Why the tautomerization of the G·C Watson-Crick base pair via the DPT does not cause point mutations during DNA replication? QM and QTAIM comprehensive analysis.

This article was downloaded by: [Florida Atlantic University] On: 10 August 2013, At: 05:01 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Biomolecular Structure and Dynamics Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tbsd20

Why the tautomerization of the G·C Watson–Crick base pair via the DPT does not cause point mutations during DNA replication? QM and QTAIM comprehensive analysis Ol’ha O. Brovarets’

a b c

& Dmytro M. Hovorun

a b c

a

Department of Molecular and Quantum Biophysics , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150, Zabolotnoho Str., 03680 , Kyiv , Ukraine b

State Key Laboratory of Molecular and Cell Biology, Research and Educational Center , 150, Zabolotnoho Str., 03680 , Kyiv , Ukraine c

Department of Molecular Biology, Biotechnology and Biophysics , Institute of High Technologies, Taras Shevchenko National University of Kyiv , 2-h, Hlushkova Ave., 03022 , Kyiv , Ukraine Published online: 02 Aug 2013.

To cite this article: Journal of Biomolecular Structure and Dynamics (2013): Why the tautomerization of the G·C Watson–Crick base pair via the DPT does not cause point mutations during DNA replication? QM and QTAIM comprehensive analysis, Journal of Biomolecular Structure and Dynamics, DOI: 10.1080/07391102.2013.822829 To link to this article: http://dx.doi.org/10.1080/07391102.2013.822829

PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions

Journal of Biomolecular Structure and Dynamics, 2013 http://dx.doi.org/10.1080/07391102.2013.822829

Why the tautomerization of the G·C Watson–Crick base pair via the DPT does not cause point mutations during DNA replication? QM and QTAIM comprehensive analysis Ol’ha O. Brovarets’a,b,c and Dmytro M. Hovoruna,b,c* a

Department of Molecular and Quantum Biophysics, Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, 150, Zabolotnoho Str., 03680, Kyiv, Ukraine; bState Key Laboratory of Molecular and Cell Biology, Research and Educational Center, 150, Zabolotnoho Str., 03680, Kyiv, Ukraine; cDepartment of Molecular Biology, Biotechnology and Biophysics, Institute of High Technologies, Taras Shevchenko National University of Kyiv, 2-h, Hlushkova Ave., 03022, Kyiv, Ukraine Communicated by Ramaswamy H. Sarma

Downloaded by [Florida Atlantic University] at 05:01 10 August 2013

(Received 9 April 2013; final version received 3 July 2013) The ground-state tautomerization of the G·C Watson–Crick base pair by the double proton transfer (DPT) was comprehensively studied in vacuo and in the continuum with a low dielectric constant (ɛ = 4), corresponding to a hydrophobic interface of protein–nucleic acid interactions, using DFT and MP2 levels of quantum-mechanical (QM) theory and quantum theory “Atoms in molecules” (QTAIM). Based on the sweeps of the electron-topological, geometric, polar, and energetic parameters, which describe the course of the G·C ↔ G⁄·C⁄ tautomerization (mutagenic tautomers of the G and C bases are marked with an asterisk) through the DPT along the intrinsic reaction coordinate (IRC), it was proved that it is, strictly speaking, a concerted asynchronous process both at the DFT and MP2 levels of theory, in which protons move with a small time gap in vacuum, while this time delay noticeably increases in the continuum with ɛ = 4. It was demonstrated using the conductor-like polarizable continuum model (CPCM) that the continuum with ɛ = 4 does not qualitatively affect the course of the tautomerization reaction. The DPT in the G·C Watson–Crick base pair occurs without any intermediates both in vacuum and in the continuum with ɛ = 4 at the DFT/MP2 levels of theory. The nine key points along the IRC of the G·C base pair tautomerization, which could be considered as electron-topological “fingerprints” of a concerted asynchronous process of the tautomerization via the DPT, have been identified and fully characterized. These key points have been used to define the reactant, transition state, and product regions of the DPT reaction in the G·C base pair. Analysis of the energetic characteristics of the H-bonds allows us to arrive at a definite conclusion that the middle N1H N3/N3H N1 and the lower N2H O2/N2H O2 parallel H-bonds in the G·C/G⁄·C⁄ base pairs, respectively, are anticooperative, that is, the strengthening of the middle H-bond is accompanied by the weakening of the lower H-bond. At that point, the upper N4H O6 and O6H N4 H-bonds in the G·C and G⁄·C⁄ base pairs, respectively, remain constant at the changes of the middle and the lower H-bonds at the beginning and at the ending of the G·C ↔ G⁄·C⁄ tautomerization. Aiming to answer the question posed in the title of the article, we established that the G⁄·C⁄ Löwdin’s base pair satisfies all the requirements necessary to cause point mutations in DNA except its lifetime, which is much less than the period of time required for the replication machinery to forcibly dissociate a base pair into the monomers (several ns) during DNA replication. So, from the physicochemical point of view, the G⁄·C⁄ Löwdin’s base pair cannot be considered as a source of point mutations arising during DNA replication. Keywords: spontaneous point replication errors in DNA; the double proton transfer; the guanine·cytosine Watson–Crick DNA base pair; DFT and MP2 QM simulations; QTAIM analysis

Introduction Spontaneous point mutations arising from the replacement of one base with another are extremely significant as they provide diversity of species (Friedberg et al., 2006; Lee, Popodi, Tang, & Foster, 2012). The understanding of the molecular mechanism of their occurence is also important for the therapy and treatment of the cancer diseases occuring due to the increased mutation rates (Vogelstein & Kinzler, 1998). Prototropic tautomer*Corresponding author. Email: [email protected] Copyright Ó 2013 Taylor & Francis

ism of the DNA bases is considered in the literature as the main reason of spontaneous point mutations in DNA (Brovarets’, Kolomiets’, & Hovorun, 2012; Goodman, 1995). However, the mechanisms of the origin of rare tautomers of the DNA bases are not currently known for certain (Brovarets’, Kolomiets’ et al., 2012). In 1963, Löwdin suggested that spontaneous point mutations can occur due to the proton tunneling along two neighboring intermolecular H-bonds that join

2

O.O. Brovarets’ and D.M. Hovorun point mutations and remains so today. From the physicochemical point of view, the advantage of the Löwdin’s mechanism lies in the fact that the tautomerization of base pairs does not significantly disturb the standard Watson–Crick base-pairing geometry. Insufficient experimental data on the double proton transfer (DPT) in the Watson–Crick base pairs have stimulated the performance of the theoretical studies, using a wide range of theoretical approaches, mostly for the isolated G·C base pair. It is known from the literature regarding the DPT in the G·C Watson–Crick base pair that the comparison of


together the nitrogen bases in the Watson–Crick DNA base pairs, in particular, in the guanine·cytosine (G·C) Watson–Crick DNA base pair, which would lead to the formation of the G⁄·C⁄ base pair (the so-called Löwdin’s base pair) involving mutagenic tautomers of bases (marked with asterisks) (Scheme 1) (Löwdin, 1963). However, Löwdin considered the short time of proton tunneling through the potential barrier comparably with the replication time of a DNA base pair as an Achilles’ heel of his model (Löwdin, 1963). At that time, the Löwdin’s mechanism was the unique explanation for the occurrence of rare tautomers of bases associated with

Scheme 1. Scheme of the spontaneous point replication errors in DNA arising by the Löwdin's mechanism (Löwdin, 1963, 1965, 1966) at the example of the G C base pair. The dotted lines indicate H-bonds (Brovarets' & Hovorun, 2013; Brovarets’, Kolo-

miets’ et al., 2012); numeration of atoms is standard (Saenger, 1984).


Tautomerization of the GC Watson-Crick base pair via the DPT the results for the isolated (Bertran, Blancafort, Noguera, & Sodupe, 2006; Florian, Leszczynski, & Scheiner, 1995; Florian & Leszczynski, 1996; Gorb, Podolyan, Dziekonski, Sokalski, & Leszczynski, 2004; Hayashi & Mukamel, 2004; Lipiński & Gorzkowska, 1983; Pérez, Tuckerman, Hjalmarson, & von Lilienfeld, 2010; Villani, 2006, 2010; Xiao, Wang, Liu, Lin, & Liang, 2012; Zoete & Meuwly, 2004), Na+ (Gorb et al., 2004) and Mg2+ (Cerón-Carrasco & Jacquemin, 2011; Cerón-Carrasco & Requena, 2012; Lipiński & Gorzkowska, 1983) coordinated, protonated (Bertran et al., 2006; Lin, Wang, Gao, & Schaefer, 2011; Noguera, Sodupe, & Bertrán, 2004, 2007), ionized (RodríguezSantiago, Noguera, Bertran, & Sodupe, 2010), microhydrated (Cerón-Carrasco et al., 2009, 2011; Villani, 2013), stacked (Cerón-Carrasco et al., 2011; Matsui, Sato, & Shigeta, 2009a; Matsui, Sato, Shigeta, & Hirao, 2009b; Nakanishi et al., 2010), joined with sugar-phosphate backbone (Cerón-Carrasco et al., 2011; Chen, Kao, & Hsu, 2009) and DNA-embedded (Chen et al., 2009; Zoete & Meuwly, 2004) G·C base pair has not revealed significant changes in the mechanism of the DPT and geometric or energetic characteristics. In opposite, Cerón-Carrasco et al. (2009, 2011) considered G·C (H2O)n complexes and noted that water plays a crucial role for the PT reaction in the hydrated G·C base pair, providing a path for the DPT in solution. Villani has also considered two naıve schemes, where the water molecules are only indirectly or directly involved in the hydrogen atom transfer (Villani, 2013). Moreover, it was demonstrated that the electric fields drastically alter the rate constants of PT and also tune the mechanism of the PT reactions in the G·C base pair (Cerón-Carrasco & Jacquemin, 2013). However, the authors did not indicate the source of the external electric field in the cell (Cerón-Carrasco & Jacquemin, 2013). Thus, one could argue that the nearest-neighbor influence upon the G·C ↔ G⁄·C⁄ tautomerization is negligibly small and the investigation of the isolated Watson–Crick base pair in order to derive information about PT processes in DNA would be biologically relevant. Qualitative analysis of the data on the DPT in the G C Watson–Crick base pair revealed that in this base pair the DPT reaction is asynchronous according to MP2 approximations (Gorb et al., 2004), while it is synchronous according to DFT approximations (CerónCarrasco et al., 2009; Gorb et al., 2004) (Table 1). Background review of existing literature on the Löwdin’s mechanism shows that important subtleties of the G·C ↔ G⁄·C⁄ tautomerization have been left without researchers’ attention and therefore should be elucidated (see Table 1): (1) The models exploring Löwdin’s mechanism do not take into account that electronic energy of the

3

reverse barrier of the G·C → G⁄·C⁄ tautomerization must exceed zero-point energy of vibrations causing this tautomerization to provide dynamic stability of the formed G⁄·C⁄ Löwdin’s mispair (Brovarets’ & Hovorun, 2012, 2013; Brovarets’, Yurenko, Dubey, & Hovorun, 2012; Brovarets’, Zhurakivsky, & Hovorun, 2013a, 2013b, 2013c). (2) It has not been clearly established yet, whether the DPT mechanism in the G·C base pair is synchronous or asynchronous. (3) In addition, no research has been found to date that surveyed the intrinsic reaction coordinate (IRC) calculations for the DPT process in the G·C Watson–Crick base pair and the dependence of geometric, polar, energetic, and electron-topological characteristics of the G·C base pair and intrapair H-bonds on the IRC. However, these data are urgently needed to clarify whether or not the tautomerization process proceeds via intermediates, namely zwitterionic. (4) Moreover, the authors of the works, devoted to the tautomerization of the G·C Watson–Crick base pair as a possible source of spontaneous point replication errors in DNA, insufficiently rely on biological knowledges associated with the molecular mechanisms of the functioning of the replication machinery at the analysis of the influence of external factors on the tautomerization processes. Researchers consider the formation of the G⁄·C⁄ mispair causing the formation of the G⁄·T and AC⁄ mismatches at the subsequent round of DNA replication as necessary and sufficient condition for the occurence of point mutations in DNA. Thus, it has been suggested in the literature that the efficient DPT in the G·C base pair is a thermodynamically possible reaction promoting the G⁄·C⁄ product contributing to spontaneous mutation with a frequency of 107 (Florian & Leszczynski, 1996; Florian et al., 1995; Gorb et al., 2004). This article seeks to remedy these problems. In order to facilitate the comparison of relative electronic and Gibbs free energies for the G·C ↔ G⁄·C⁄ transition available in current literature, we have collected these data in Table 1. We are convinced that the G⁄·C⁄ Löwdin’s base pair must meet the following requirements to be a source of spontaneous point replication errors in DNA (Brovarets’, Kolomiets’ et al., 2012): (1) The G⁄·C⁄ Löwdin’s mispair should be dynamically stable (Brovarets’ & Hovorun, 2012, 2013; Brovarets’, Kolomiets’ et al., 2012; Brovarets’, Yurenko et al., 2012; Brovarets’ et al.,

4

O.O. Brovarets’ and D.M. Hovorun

Table 1. Available literature data on the energetic characteristics for the G·C ↔ G⁄·C⁄ tautomerization via the DPT obtained at the different levels of QM theory. Energies are given in kcal/mol. G·C ↔ G⁄·C⁄


References

Level of QM theory

ɛ

ΔE ΔΔETS ΔΔE ΔG ΔΔGTs ΔΔG

Lipiński & Gorzkowska (1983) MO LCAO SCF CI (INDO/L) 1 35.05 Florian et al. (1995) HF/MINI-1 1 0.5 Florian & Leszczynski (1996) HF/MINI-1 1 0.5 Florian et al. (1995) HF/MINI-1 1 1.8 Guallar, Douhal, Moreno, & Lluch (1999) HF/6-31G(d) 1 14.8 Florian & Leszczynski (1996) HF/6-31G(d) 1 11.14 HF/6-31G(d,p)//HF/6-31G(d) 1 9.6 MP2/6-31G(d,p)//HF/6-31G(d) 1 9.0 Pérez et al. (2010) HF/def-TZVPP 1 13.50 Gorb et al. (2004) B3LYP/6-31G(d) 1 10.8 Hayashi & Mukamel (2004) B3LYP/6-31G(d) 1 10.75 Noguera et al. (2004) B3LYP/6-311++G(d,p)//B3LYP/6-31G(d,p) 1 9.80 Zoete & Meuwly (2004) B3LYP/6-311++G(d,p) 1 10.0 Noguera et al. (2007); Bertran et al. (2006) B3LYP/6-311++G(d,p) 1 9.8 Rodríguez-Santiago et al. (2010) B3LYP/6–311++G(d,p) 1 10.2 Hayashi & Mukamel (2004) B3LYP/6–31G(d) 78.39b 9.79 Noguera et al. (2004) B3LYP/6-311++G(d,p)//B3LYP/6-31G(d,p) Waterc 13.8 Pérez et al. (2010) B3LYP/def-TZVPP 1 11.39 BLYP/def-TZVPP 1 11.16 Zoete & Meuwly (2004) SCC-DFTB 1 5.8 Pérez et al. (2010) CC2/def-TZVPP 1 10.24 PBE0/def-TZVPP 1 10.95 PBE/def-TZVPP 1 10.63 Gorb et al. (2004) MP2/6-31G(d) 1 9.7 MP2/cc-pVDZ//MP2/6-31G(d) 1 6.7 MP2/cc-pVTZ//MP2/6-31G(d) 1 7.6 MP2/infinite//MP2/6-31G(d) 1 7.8 Pérez et al. (2010) MP2/def-TZVPP 1 9.37 Cerón-Carrasco et al. (2009) MP2/infinite//BP86/6-311++G(d,p) 1 7.80 Xiao et al. (2012) Ab initio constrained molecular dynamics 1 –

52.58 3.3 3.8 – 20.8 31.00 27.0 14.6 28.15 17.3 – 14.80 16.1 14.8 15.8 11.22 18.9 16.17 14.31 18.8 12.91 13.95 11.18 17.8 14.0 13.8 13.3 13.41 12.45 –

17.53 2.8 3.3 – 6.0 19.86 17.4 5.6 14.65 6.5 – 5.00 6.1 – 5.6 1.43 5.1 4.78 3.15 13.0 2.67 3.00 0.55 8.1 7.3 6.2 5.5 4.04 4.65 –

– – – – – – – – – 11.1 – – – – – – – – – – – – – 9.7 6.7 7.6 7.8 – 8.28 9.4

– – – – – – – – – 13.4 – – – – – – – – – – – – – 14.6 10.8 10.6 10.2 – 9.72 13.6

– – – – – – – – – 2.3 – – – – – – – – – – – – – 4.9 4.1 3.0 2.4 – 1.44 4.2

Notes: ΔE – the relative electronic energy of the Löwdin’s base pair; ΔΔETS – the activation electronic energy for the forward reaction of tautomerization; ΔΔE = ΔΔETSΔE – the activation electronic energy for the reverse reaction of tautomerization; ΔG – the Gibbs free energy of the Löwdin’s base pair (T = 298.15 K); ΔΔGTS – the Gibbs free energy of activation for the forward reaction of tautomerization (T = 298.15 K); ΔΔGΔΔGTSΔG – the Gibbs free energy of activation for the reverse reaction of tautomerization (T = 298.15 K).aThe Onsager reaction field model (1936); bthe SCRF method (Kirkwood, 1934; Onsager, 1936; Wiberg & Wong, 1993; Wong, Frisch, & Wiberg, 1991a; Wong, Wiberg, & Frisch, 1991b); cthe PCM proposed by Miertus et al. (1981; Miertus & Tomasi, 1982), where the solvent is viewed as a continuous dielectric medium of uniform dielectric constant ɛ.

2013a, 2013b, 2013c; Hovorun, Gorb, & Leszczynski, 1999), that is, the electronic energy of the back-reaction barrier of the G·C → G⁄·C⁄ tautomerization should exceed zero-point energy associated with the vibrational mode, which frequency becomes imaginary in the transition state (TS) of the tautomerization reaction. At this point, the ΔΔG Gibbs free energy of activation for the reverse reaction of tautomerization should be positive (ΔΔG > 0). (2) A thermodynamic population of the G⁄·C⁄ Löwdin’s mispair should be within the rate of spontaneous point mutations 1011/108 (Friedberg et al., 2006; Lee et al., 2012). (3) The electronic energy of interaction of the bases in the G⁄·C⁄ Löwdin’s mispair should not exceed

the dissociation energies of the G·C Watson–Crick DNA base pair in order not to slow down the work of the replication machinery (Brovarets’, Kolomiets’ et al., 2012). (4) The lifetime of the G⁄·C⁄ Löwdin’s mispair should exceed the time required for the replication machinery to forcibly dissociate a base pair into the monomers (several ns (Brovarets’ & Hovorun, 2013; Brovarets’, Kolomiets’ et al., 2012)) during the DNA replication. (5) The lifetime of the G⁄ and C⁄ mutagenic tautomers formed due to the dissociation of the G⁄·C⁄ base pair should be large enough comparably with the time taken by the DNA polymerase to incorporate one complementary nucleotide (104 s (Kornberg & Baker, 1992)) to promote

Tautomerization of the GC Watson-Crick base pair via the DPT


point mutations. This requirement is satisfied as it was established that the lifetime of the mutagenic tautomers of the DNA bases (Govorun et al., 1992) by 3–10 orders exceeds typical time of the DNA replication in the cell (103 s) (Brovarets’ & Hovorun, 2010a, 2010b, 2011b; Brovarets’, Kolomiets’ et al., 2012). (6) The time τ99.9% (Atkins, 1998) necessary to reach 99.9% of the equilibrium concentration of the G⁄·C⁄ Löwdin’s mispair and the G·C Watson– Crick base pair in the system should be considerably less than the time taken to complete a round of DNA replication (103 s (Kornberg & Baker, 1992)). And so, one important message of this paper is to test, if the G⁄·C⁄ Löwdin’s base pair satisfies all the above-mentioned physicochemical requirements. In the course of the theoretical investigations of the mechanism of the DPT in the G·C base pair in vacuo and in the medium of a low dielectric constant (ɛ = 4) corresponding to a hydrophobic interfaces of protein– nucleic acid interactions (Bayley, 1951; Dewar & Storch, 1985; García-Moreno et al., 1997; Mertz & Krishtalik, 2000; Petrushka, Sowers, & Goodman, 1986) using DFT and MP2 quantum-mechanical (QM) methods, it was established that for the G⁄·C⁄ Löwdin’s base pair, all requirements are satisfied except the 4th, since the G·C base pair is a too short-lived structure to be responsible for genetic instabilities. In this paper, we also had in mind the construction and analysis of the sweeps of the several types of properties along the IRC, namely, the electronic energy, the first derivative dE/dIRC of the electronic energy with respect to the IRC; the dipole moment of the G·C base pair, which is tautomerized; intermolecular H-bond distances and H-bond angle; the electron density, the Laplacian of the electron density, the ellipticity, and the energy at the (3,1) bond critical points (BCPs) of the intrapair H-bonds; distance between the H1 and H9 glycosidic protons and glycosidic angles. We established that, strictly speaking, the DPT in the G·C base pair in vacuum and in the continuum with ɛ = 4 as at the DFT, so at the MP2 levels of theory is a concerted asynchronous process, in which protons move with a small time gap in vacuum, while it noticeably increases in the continuum with ɛ = 4. At this, first the most acidic proton, localized at the N1 nitrogen atom of the G base, initially migrates along the N1H N3 Hbond to the N3 nitrogen atom of the C base inducing the formation of the unstable G·C+ zwitterion. This migration in turn provokes the concerted transition of the proton, localized at the N4 nitrogen atom of the C base, to the O6 oxygen atom of the G base. These migrations finally lead to the formation of the G⁄·C⁄ mispair. In the

5

continuum with a low dielectric constant (ɛ = 4), the mechanism of the DPT reaction in the G·C base pair remains the same, but the time gap between proton transfers noticeably increases. The nine key points along the IRC of the G·C base pair tautomerization, which could be considered as electron-topological “fingerprints” of a concerted asynchronous process of the tautomerization via the DPT, have been identified and fully characterized. These key points have been used to define the reactant, transition state, and product regions of the DPT in the G·C base pair. Analysis of the energetic characteristics of the H-bonds allows us to make a definite conclusion that the middle N1H N3 and the lower N2H O2 parallel H-bonds in the G·C base pair and the middle N3H N1 and the lower N2H O2 parallel H-bonds in the G⁄·C⁄ base pair are anticooperative, that is, the strengthening of the middle H-bond is accompanied by the weakening of the lower H-bond. It should be noted that the upper N4H O6 and O6H N4 H-bonds in the G·C and G⁄·C⁄ base pairs, respectively, remain constant at the changes of the middle and the lower H-bonds at the beginning and at the ending of the G·C ↔ G⁄·C⁄ tautomerization. As a result of research, we established that the G⁄·C⁄ Löwdin’s base pair satisfies all the requirements presented to it to cause point mutations in DNA except its lifetime, which appeared an Achilles' heel of the Löwdin’s model as was supposed by Löwdin himself. The point is that its lifetime (2.90·1014 s obtained at the MP2/aug-cc-pVTZ//MP2/6-311++G(d,p) level of theory in the continuum with ɛ = 4) is much less than the time required for a replication machinery to forcibly dissociate a base pair into monomers (several ns (Brovarets’ & Hovorun, 2013; Brovarets’, Kolomiets’ et al., 2012)) during DNA replication. It means that the G⁄·C⁄ mispair “escapes from the hands” of the replication machinery due to its transformation to the G·C Watson–Crick base pair and further dissociation into the G and C monomers. This situation cannot be changed taking into account the effect of more polar environment, as dipole moment of the TS exceeds the dipole moment of the G⁄·C⁄ mispair. It should be noted that the increasing of polarity of the environment is usually accompanied by the reduction of the relative Gibbs free energies of both the base pairs and the TS of their interconversion. We realize that our computational model of point mutations arising via the DPT is rather simplified and thus does not take into consideration some additional factors, e.g. anisotropy of the environment, which is characteristic for real biomolecular systems; however, the analysis of these factors provided by us earlier (Brovarets’, Yurenko et al., 2012) indicates that they most likely play the secondary role. So, based


6


on the aforementioned physicochemical reasons, we arrived at the conclusion that the G⁄·C⁄ Löwdin’s base pair would not cause point replication errors in DNA. However, the biological implication of the G⁄·C⁄ mispair with Watson–Crick geometry can consist in the fact that its formation in some way in the active center of DNA polymerase due to the specific interaction between the G⁄ and C⁄ mutagenic tautomers of the bases in the template strand and in the incoming nucleotide prevents formation of genetic mutations, since the G⁄·C⁄ Löwdin’s base pair in the DNA double helix quickly recombines into the G·C Watson–Crick base pair, thereby escaping from the undesirable mutations. The other biological importance of the G⁄·C⁄ Löwdin’s base pair lies in the fact that this mispair possesses conformational behavior similar to the A·T Watson-Crick base pair (Brovarets’, 2013a, 2013b). We believe that these findings provide not only a biological knowledge of prime importance, but also the great stimulus to discover a novel scenario of the mutagenic tautomerization of the Watson–Crick DNA base pairs. Computational methodology All calculations have been carried out with the Gaussian’09 program suite (Frisch et al., 2010). Geometries and harmonic vibrational frequencies of the base pairs and the TS of their tautomerization were obtained using Density Functional Theory (DFT) with the B3LYP hybrid functional (Tirado-Rives & Jorgensen, 2008), which includes Becke’s three-parameter exchange functional (B3) (Parr & Yang, 1989) combined with Lee, Yang, and Parr’s (LYP) correlation functional (Lee, Yang, & Parr, 1988), and MP2 method (Frisch, HeadGordon, & Pople, 1990) in connection with Pople’s 6-311++G(d,p) basis set (hereinafter in this article referred to as “DFT” and “MP2”, respectively). Scaling factors of 0.9580 and 0.9531 (Brovarets’ & Hovorun, 2013) have been used in the present work at the B3LYP and MP2 levels of theory, respectively, to correct the harmonic frequencies of all the studied base pairs. MP2 and DFT levels of theory have been successfully applied on similar systems recently studied and has been verified to give accurate normal mode frequencies, barrier heights, characteristics of intra- and intermolecular H-bonds, and geometries (Brovarets’, 2013a, 2013b; Brovarets’ & Hovorun, 2010a, 2010b, 2010c; Brovarets’, Yurenko et al., 2012; Brovarets’, Yurenko, & Hovorun, 2013; Lozynski, Rusinska-Roszak, & Mack, 1998; Matta, 2010; Pelmenschikov, Hovorun, Shishkin, & Leszczynski, 2000; Platonov et al., 2005; Shishkin, Pelmenschikov, Hovorun, & Leszczynski, 2000). Moreover, an excellent agreement between computational and experimental NMR, UV, and IR (Brovarets’ & Hovorun,

2011a, 2011b; Kondratyuk, Samijlenko, Kolomiets’, & Hovorun, 2000; Samijlenko, Krechkivska, Kosach, & Hovorun, 2004; Samijlenko, Yurenko, Stepanyugin, & Hovorun, 2012) spectroscopic data evidences that the MP2 and DFT methods employed for geometry optimization are reliable. Furthermore, it should be noted that two methods used in this study gave us the opportunity to compare the results obtained using them. In order to take into account the impact of the surrounding effect on the tautomerization of the investigated complexes, we have repeated the geometry optimizations at the MP2 and DFT levels of theory using the Conductor-Like Polarizable Continuum Model (CPCM) (Barone & Cossi, 1998; Cossi, Rega, Scalmani, & Barone, 2003), choosing the continuum with a dielectric constant ɛ = 4 typical for the hydrophobic interiors of proteins (García-Moreno et al., 1997) and protein–nucleic acid interfaces (Bayley, 1951; Dewar & Storch, 1985; Mertz & Krishtalik, 2000; Petrushka et al., 1986). To consider electronic correlation effects as accurately as possible, we followed the geometry optimizations with single-point energy calculations in vacuum and in the continuum with a low dielectric constant (ɛ = 4) at the MP2 level of theory using a wide variety of basis sets, in particular, Pople’s basis sets of valence triple-ζ (VTZ, i.e. 6-311G type) quality (Frisch, Pople, & Binkley, 1984; Hariharan & Pople, 1973; Krishnan, Binkley, Seeger, & Pople, 1980), as well as Dunning’s cc-type basis sets (Dunning, 1989; Kendall, Dunning, & Harrison, 1992), augmented with polarization and/or diffuse functions. They are: 6-311++X, where X = G(d,p), G(2df,pd), G(3df,2pd), as well as cc-pVXZ (X = D, T, Q), aug-cc-pVXZ (X = D, T) (altogether 8 basis sets). The correspondence of the stationary points to minimum on the potential energy landscape or TS has been checked by the absence or the presence, respectively, of one and only one imaginary frequency corresponding to the normal mode that identifies the reaction coordinate. TSs were located by means of Synchronous Transitguided Quasi-Newton (STQN) method (Peng & Schlegel, 1993; Peng, Ayala, Schlegel, & Frisch, 1996). Since the stationary points were located, the reaction pathway was established by following the intrinsic reaction coordinate (IRC) in the forward and reverse directions from each TS using the Hessian-based predictor–corrector (HPC) integration algorithm (Hratchian & Schlegel, 2004, 2005a, 2005b) 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. We have investigated the evolution of the energetic, geometric, polar, and electron-topological characteristics of the H-bonds and base pairs along the reaction


Tautomerization of the GC Watson-Crick base pair via the DPT pathway establishing them at each point of the IRC in vacuum and in the continuum with ɛ = 4. The electronic interaction energies in vacuum Eint have been computed at the MP2/6-311++G(2df,pd) level of theory for the geometries optimized at the DFT and MP2 levels of theory as the difference between the total energy of the base pair and the energies of the isolated monomers. In each case, the interaction energy was corrected for the basis set superposition error (BSSE) (Boys & Bernardi, 1970; Gutowski, Van Lenthe, Verbeek, Van Duijneveldt, & Chalasinski, 1986) through the counterpoise procedure (Sordo, Chin, & Sordo, 1988; Sordo, 2001). The interaction energies in solution Esol have been calculated as the sum of gas phase interaction energies and a solvation correction term (Brovarets’ & Hovorun, 2013): Esol ¼ Eint þ ðECOSMO EGAS PHASE Þ; where ECOSMO and EGAS PHASE are the BSSE uncorrected interaction energy values in solution and gas phase, respectively, evaluated for the geometries optimized in solution. The Gibbs free energy G values for all structures were obtained in the following way: G ¼ Eel þ Ecorr ; where Eel – the electronic energy, while Ecorr – thermal correction. We applied the standard TS theory (Atkins, 1998) to estimate activation barriers of the tautomerization reactions. The time τ99.9% necessary to reach 99.9% of the equilibrium concentration of the G⁄·C⁄ Löwdin’s mispair and the G·C Watson–Crick base pair in the system of reversible first-order forward (kf) and reverse (kr) reactions can be calculated from (Atkins, 1998):

s99:9% ¼

ln 103 kf þ kr

and the lifetime τ of the complexes is given by 1/kf,r. The equilibrium constants were calculated using the standard equation K = exp(ΔG/RT) (Atkins, 1998), where ΔG is the relative Gibbs free energy of the product, T is the absolute temperature, and R is the universal gas constant. To calculate the values of rate constants kf and kr:

kf ;r ¼ C

7

f ;r kB T DDG e RT h

we applied the standard TS theory (Atkins, 1998), in which quantum tunneling effects are accounted by the Wigner’s tunneling correction (Wigner, 1932) that is adequate for PT reactions (Brovarets’ & Hovorun, 2010a, 2010b; Brovarets’, Zhurakivsky, & Hovorun, 2010; Brovarets’, Yurenko et al., 2012; Brovarets’ et al., 2013a, 2013b, 2013c): 2 1 hmi C¼1þ ; 24 kB T where kB – Boltzmann’s constant, h – Planck’s constant, ΔΔGf,r – Gibbs free energy of activation for the PT reaction, and νi – the magnitude of the imaginary frequency associated with the vibrational mode at the TS that connects reactants and products. Bader’s quantum theory “Atoms in molecules” (QTAIM) was applied to analyze electron density (Bader, 1990). The topology of the electron density distribution was analyzed using program package AIMAll (Keith, 2010) with all the default options. The presence of a 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 H-bond formation (Bader, 1990). Wave functions were obtained at the level of theory used for geometry optimization. The energies of the H-bonds at the investigation of the corresponding sweeps were evaluated by the empirical Espinosa–Molins–Lecomte (EML) formula (Espinosa, Molins, & Lecomte, 1998; Mata, Alkorta, Espinosa, & Molins, 2011) based on the electron density distribution at the (3,1) BCPs of the H-bonds: EHB ¼ 0:5 V ðrÞ; where V(r) is the value of a local potential energy at the (3,1) BCP. The energy of the N2H O2 and N3+H N1 H-bonds in the TSGC$G C were calculated by the Nikolaienko–Bulavin–Hovorun formula (Nikolaienko, Bulavin, & Hovorun, 2012): EHB ¼ 2:03 þ 225 q; where ρ is the electron density at the (3,1) BCP of the H-bond. The energies of the conventional H-bonds were evaluated by the empirical Iogansen’s formula (Iogansen, 1999):

8

O.O. Brovarets’ and D.M. Hovorun EHB ¼ 0:33

pffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi Dm 40;

where Δν – the magnitude of the redshift (relative to the free molecule) of the stretching mode of H-bonded AH groups in the AH B H-bond (A and B – N, O). The partial deuteration was applied to minimize the effect of vibrational resonances (Brovarets’ & Hovorun, 2010a, 2010b; Brovarets’ & Hovorun, 2013; Pelmenschikov et al., 2000). The atomic numbering scheme for the purine and pyrimidine bases is conventional (Saenger, 1984).


Results and their discussion The obtained results are collected in Tables 2–5 and presented in Figures 1–15 (see also Figures S1–S7 in the Supporting Information). Their analysis leads to the following conclusions. The G·C Watson–Crick and the G⁄·C⁄ Löwdin’s DNA base pairs are stabilized by three intermolecular H-bonds each: their basic physicochemical characteristics are presented in Table 2. Notably that at the G·C → G⁄·C⁄ tautomerization, the total energy of the intrapair H-bonds increases in 1.11/1.19 and in 1.09/1.13 times (ɛ = 1/4) at the DFT and MP2 levels of theory, respectively (Tables 2 and 3). Within the framework of the CPCM model (Barone & Cossi, 1998; Cossi et al., 2003), it was demonstrated that total energy of the intrapair H-bonds slightly strengthen in the G⁄·C⁄ base pair (19.62/18.65 kcal/mol in vacuum) under the transition from vacuum (ɛ = 1) to the continuum with a low dielectric constant (ɛ = 4) by 1.53/0.94 kcal/mol, while this value remains almost the same in the G·C base pair (17.79/17.44 kcal/mol in vacuum) (Tables 2 and 3). Analysis of the energetic characteristics listed in Figures 8(a) and 6Sc allows us to make a definite conclusion that the middle N1H N3 and the lower N2H O2 parallel H-bonds in the G·C base pair and the middle N3H N1 and the lower N2H O2 parallel H-bonds in the G⁄·C⁄ base pair are anticooperative, that is, the strengthening of the middle H-bond is accompanied by the weakening of the lower H-bond. It should be noted that the upper N4H O6 and O6H N4 H-bonds in the G·C and G⁄·C⁄ base pairs, respectively, remain constant under the changes of the middle and the lower H-bonds at the beginning and at the ending of the G·C → G⁄·C⁄ tautomerization. We obtained the following numerical relations of the H-bonds cooperativity (Brovarets’ & Hovorun, 2013; Mishchuk, Potyagaylo, & Hovorun, 2000) in the G·C and G⁄·C⁄ base pairs: dEN1H N3/dEN2H O2 = -1.53/1.00 and dEN3H N1/ dEN2H O2 = 2.36/1.00, respectively, obtained at the DFT level of theory in vacuum.

In the G·C Watson-Crick DNA base pair, the strongest H-bond in vacuum is the upper N4H O6 H-bond, exposed in the major groove of DNA (6.78/ 6.40 kcal/mol at the DFT/MP2 levels), while in the continuum with ɛ = 4 – the middle N1H N3 H-bond (6.39/6.62 kcal/mol at the DFT/MP2 levels). In the G⁄·C⁄ Löwdin’s base pair, the strongest H-bond both in vacuum and in the continuum with ɛ = 4 is the upper O6H N4 H-bond, exposed in the major groove of DNA, which energy is 8.72/10.42 and 8.39/9.57 kcal/ mol (ɛ = 1/4) at the DFT and MP2 levels of theory, respectively. So, in the G⁄·C⁄ Löwdin’s base pair, the strongest O6H N4 H-bond can be considered as “a primary actor”. The classical geometric criteria for the identification of H-bonds are satisfied for all AH B H-bonds; physicochemical parameters are presented in Table 2. The dH B distances are less than the sum of Bondi’s van der Waals radii (Bondi, 1964) of H (1.2 Å) and B (O (1.52 Å) and N (1.55 Å)) atoms. An elongation of the proton donor group AH upon the conventional H-bonding is positive in all cases (for more details, see Table 2) and the angle of H-bonding is around 175°. The spectroscopic data collected in Table 2 confirm geometric results. The shift in frequency of the stretching mode of the AH donor group (the difference between the frequency for the AH group in monomer and the frequency of this group involved in H-bonding) is positive (shift to the red) for all H-bonds. All studied intrapair H-bonds without exceptions show positive values of the Laplacian of the electron density Δρ at the BCPs of the H B H-bonds and the values of the electron density ρ at these BCPs, usually treated as a measure of hydrogen bonding strength (Grabowski, 2011; Gutowski et al., 1986), are situated within the range 0.022/0.051/0.020/0.060 and 0.020/ 0.048/0.018/0.056 a.u. (ɛ = 1/ɛ = 4) at the DFT and MP2 levels, respectively. At that the maximum value of the ρ both in vacuum and in the continuum with ɛ = 4 corresponds to the strongest O6H N4 H-bond in the G⁄·C⁄ base pair. The comparison of the electronic energy of the reverse barrier of the tautomerization of the G⁄·C⁄ Löwdin’s mispair with the zero-point energy EZPE (Table 4) of the corresponding vibrational modes, which frequencies become imaginary in the TSG·C↔G⁄·C⁄ indicates that the G⁄·C⁄ mispair is dynamically (vibrationally) stable in vacuum. This statement is objective and does not depend on the chosen QM level of theory (Table 4). It should be also noted that the ΔΔG Gibbs free energy of activation for the reverse reaction of the G·C → G⁄·C⁄ tautomerization is less than kT in vacuum (ΔΔG < kT). However, we established that the G⁄·C⁄ Löwdin’s mispair loses its dynamical stability at the transition from the vacuum to the continuum with ɛ = 4.

G·C↔G·C⁄

N4H O6 N1H N3 N2H O2 O6H N4 N3H N1 N2H O2 N3H N1 N2H O2

N4H O6 N1H N3 N2H O2 O6H N4 N3H N1 N2H O2 N2H O2 0.031 0.033 0.030 0.060 0.036 0.021 0.049 0.020

0.037 0.033 0.027 0.051 0.038 0.022 0.026

DFT

ρa

0.029 0.036 0.026 0.056 0.037 0.019 0.052 0.018

0.034 0.034 0.023 0.048 0.039 0.020 0.023

MP2

0.107 0.087 0.102 0.098 0.090 0.076 0.093 0.072

0.120 0.088 0.094 0.103 0.089 0.080 0.096

DFT

0.114 0.107 0.104 0.121 0.107 0.078 0.114 0.073

0.131 0.105 0.096 0.122 0.107 0.081 0.094

MP2

Δρb

4.52 7.00 5.92 5.22 6.47 6.03 6.21 5.91

3.71 6.93 5.77 5.27 6.36 5.73 5.25

DFT

100

6.12 8.51 7.72 5.91 8.12 7.58 7.37 7.42

5.24 8.71 7.35 6.00 7.86 7.22 6.79

MP2

ɛc

2.873 2.955 2.897 2.665 2.917 3.032 2.820 3.054

2.809 2.954 2.936 2.720 2.910 3.014 2.935

DFT

2.880 2.885 2.927 2.664 2.878 3.052 2.765 3.080

2.809 2.908 2.961 2.712 2.869 3.039 3.141

MP2

dA…Bd

1.846 1.921 1.874 1.650 1.879 2.018 1.762 2.044

1.774 1.922 1.915 1.714 1.865 2.001 1.927

DFT

1.856 1.846 1.906 1.653 1.841 2.035 1.704 2.067

1.777 1.873 1.941 1.709 1.823 2.023 2.140

MP2

dH…Be

178.1 177.9 179.4 171.9 175.8 176.9 175.0 174.1

178.8 177.1 178.4 172.1 176.7 177.3 171.8

DFT

176.3 177.2 175.7 172.9 174.8 179.0 173.7 175.3

177.7 175.8 175.5 173.3 175.7 177.8 171.9

MP2

\AH Bf

0.019 0.022 0.014 0.053 0.028 0.007 – –

0.027 0.020 0.012 0.038 0.034 0.007 –

DFT

0.014 0.024 0.011 0.046 0.026 0.006 – –

0.021 0.021 0.009 0.034 0.031 0.006 –

MP2

ΔdAHg

344.3 415.1 328.6 1036.1 514.5 155.0 – –

461.8 359.2 279.9 738.8 590.0 131.6 –

DFT

Δνh

322.6 442.6 289.6 880.5 494.5 121.7 – –

416.5 385.9 260.5 685.8 559.6 108.8 –

MP2

5.76 6.39 5.61 10.42 7.19 3.54 9.00⁄ 2.47⁄

6.78 5.90 5.11 8.72 7.74 3.16 3.82⁄

DFT

5.55 6.62 5.21 9.57 7.04 2.98 9.67⁄ 2.02⁄

6.40 6.14 4.90 8.39 7.52 2.74 3.15⁄

MP2

EHBi

Notes: aThe electron density at the BCP (a.u.); bThe Laplacian of the electron density at the BCP (a.u.); cThe ellipticity at the BCP; dThe distance between A and B atoms in the AH B H-bond (Å); e The distance between H and B atoms in the AH B H-bond (Å); fThe H-bond angle (degree); gElongation of the donating group AH upon H-bonding (Å); hThe red shift (positive value) or the blue shift (negative value) of the stretching vibrational mode of the H-bonded AH group (cm1); iThe H-bond energy calculated by Iogansen’s (1999) and Nikolaienko, Bulavin, and Hovorun (2012) (marked with an asterisk) formulae (kcal/mol).

TS

G⁄C⁄

ɛ=4 G·C

TSG·C↔G⁄·C⁄

G⁄·C⁄

ɛ=1 G·C

AH B Н-bond

Table 2. Electron-topological, struconds in the G·C Watson–Crick and G⁄·C⁄ Löwdin’s DNA base pairs and TSG·C↔G⁄·C⁄, obtained at the B3LYP/6-311++G(d,p) (DFT) and MP2/6-311++G(d,p) (MP2) levels of theory in vacuo and in the continuum with a low dielectric constant (ɛ=4).


Tautomerization of the GC Watson-Crick base pair via the DPT 9

10


Table 3. Interbase interaction energies (in kcal/mol) for the studied DNA base pairs in vacuo and in the continuum with a low dielectric constant (ɛ = 4). DNA base pair

-ΔEint

ΣEHB

ΣEHB/|ΔEint|, %

ΔGint

17.79 19.62

60.8 85.5

15.97 10.09

17.44 18.65

62.6 85.2

14.17 8.28

17.76 21.15

82.8 101.2

7.82 8.47

17.38 19.59

85.3 99.9

6.54 6.09

ɛ=1 MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) G·C 29.28 G⁄·C⁄ 22.94 MP2/6-311++G(2df,pd)//MP2/6-311++G(d,p) G·C 27.87 G⁄·C⁄ 21.88


ɛ=4 MP2/6-311++G(2df,pd)//B3LYP/6-311++G(d,p) G·C 21.44 G⁄·C⁄ 20.89 MP2/6-311++G(2df,pd)//MP2/6-311++G(d,p) G·C 20.38 G⁄·C⁄ 19.61

Notes: ΔEint – the BSSE corrected electronic interaction energy; ΣEHB – the total energy of the intermolecular H-bonds; ΔGint – the BSSE corrected Gibbs free energy of interaction (T = 298.15 K).

Moreover, the ΔΔG Gibbs free energy of activation for the reverse reaction of the G·C → G⁄·C⁄ tautomerization becomes a negative value (ΔΔG < 0) at this transition. As a consequence (Table 4), the lifetime of the G⁄·C⁄ Löwdin’s mispair as in vacuum (1.57 1013 s) and especially in the continuum with ɛ = 4 (2.90 1014 s) is by the many orders less than the time required for the replication machinery to forcibly dissociate a base pair into the monomers (several ns (Brovarets’ & Hovorun, 2013; Brovarets', Kolomiets' et al., 2012)) during DNA replication. This fact leaves no chance for the G·C → G⁄·C⁄ tautomerization to be a source of the point mutations in DNA. The small value (kT at room temperature) of the ΔΔG Gibbs free energy of activation for the reverse reaction of the G·C → G⁄·C⁄ tautomerization in vacuum or the negative value of the ΔΔG in the continuum with ɛ = 4, which causes very short lifetime of the G⁄·C⁄ mispair in comparison with the time required for the replication machinery to forcibly dissociate a base pair into the monomers during DNA replication are considered by the authors as “the quantum protection” of the G·C Watson–Crick base pair from its spontaneous mutagenic tautomerization through the DPT. The similar effect was discovered recently for the mutagenic tautomers of the DNA bases, hypothetically generated by the DNA-binding proteins of replisome (Brovarets’, Yurenko et al., 2012), the adenine⁄·thymine⁄ (A⁄·T⁄) Löwdin’s mispair (Brovarets’ & Hovorun, 2013), the hypoxanthine⁄·hypoxanthine⁄ (H⁄·H⁄) homodimer, the H·T⁄ mispair and the H⁄·C⁄ mispair (Brovarets’ & Hovorun,

2012), which have no impact on the total spontaneous mutation rate due to their dynamical instability. The equilibrium constant of the Löwdin’s base pair is not less than the rate of spontaneous point mutations (Table 4). It should be noted that both the G·C and G⁄·C⁄ base pairs studied here are thermodynamically stable structures (ΔGint < 0) (Table 3). Notably, the H-bonds in the G⁄·C⁄ base pair make greater contribution to the stabilization of this base pair than to the G·C base pair, that is the ΣEHB/|Eint| ratio is higher for the G⁄·C⁄ base pair than for the G·C base pair. It was established that the interaction energy for the G·C and G⁄·C⁄ base pairs increases at the transition from vacuum to the continuum with ɛ = 4 (in comparison with vacuum). The electronic interaction energy of the G⁄·C⁄ Löwdin’s mispair is less than the corresponding value for the G·C Watson–Crick DNA base pair both in vacuum and in the continuum with ɛ = 4 (Table 3). We have performed the IRC calculations both in vacuum and in the continuum with ɛ = 4 corresponding to a hydrophobic interface of protein–nucleic acid interactions (Bayley, 1951; Brovarets’ & Hovorun, 2012; Brovarets’, Yurenko et al., 2012; Brovarets’ et al., 2013a; Dewar & Storch, 1985; García-Moreno et al., 1997; Mertz & Krishtalik, 2000; Petrushka et al., 1986) to map out a reaction pathway of the G·C ↔ G⁄·C⁄ transformation and to test whether the optimized TS structure is connected to the reactant (the G·C base pair) and the product (the G⁄·C⁄ base pair) of the tautomerization reaction starting from the TS downhill both in forward and reverse directions (Figures 2 and 2Sa).

a

1 4 1 4 1 4 1 4 1 4 1 4 1 4 1 4 1 4

ɛ 8.22 9.44 8.09 8.82 8.99 9.49 9.22 9.71 7.87 8.22 8.69 9.17 9.24 9.73 9.23 9.76 9.22 9.71

ΔG 7.87 8.84 7.09 8.41 8.00 9.08 8.23 9.30 6.88 7.81 7.70 8.75 8.24 9.32 8.24 9.35 8.22 9.30

ΔE 9.05 9.10 9.65 8.91 9.15 8.78 9.75 9.23 9.36 8.99 9.36 9.07 9.86 9.39 9.58 9.09 9.69 8.97

ΔΔGTS 13.02 10.83 13.24 11.04 12.75 10.91 13.34 11.36 12.96 11.12 12.96 11.19 13.46 11.51 13.17 11.22 13.28 11.10

ΔΔETS 0.83 0.34 1.56 0.10 0.16 0.70 0.53 0.48 1.49 0.78 0.67 0.10 0.62 0.34 0.35 0.66 0.47 0.65

ΔΔG 5.15 1.99 6.15 2.63 4.75 1.83 5.12 2.06 6.08 3.31 5.26 2.44 5.21 2.19 4.94 1.87 5.06 1.86

kcal/mol 1800.8 695.6 2151.2 920.5 1661.5 641.4 1790.6 720.1 2126.6 1158.5 1840.7 852.5 1823.5 767.1 1727.1 655.6 1770.1 655.5

cm1 2951.8 2684.9 3008.4 2813.6 3008.4 2813.6 3008.4 2813.6 3008.4 2813.6 3008.4 2813.6 3008.4 2813.6 3008.4 2813.6 3008.4 2813.6

ν 1475.9 1342.4 1504.2 1406.8 1504.2 1406.8 1504.2 1406.8 1504.2 1406.8 1504.2 1406.8 1504.2 1406.8 1504.2 1406.8 1504.2 1406.8

EZPE

12

1.04 10 4.94 1014 9.84 1013 1.05 1013 9.32 1014 2.75 1014 1.73 1013 4.01 1014 8.74 1013 3.31 1013 2.21 1013 7.59 1014 2.03 1013 5.03 1014 1.28 1013 2.94 1014 1.57 1013 2.90 1014

τ

12

9.61 10 4.55 1013 9.06 1012 9.70 1013 8.58 1013 2.53 1013 1.60 1012 3.70 1013 8.10 1012 3.05 1012 2.00 1012 6.99 1013 1.90 1012 4.63 1013 1.18 1012 2.71 1013 1.45 1012 2.69 1013

τ99.9%

9.42 107 1.19 107 1.17 106 3.40 107 2.53 107 1.10 107 1.73 107 7.52 108 1.68 106 9.38 107 4.21 107 1.89 107 1.67 107 7.30 108 1.70 107 6.98 108 1.74 107 7.50 108

К

Notes: ΔG – the relative Gibbs free energy of the Löwdin’s base pair (ΔGG·C = 0; T = 298.15 K), kcal/mol; ΔE – the relative electronic energy of the Löwdin’s base pair, kcal/mol; ΔΔGTS – the Gibbs free energy of activation for the forward reaction of tautomerization (T = 298.15 K), kcal/mol; ΔΔETS – the activation electronic energy for the forward reaction of tautomerization, kcal/mol; ΔΔGTSΔG – the Gibbs free energy of activation for the reverse reaction of tautomerization (T = 298.15 K), kcal/mol; ΔΔE = ΔΔETSΔE – the activation electronic energy for the reverse reaction of tautomerization; ν – the frequency of the vibrational mode of the tautomerized complex which becomes imaginary in the TSG·C↔G⁄·C⁄ of the tautomerization, obtained at the level of geometry optimization, cm1; EZPE – zero-point vibrational energy associated with this normal mode, cm1; τ – the lifetime of the Löwdin’s base pair, s; τ99.9% – the time necessary to reach 99.9% of the equilibrium concentration of the G·C Watson–Crick base pair and the G⁄·C⁄ Löwdin’s base pair, s; К – the equilibrium constant of the G·C → G⁄·C⁄ tautomerization. aData are taken from Ref. (Brovarets’, Kolomiets’ et al., 2012).

MP2/aug-cc-pVTZ//MP2/6–311++G(d,p)

MP2/aug-cc-pVDZ//MP2/6–311++G(d,p)

MP2/cc-pVQZ//MP2/6-311++G(d,p)

MP2/cc-pVTZ//MP2/6-311++G(d,p)

MP2/cc-pVDZ//MP2/6-311++G(d,p)

MP2/6-311++G(3df,2pd)//MP2/6–311++G(d,p)

MP2/6-311++G(2df,pd)//MP2/6–311++G(d,p)

MP2/6-311++G(d,p)

MP2/6-311++G(2df,pd)//B3LYP/6–311++G(d,p)

QM method

ΔΔE

Table 4. Energetic and kinetic characteristics of the G·C ↔ G⁄·C⁄ mutagenic tautomerization via the DPT in vacuo and in the continuum with a low dielectric constant (ɛ = 4), obtained at the different levels of QM theory.


Tautomerization of the GC Watson-Crick base pair via the DPT 11

12


Table 5.Table parameters for the G·C Watson–Crick and the G⁄·C⁄ Löwdin’s DNA base pairs and TSG·C↔G⁄·C⁄, obtained at the B3LYP/6-311++G(d,p) (DFT) and MP2/6-311++G(d,p) (MP2) levels of theory in vacuo and in the continuum with a low dielectric constant (ɛ = 4). α1, degree

R(H1-H9), Å Complex

α2, degree

DFT

MP2

DFT

MP2

DFT

MP2

C

10.213 10.284 10.064

10.149 10.199 9.993

53.1 51.9 51.1

52.9 51.9 51.1

55.1 51.9 52.2

55.4 53.0 52.0

G C G⁄·C⁄ TSG·C↔G⁄·C⁄

10.213 10.327 10.285

10.123 10.234 10.195

52.6 50.8 49.7

52.7 50.9 49.7

55.2 51.2 49.9

55.2 51.8 50.3

ɛ=1 G C G⁄·C⁄ TSG·C↔G⁄

⁄


ɛ=4

Notes: R(H1–H9) – the distance between the H1 and H9 glycosidic protons; α1 = N9H9-H1(N1) and α2 = N1H1-H9(N9) – the angles subtended at the glycosidic nitrogen atoms N9(purine) and N1(pyrimidine), respectively; Н – the proton located at the glycosidic nitrogen atom.

(a)

(b)

Figure 1. Geometric structures of the nine key points describing the evolution of the G·C ↔ G⁄·C⁄ tautomerization via the DPT along the IRC (see also Figure S1 in the Supporting Information) obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4). Coordinates of the nine key points are presented in Bohr for each structure. The dotted lines indicate AH B H-bonds, while continuous lines show covalent bonds (their lengths are presented in angstroms). Carbon atoms are in light-blue, nitrogen in dark-blue, hydrogen in grey, and oxygen in red.

Figure 2. Profiles of the electronic energy E along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).



13

Figure 3. Profiles of the first derivative of the electronic energy with respect to the IRC dE/dIRC along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

Figure 4. Profiles of the dipole moment μ along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

Formally, an additional minimum corresponding to the G·C+ zwitterionic base pair is observed at the DFT and MP2 levels of theory in the continuum with ɛ = 4 (Figure 2(b)). However, the G·C+ zwitterionic base pair cannot be considered as a dynamically stable intermediate, because the electronic energy of the reverse barrier of the G·C ↔ G·C+ transition has the negative value (69.5 cm1 at the MP2/6-311++G(2df,pd)//B3LYP/6311++G(d,p) level of theory and 20.4 cm1 at the MP2/6-311++G(2df,pd)//MP2/6-311++G(d,p) level of theory in the continuum with ɛ = 4). Details of the proton movements along the IRC of the G·C ↔ G⁄·C⁄ tautomerization reaction show a concerted process characterized by concerted motions of two protons along the upper and middle H-bridges between

the G and C bases. Moreover, we established that the DPT in the G·C base pair in vacuum as at the DFT, so that the MP2 levels of theory is, strictly speaking, a concerted asynchronous process, in which protons move with a small time gap in vacuum, while the time gap noticeably increases in the continuum with ɛ = 4. Our results concerning the physicochemical mechanism of the G·C ↔ G⁄·C⁄ tautomerization via the DPT (concerted asynchronous process, in which limiting stage is the final proton transfer along the intermolecular N4H O6 Hbond) are completely consistent with previous ab initio constrained molecular dynamics simulations (Xiao et al., 2012). At this, first, the most acidic proton, localized at the N1 nitrogen atom of the G base, initially migrates along the N1H N3 H-bond to the N3 nitrogen atom of


14


Figure 5. Profiles of the electron density ρ at the BCPs of the H-bonds along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

Figure 6. Profiles of the Laplacian of the electron density Δρ at the BCPs of the H-bonds along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

the C base inducing the formation of the unstable G·C+ zwitterion (Figure 1 and 1S). This migration in turn provokes the concerted transition of the proton, localized at the N4 nitrogen atom of the C base, to the O6 oxygen atom of the G base. These migrations finally lead to the formation of the G⁄·C⁄ mispair. In the continuum with a low dielectric constant (ɛ = 4), the mechanism of the DPT reaction in the G·C base pair remains the same. We revealed for the G·C ↔ G⁄·C⁄ tautomerization reaction that the TSG·C↔G⁄·C⁄ is strongly shifted toward the product – the G⁄·C⁄ Löwdin’s mispair and is more similar to it. Therefore, we can suggest that the G·C ↔ G⁄·C⁄ tautomerization is endothermic reaction, obeying the Hammond’s postulate (Hammond, 1955), which states that endothermic reactions have a product-like TS.

We distinguished nine key points, three of which are stationary – the G·C and G⁄·C⁄ base pairs and TSGC ↔ G⁄C⁄, to describe clearly the evolution of the G·C ↔ G⁄·C⁄ tautomerization along the reaction coordinate and to characterize the IRC pathway as comprehensively as possible (Figures 1 and 1S). These nine key points have been used to define step by step the mechanism of the G·C ↔ G⁄·C⁄ tautomerization. Their energetic, geometric, electron-topological, and polar characteristics are exhaustively presented in Figures 2–15 and 2S–7S. The key point 1: The starting structure along the IRC pathway is the G·C Watson–Crick base pair. It is stabilized by the N4H O6, N1H N3 and N2H O2 H-bonds (Table 2, Figures 1 and S1).



15

Figure 7. Profiles of the ellipticity ɛ at the BCPs of the H-bonds along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

Figure 8. Profiles of the energy of the H-bonds EHB, calculated by the EML formula (Espinosa et al., 1998; Mata et al., 2011), at the BCPs of the H-bonds along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

The key point 2: The structure of the base pair corresponding to the situation, in which the N1–H chemical bond of the G base is significantly weakened and the N3 H H-bond actually becomes the N3–H covalent bond (Figures 1 and S1). A characteristic feature of this structure is a zero value of the Δρ at the BCP of the N3 H H-bond (Figures 6 and 4Sb). The maximum value of the energy of the N3 H H-bond is attained at this key point (Figures 8 and 4Sd). It should be noted that on the graphs the energy of the H-bonds corresponding to the value Δρ P 0 (Figures 6 and 4Sb) is only shown. The key point 3: This structure is characterized by the equivalent loosened N1–H and N3–H covalent bonds that have equal values of the electron density, the

Laplacian of the electron density at the BCPs, and the dN1H/N3H distances. χ-like dependencies of these electron-topological and geometric characteristics are observed for the loosened N1–H–N3 bridge (Figures 5, 6, 13, 4Sa, 4Sb, and 5Sb). The key point 4: The structure corresponding to the situation, in which the unstable G·C+ zwitterionic base pair begins to form, i.e. when the N1–H covalent bond begins to acquire characteristics of the N1 H H-bond. A characteristic feature of this structure is a zero value of Δρ at the BCP of the N1 H H-bond (Figures 6 and 4Sb). The maximum value of the energy of the N1 H H-bond is attained at this key point (Figures 8 and 4Sd). The key point 5: The TS of the G·C ↔ G⁄·C⁄ tautomerization via the DPT (Figures 1 and S1).


16


Figure 9. Profiles of the electron density ρ (N2H O2) at the BCP of the N2H O2 H-bond along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

Figure 10. Profiles of the Laplacian of the electron density Δρ (N2H O2) at the BCP of the N2H O2 H-bond along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

The key point 6: The structure corresponding to the situation, in which the N4–H chemical bond of the C base is significantly weakened and the O6 H H-bond actually becomes the O6–H covalent bond. A characteristic feature of this structure is a zero value of Δρ at the BCP of the O6 H H-bond (Figures 6 and 4Sb). The maximum value of the energy of the O6 H H-bond is attained at this key point (Figures 8 and 4Sd). The key point 7: The structure possessing two equivalent loosened O6–H and N4–H covalent bonds that have equal values of the electron density, the Laplacian of the electron density at the BCPs, and the dO6H/N4H distances. χ-like dependencies of these electron-topological and

geometric characteristics are observed for the loosened O6–H–N4 bridge (Figures 5, 6, 13, 4Sa, 4Sb, and 5Sb). The key point 8: The structure corresponding to the situation, in which the mispair containing mutagenic tautomers begins to form, i.e. when the significantly weakened N4–H covalent bond begins to acquire characteristics of the N4 H H-bond (Figures 1 and S1). A characteristic feature of this structure is a zero value of the Δρ at the BCP of the N4 H H-bond (Figures 6 and 4Sb). The maximum value of the energy of the N4 H H-bond is attained at this key point (Figures 8 and 4Sd). The key point 9: The final structure – the tautomerized (Löwdin’s) G⁄·C⁄ base pair. It is stabilized by the



17

Figure 11. Profiles of the energy of the N2H O2 H-bond EHB (N2H O2), calculated by the EML formula (Espinosa et al., 1998; Mata et al., 2011), at the BCP of the N2H O2 H-bond along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

Figure 12. Profiles of the distance dA B between the electronegative A and B atoms of the AH B H-bonds along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

O6H N4, N3H N1, and N2H O2 H-bonds (Table 2, Figures 1 and S1). These nine key points have been used to define the reactant, transition state, and product regions of the DPT in the G·C base pair (Brovarets’ & Hovorun, 2013; Brovarets’ et al., 2013a, 2013b, 2013c; Burda, Toro-Labbé, Gutiérrez-Oliva, Murray, & Politzer, 2007; Politzer, Murray, & Jaque, 2013; Yepes, Murray, Politzer, & Jaque, 2012). In the reactant region, the G and C nucleotide bases do not lose their chemical individuality and acquire such mutual deformation and orientation that eventually lead to the chemical reaction, namely to the DPT. It quite logically follows from our data (sweeps) on Δρ that the

reactant region begins at the key point 1, corresponding to the G·C base pair, and ends at the key point 2, where Δρ at the BCP of the N3 H H-bond equals zero and after passing which bases lose their chemical individuality and the DPT chemical reaction starts. The transition state region, where molecular rearrangements occur, is located between the key points 2 and 8. Similarly, basing on the sweeps of Δρ, we came to the conclusion that the product region, where the G⁄ and C⁄ mutagenic tautomers do not lose their chemical individuality and the relaxation to the final G⁄·C⁄ Löwdin’s base pair occurs, encompasses the key point 8, at which Δρ at the BCP of the N4 H H-bond is equal to zero, and the final key point 9.


18


Figure 13. Profiles of the distance dAH/HB between the hydrogen and electronegative A or B atoms of the AH B H-bonds along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

Figure 14. Profiles of the distance R (H1-H9) between the H1 and H9 glycosidic protons along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

Interestingly, that exactly at the key points 2 and 8, the dE/dIRC, first derivative of the electron energy with respect to the IRC, gets the extreme values in the appropriate vicinity (Brovarets’ & Hovorun, 2013; Brovarets’ et al., 2013a, 2013b, 2013c; Burda et al., 2007; Politzer et al., 2013; Yepes et al., 2012) (Figures 3 and 2Sb). We have established that the electronic energy necessary to bring the donor and acceptor atoms as close to one another as possible to activate the DPT reaction, that is the energy difference between the key points 2 and 1, is 11.04/9.28/10.16 kcal/mol obtained at the DFT(ɛ = 1)/ DFT(ɛ = 4)/MP2(ɛ = 1) levels of theory, respectively. A relatively small amount of energy (4.08/1.51/4.25 kcal/ mol estimated at the DFT(ɛ = 1)/DFT(ɛ = 4)/MP2(ɛ = 1) levels of theory, respectively), that is, the energy differ-

ence between the key points 8 and 9, is released upon the base pair, corresponding to the key point 8, relaxation to the G⁄·C⁄ mispair, corresponding to the key point 9. Characteristic structural feature of the G·C ↔ G⁄·C⁄ tautomerization is that it noticeably disturbs the Watson– Crick-like geometry of the G·C base pair (see Figures 14, 15, and 7S), namely, the DPT reaction is accompanied by a contraction of the distance between the G and C bases, changes of the glycosidic parameters of the base pair (Table 5), and geometric characteristics of the H-bonds, especially dA B and dAH/HB distances (Table 2, Figures 12, 13, and 5S), vividly demonstrating the “breathing” of the G·C base pair throughout the tautomerization process.



19

Figure 15. Profiles of the α1 = N9H9-H1(N1) and α2 = N1H1-H9(N9) glycosidic angles along the IRC of the G·C ↔ G⁄·C⁄ tautomerization obtained at the DFT level of theory (a) in vacuo and (b) in the continuum with a low dielectric constant (ɛ = 4).

It is connected with the change of distances between the pairs of electronegative atoms: N4 and O6, N3 and N1, N2 and O2 (Figures 12 and 5Sa). It should be noted that in vacuum and in the continuum with ɛ = 4, the O6 N4 and N3 N1 distances monotonically decrease reaching their minima at the key point 2 and then nonmonotonically increase. The G·C ↔ G⁄·C⁄ tautomerization is represented by the nonmonotonical changes of α1 (N9H9–H1(N1)) and α2 (N1H1–H9(N9)) glycosidic angles and the R(H1–H9) distance between the H1 and H9 glycosidic atoms (Figures 14, 15, and 7S). The dipole moment μ shows significant changes in vacuum and nonmonotonic Ω-like dependence in the continuum with ɛ = 4 along the IRC of the tautomerization of the G·C base pair (Figures 4 and 3S). Comparatively large changes in the dipole moment (5.18/4.92/ 7.09 ÷ 7.66/6.24/13.43 D obtained at the DFT(ɛ = 1)/DFT (ɛ = 4)/MP2(ɛ = 1) levels of theory, respectively) along the reaction coordinate indicate that the DPT is accompained with strong electronic reordering of the system along the IRC of the tautomerization (Figures 4 and 3S). The profile of the dipole moment gives a qualitative conception of the charge separation at the tautomerization of the G·C base pair along the IRC. This observation is informative for fundamental understanding of the particular influence of solvation on the tautomerization of the base pair. The middle N1H N3 and the upper N4H O6 H-bonds in the G·C Watson–Crick base pair exist within the structures 1–2 and 1–4 (Figures 8 and 4Sd), respectively, coherently becoming stronger during the tautomerization process. The middle N3H N1 and the upper O6H N4 H-bonds in the Löwdin’s G⁄·C⁄ base pair exist within the structures 6–9 and 8–9 (Figures 8 and 4Sd), respectively, coherently becoming weaker during the

tautomerization process. The solvent effect practically has no impact on the energy of the H-bonds (Figure 4Sd). The third N2H O2 H-bond in the G·C DNA base pair assists its tautomerization via the DPT. The profiles of the EHB, ρ and Δρ of the N2H O2 H-bond are presented at Figures 9–11, and 6S. Profile of the N2H O2 H-bond energy has two maxima and three minima (Figures 11 and 6Sc). It draws attention to the fact that the maximum value of the energy of the N2H O2 H-bond along the IRC is realized exactly at the points, where maximum values of the electron density ρ and the Laplacian of the electron density Δρ are implemented (Figures 9, 10, 11 and 6Sc). The geometric characteristics of the N2H O2 H-bond – N2 O2, N2H and HO2 distances nonmonotonically increase along the IRC in vacuum and in the continuum with ɛ = 4 (Figures 12, 13 and 5S). We established that the time τ99.9% necessary to reach 99.9% of the equilibrium concentration of the G⁄·C⁄ Löwdin’s mispair and the G·C Watson–Crick base pair in the system is less by many orders than the time taken to complete a round of DNA replication (

DPT tautomerisation of the wobble guanine·thymine DNA base mispair is not mutagenic: QM and QTAIM arguments.

Is the DPT tautomerization of the long A·G Watson-Crick DNA base mispair a source of the adenine and guanine mutagenic tautomers? A QM and QTAIM response to the biologically important question.

QTAIM combined atomistic investigation.

QTAIM investigation.

DNA Mutations May Not Be the Cause of Cancer.

Does the tautomeric status of the adenine bases change upon the dissociation of the A*·A(syn) Topal-Fresco DNA mismatch? A combined QM and QTAIM atomistic insight.

Caffeine overcomes a restriction point associated with DNA replication, but does not accelerate mitosis.

NMR analysis of base-pair opening kinetics in DNA.

Mutations and DNA replication.

Initiation of adenovirus DNA replication does not occur via a hairpin mechanism.

Base pair opening pathways in B-DNA.

Deletion mutations and the replication of DNA in bacteriophage lambda.

Guanine- 5-carboxylcytosine base pairs mimic mismatches during DNA replication.

Base J glucosyltransferase does not regulate the sequence specificity of J synthesis in trypanosomatid telomeric DNA.

Base pair buckling can eliminate the interstrand purine clash at the CpG steps in B-DNA caused by the base pair propeller twisting.

DNA binding of the nonintercalative ligands SN-6132, SN-6131 and SN-6113: minor variations of the ligand structure may cause changes in the base pair preference.

Child health training: why does the College not act?

The physicochemical essence of the purine·pyrimidine transition mismatches with Watson-Crick geometry in DNA: A·C* versa A*·C. A QM and QTAIM atomistic understanding.

Tautomerization-dependent recognition and excision of oxidation damage in base-excision DNA repair.

Natural-like replication of an unnatural base pair for the expansion of the genetic alphabet and biotechnology applications.

Inactivation of the intrinsic muscle clock does not cause sarcopenia.

DNA-sequence recognition by CAP: role of the adenine N6 atom of base pair 6 of the DNA site.

APOBEC-induced mutations in human cancers are strongly enriched on the lagging DNA strand during replication.

Protein C deficiency: identification of a novel two-base pair insertion and two point mutations in exon 7 of the protein C gene in Spanish families.