCOMPANIED BY THE SUBSTANTIAL CHANGES OF THEIR GEOMETRY (MP2/cc-pVQZ//B3LYP/ G(d,p) LEVEL OF THEORY, ε=1) Tautomeric conversion G E G TS E TS G E τ 99.9% a A Т(WC) A* Т (w) A Т(WC) A Т* (w) G C(WC) G C* (w) G C(WC) G* C (w) A C(w) A C*(WC) G T(w) G* T(WC) A A(w) A* A(WC) G G(w) G* G(WC) A G(WC) A G* (w) A G(WC) A* G (w) C T(WC) C* T (w) C T(WC) C T* (w) T T(w) T T*(WC) C C(w) C C*(WC) Note: for designations refer to Table 2. a The time necessary to reach 99.9% of the equilibrium concentration between the reactant and the product of the tautomerisation reaction, s. For the first time it was proposed novel theoretical approach to the elucidation of the microstructural mechanisms of the incorporation and replication errors arising at the DNA replication. It was discovered for the first time the intrinsic ability of the purine pyrimidine (A T [59], G C [59], G T [60], [61] and A C [60], [62]), INTERNATIONAL YOUTH SCIENCE FORUM LITTERIS ET ARTIBUS, NOVEMBER 2016, LVIV, UKRAINE 25

IRC, Bohr IRC, Bohr IRC, Bohr A T(WC) A T*

A C*(WC) IRC, Bohr IRC, Bohr IRC, Bohr G T(w)

Bohr IRC, Bohr IRC, Bohr A G(WC) A G* (w)/a*

on the potential energy hypersurface of the

the DPT, accompanied by the shifting of the

spontaneous transitions and transversions

(B3LYP/6-311++G(d,p) level theory, ε=1) IRC,

4 IRC, Bohr IRC, Bohr IRC, Bohr A T(WC) A T* (w)/a* T (w) G C(WC) G C* (w)/g* C (w) A C(w) A C*(WC) IRC, Bohr IRC, Bohr IRC, Bohr G T(w) G* T(WC) A А(w) A* А(WC) G G(w) G* G(WC) IRC, Bohr IRC, Bohr IRC, Bohr A G(WC) A G* (w)/a* G (w) C T(WC) C* T (w)/c T* (w) T T(w) T T*(WC) Fig. 3. Energetic profiles and stationary structures on the potential energy hypersurface of the biologically important transformations via the DPT, accompanied by the shifting of the bases relative to each other within a base pair into the minor or major DNA groove sides, leading to the occurrence of the spontaneous transitions and transversions incorporation and replication errors (B3LYP/ G(d,p) level theory, ε=1) IRC, Bohr C C(w) C C*(WC) 26 INTERNATIONAL YOUTH SCIENCE FORUM LITTERIS ET ARTIBUS, NOVEMBER 2016, LVIV, UKRAINE

a) 2AP T(WC) 2AP T*(w) 2AP T(WC) (ΔG=0.00) TS 2AP+ T- 2AP T(WC) 2AP T*(w) (ν i=134.2 i cm -1 ) (ΔG=18.66) 2AP T*(w) (ΔG=8.62) ΔE int=-14.

18) b) C 2AP(w) C* 2AP(WC) C 2AP(w) S C- 2AP+ C 2AP(w) C* 2AP(W (ν i=146.9 i cm -1 ) (ΔG=0.00) (ΔG=21.95) (ΔE int=-15.19 / ΔG int=-3.

35) A 2AP(w) (ΔG=0.00) TS A- 2AP+ A 2AP(w) A* 2AP(WC (ν i=117.0 i cm -1 ) (ΔG=29.85) A* 2AP(WC) (ΔG=13.71) (ΔE int=-12.35 / ΔG int=0.

65) A* 2AP syn (ΔG=12.88) (ΔE int=-10.47 / ΔG int=0.40) (ΔE def=2.59 / 4.50) d) 2AP* G(w) 2AP G*(w) 2AP G(WC) 2AP G syn 2AP* G(w) (ΔG=0.

1 i cm -1 ) (ΔG=-9.37) (ΔG=7.33) (ΔE int=-12.04 / ΔG int=-0.15) E int=-114.69 / ΔG int=-101.1 2AP G*(w) (ΔG=-10.70) (ΔE int=-15.80 / ΔG int=-4.

Reaction pathways of the biologically important tautomerisations and conformational transitions of the investigated structures containing canonical

tautomer of the 2AP (B3LYP/6-311++G(d,p) level of theory, ε=1).

A [[63]], G G [[63]] and A G [[64]]) and pyrimidine pyrimidine (С С [[65]], Т Т [[65]] and С T [[64]]) DNA base mispairs to perform wobble

understanding of the microstructural mechanisms of the spontaneous transitions and transversions (Fig. 3, Table 3).

zwitterionic transition states of the type (protonated base) (deprotonated base).

towards both minor and major DNA grooves and vice versa.

the intrinsic reaction coordinate of the unique patterns of the specific intermolecular interactions, including H-bonds, loosened covalent bridges

Microstructural mechanism of the mutagenic action of 2-aminopurine (2АР) causing replication errors consists in the generation with the highest

base pair due to the A Т(WC) A Т*(w) tautomerisation [[66]].

mispair, which is tautomerised into the Watson-Crick-like base mispair С* 2АР(WC), that quite easily in the process of the thermal fluctuations

By estimating the probability ratio Р А 2АР /Р А А =40.

5 a) 2AP T(WC) 2AP T*(w) 2AP T(WC) (ΔG=0.00) TS 2AP+ T- 2AP T(WC) 2AP T*(w) (ν i=134.2 i cm -1 ) (ΔG=18.66) 2AP T*(w) (ΔG=8.62) ΔE int= / ΔG int=-2.88) ΔE int= / ΔG int= (δe int= / ΔG int=-9.18) b) C 2AP(w) C* 2AP(WC) C 2AP(w) S C- 2AP+ C 2AP(w) C* 2AP(W (ν i=146.9 i cm -1 ) (ΔG=0.00) (ΔG=21.95) (ΔE int= / ΔG int=-3.70 (ΔE int= / ΔG int= c) A 2AP(w) A* 2AP(WC) A* 2AP syn C* 2AP(WC) (ΔG=1.85) (ΔE int= / ΔG int=-3.05) (ΔE def=0.21 / 0.35) A 2AP(w) (ΔG=0.00) TS A- 2AP+ A 2AP(w) A* 2AP(WC (ν i=117.0 i cm -1 ) (ΔG=29.85) A* 2AP(WC) (ΔG=13.71) (ΔE int= / ΔG int=0.43) ΔE int= / ΔG int= (ΔE int=-9.82 / ΔG int=1.29) TS A* 2AP(WC) A* 2APsyn (ν i=18.0 i cm -1 ) (ΔG=20.25) (ΔE int=-2.45 / ΔG int=6.65) A* 2AP syn (ΔG=12.88) (ΔE int= / ΔG int=0.40) (ΔE def=2.59 / 4.50) d) 2AP* G(w) 2AP G*(w) 2AP G(WC) 2AP G syn 2AP* G(w) (ΔG=0.00) ΔE int= / ΔG int= TS 2AP* G(w) 2AP G*(w) (ν i= i cm -1 ) (ΔG=-0.11) S 2AP+ G- 2AP G*(w) 2AP G(W 2AP G(WC) (ν i=130.1 i cm -1 ) (ΔG=-9.37) (ΔG=7.33) (ΔE int= / ΔG int=-0.15) E int= / ΔG int= AP G*(w) (ΔG=-10.70) (ΔE int= / ΔG int=-4.25) 2AP G syn (ΔG=6.31) (ΔE int=-4.07/δg int=4.89) Fig. 4. Reaction pathways of the biologically important tautomerisations and conformational transitions of the investigated structures 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/ G(d,p) level of theory, ε=1). The base, belonging to the template strand of DNA, is situated on the left, while the base of the incoming nucleotide on the right purine purine (A A [[63]], G G [[63]] and A G [[64]]) and pyrimidine pyrimidine (С С [[65]], Т Т [[65]] and С T [[64]]) DNA base mispairs to perform wobble Watson-Crick tautomeric transitions via the sequential intrapair DPT and subsequent shifting of the bases relative each other, that are crucial for understanding of the microstructural mechanisms of the spontaneous transitions and transversions (Fig. 3, Table 3). It was revealed that these non-dissociative tautomerisations via the sequential DPT are controlled by the highly stable, highly polar and zwitterionic transition states of the type (protonated base) (deprotonated base). These interconverions are accompanied by a significant rebuilding of the base mispairs with Watson-Crick architecture into the mismatches wobbled towards both minor and major DNA grooves and vice versa. Moreover, it was established that these tautomerisation reactions occur non-dissociatively and are accompanied by the consequent replacement along the intrinsic reaction coordinate of the unique patterns of the specific intermolecular interactions, including H-bonds, loosened covalent bridges and, in some cases, attractive van der Waals contacts. Microstructural mechanism of the mutagenic action of 2-aminopurine (2АР) causing replication errors consists in the generation with the highest probability of the mutagenic tautomer T* according to the 2AP Т(WC) 2AP Т*(w) reaction, rather then it takes place in the Watson-Crick A Т(WC) DNA base pair due to the A Т(WC) A Т*(w) tautomerisation [[66]]. At this, the mutagenic effect is achieved due to the greater stability of the 2AP Т*(w) complex in comparison with the analogical A Т*(w) base pair (Fig. 4a, Table 4). We have shown for the first time that 2AP very effectively produces incorporation errors by binding with cytosine (C) into the wobble С 2АР(w) mispair, which is tautomerised into the Watson-Crick-like base mispair С* 2АР(WC), that quite easily in the process of the thermal fluctuations acquires enzymatically-competent conformation (estimated ratio of probabilities Р С 2АР /Р С А = ) (Fig. 4b, Table 4) [[67]]. By estimating the probability ratio Р А 2АР /Р А А =40.5 we came to the conclusion that 2AP in the case of structural transformations А 2АР(w) А* 2АР(WC) А* 2АР syn (Fig. 4c, Table 4) causes transversion, when pyrimidine base (in this case T) is substituted by the purine, in particular A [[67]]. It was proven for the first time that 2AP* as a base of the incoming nucleotide may produce also another transversion, when 2AP* mutagenic tautomer pairs with G base and formed mispair converts according the route of the tautomeric and conformational transformations G 2AP*(w) G* 2AP(w) G 2AP(WC) G 2AP syn (Fig. 4d, Table 4) [[68]]. Estimated ratio of probabilities Р G 2АР* /Р G А* = points that this route of structural transformation 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 [[68]]. INTERNATIONAL YOUTH SCIENCE FORUM LITTERIS ET ARTIBUS, NOVEMBER 2016, LVIV, UKRAINE 27

6 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 (MP2/aug-cc-pVDZ//B3LYP/ G(d,p) LEVEL OF THEORY, ε=1) TABLE 4 Tautomerisation / Conformational transition a ν i G E G TS E TS G E τ 99.9% τ 2AP T(WC) 2AP T*(w) A T(WC) A T*(w) C 2AP(w) C* 2AP(WC) C A(w) C* A(WC) A 2AP(w) A* 2AP(WC) A* 2AP(WC) A* 2AP syn A A(w) A A*(WC) A A*(WC) A* A(WC) A* A(WC) A* A syn (TF) G 2AP*(w) G* 2AP(w) G* 2AP(w) G 2AP(WC) G 2AP(WC) G 2AP syn G A*(w) G A(WC) G A(WC) G A syn C 2AP*(w) C 2AP(w) Note: for designations refer to Table 2. a Imaginary frequency at the TSs of the interconversions, cm -1. Conclusions Obtained results allow us to explain the number of biological experiments available in the literature, but remained yet without proper theoretical justification: Obtained numerical estimations of the frequencies of the mispairs occurrence satisfactorily explain experimental data: ( ) G T/T G >> A C/C A >> C T/T C > A A > G A/A G >> G G C C (10-6 ) [69]. Established A C(w) A C*(WC) and G T(w) G* T(WC) wobble(w) Watson-Crick(WC) transformations via the sequential DPT allow us to explain the way of the acquisition by the A C(w)/G T(w) wobble mispairs of the Watson-Crick geometry in the active center of the high-fidelity DNA-polymerase or DNA duplex and to interpret X-ray [14, 15] and NMR [16] experiments. Presented approach allows us to clarify microstructural mechanisms of the mutations induced by the classical mutagens, in particular 2-aminopurine, for which induced frequencies agree well with the experimental data. Acknowledgements. The author 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 years, by the Scholarship of the President of Ukraine for young scientists for the years and by the FEBS organisation (YTF grant for visiting FEBS Workshop on Chromatin Proteomics (October 3-8, 2016, Crete, Greece)) given to O.O.B. The author sincerely thanks 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 manuscript preparation. References [1] Cooper, G.M. The Cell (2 nd edition). A molecular approach. Boston University. Sunderland (MA): Sinauer Associates, [2] Freese, E.B. On the molecular explanation of spontaneous and induced mutations. Brookhaven Symp. Biol., 1959, 12, [3] von Borstel, R.C. Origins of spontaneous base substitutions. Mutation Research, 1994, 307, [4] Friedberg, E.C., Walker, G.C., Siede, W., Wood, R.D., Schultz, R.A., & Ellenberger, T. DNA repair and mutagenesis. Washington D.C.: ASM Press, [5] Drake, J.W., Charlesworth, B., Charlesworth, D., & Crow, J.F. Rates of spontaneous mutation. Genetics, 1998, 148, [6] Lee, H., Popodi, E., Tang, H., & Foster, P.L. Rate and molecular spectrum of spontaneous mutations in the bacterium Escherichia coli as determined by wholegenome sequencing. Proc. Natl. Acad. Sci. USA, 2012, 109, E2774-E2783. [7] Lynch, M. Rate, molecular spectrum, and consequences of human mutation. Proc. Natl. Acad. Sci. USA, 2010, 107, [8] Fijalkowska, I.J., Schaaper, R.M., & Jonczyk, P. DNA replication fidelity in Escherichia coli: a multi-dna polymerase affair. FEMS Microbiol. Rev., 2012, 36, INTERNATIONAL YOUTH SCIENCE FORUM LITTERIS ET ARTIBUS, NOVEMBER 2016, LVIV, UKRAINE

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Electronic Supplementary Information. DPT tautomerisation of the wobble guanine thymine DNA base mispair is not mutagenic: QM and QTAIM arguments 1

Electronic Supplementary Information DPT tautomerisation of the wole guanine thymine DNA ase mispair is not mutagenic: QM and QTAIM arguments 1 Ol ha O. Brovarets a,, Roman O. Zhurakivsky a and Dmytro