**3.7. Complete set of incorrect DNA base pairs responsible for the origin of spontaneous transitions and transversions in DNA**

For the first time, we outline a complete set of the 12 incorrect DNA base pairs representing a primary cause of spontaneous point mutations and determining both incorporation and replication errors: А·С\*/С\*·A, G\*·T/T·G\*, G·Asyn, A\*·G\*syn, A\*·Asyn, G·G\*syn, C·T/T·C, C\*·C/C·C\* and T\*·T/T·T\* (three of these mispairs—G·Asyn, C·T and T·C—consist exclusively of the canonical tautomers of the DNA bases) (**Figure 7**). Precisely, these mismatches, which quite easily in the process of the thermal fluctuations acquire enzymatically competent conformations and do not cause steric constraints in the recognition pocket of the high-fidelity replication DNA polymerase (**Table 3**), should be experimentally observed in the closed conformation of the latter.

#### **3.8. Key microstructural mechanisms of the 2-aminopurine (2AP) mutagenicity**

Based on the mechanisms of the spontaneous point mutations [33, 34, 51–66, 68–74, 78], we established physico-chemical mechanisms of the mutagenic action of the classical mutagen

Renaissance of the Tautomeric Hypothesis of the Spontaneous Point Mutations in DNA: New Ideas… http://dx.doi.org/10.5772/intechopen.77366 43

**Figure 6.** Profiles of: **(a)** the relative electronic energy ΔE, **(b)** the first derivative of the electronic energy with respect to the IRC (dE/dIRC), **(c)** the dipole moment μ, **(d)** the electron density ρ, (**e**) the Laplacian of the electron density Δρ, (**f**) the energy of the intermolecular H-bonds EHB estimated by the EML formula [79] at the (3,-1) BCPs, (**g**) the distance dA···B between the electronegative A and B atoms, (**h**) the distance dAH/HB between the hydrogen and electronegative A or B atoms and (**i**) the angle ∠AH···B of the covalent and hydrogen bonds along the IRC of the 2AP·T(WC)↔2AP·T\*(w) tautomerization obtained at the B3LYP/6–311++G(d,p) level of theory, ε = 1.

This methodology enables to make an objective conclusion about the character of tautomerization (concerted, synchronous or asynchronous), quantitatively estimate the cooperativity of the specific intermolecular interactions (namely, H-bonds, in particular nonclassical CH···O/N or dihydrogen AH···HB H-bonds, loosened A-H-B covalent bridges and attractive A···B van der Waals contacts), sequentially changing each other along the IRC of the tautomerization, and trace how these interactions are grouped into the patterns (from 9 to 15) and how they

**Figure 5.** Geometric structures of the nine key points with their IRC coordinated describing the evolution of the 2AP·T(WC)↔2AP·T\*(w) tautomerization *via* the single PT and sequential shifting of the bases relative to each other within the base pair into the minor groove side of the DNA helix along the IRC obtained at the B3LYP/6–311++G(d,p) level of theory, ε = 1 [79] tautomerization). At this point, three KPs correspond to the two abovementioned local minima (the first and the last KPs–reagent and product, respectively) and transition state of the tautomerization. Other KPs include two KPs, for which migrating proton is localized midway between the electronegative atoms involved in the specific contact and are characterized by the loosened A-H-B covalent bridge, and also four key points, in which the 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: Δρ = 0.

For the first time, we outline a complete set of the 12 incorrect DNA base pairs representing a primary cause of spontaneous point mutations and determining both incorporation and replication errors: А·С\*/С\*·A, G\*·T/T·G\*, G·Asyn, A\*·G\*syn, A\*·Asyn, G·G\*syn, C·T/T·C, C\*·C/C·C\* and T\*·T/T·T\* (three of these mispairs—G·Asyn, C·T and T·C—consist exclusively of the canonical tautomers of the DNA bases) (**Figure 7**). Precisely, these mismatches, which quite easily in the process of the thermal fluctuations acquire enzymatically competent conformations and do not cause steric constraints in the recognition pocket of the high-fidelity replication DNA polymerase (**Table 3**), should be experimentally observed in the closed

successively substitute each other along the IRC of tautomerization.

**spontaneous transitions and transversions in DNA**

conformation of the latter.

42 Mitochondrial DNA - New Insights

**3.7. Complete set of incorrect DNA base pairs responsible for the origin of** 

**3.8. Key microstructural mechanisms of the 2-aminopurine (2AP) mutagenicity**

Based on the mechanisms of the spontaneous point mutations [33, 34, 51–66, 68–74, 78], we established physico-chemical mechanisms of the mutagenic action of the classical mutagen

**Figure 7.** Geometrical structures of the 12 incorrect DNA base mispairs causing spontaneous point incorporation and replication errors (B3LYP/6–311++G(d,p) level of theory, ε = 1).


a The distance between the glycosidic protons at the N1/N9 atoms, Å.

b The glycosidic angles for the bases situated on the left and right within the base pair, respectively, degree.

c The glycosidic angles for the bases situated on the left and right within the base pair, respectively, degree.

<sup>d</sup>The electronic energy of deformation, necessary to apply to the mismatch to acquire the sizes of the A·T (in the left column) and G·C (in the right column) Watson-Crick DNA base pairs.

e The electronic energy of interaction.

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 = 298.15 K).

**Table 3.** Selected structural and energetic (in kcal·mol−1) characteristics of the canonical and noncanonical DNA base pairs, responsible for the origin of the spontaneous transitions and transversions (MP2/6-311++G(2df,pd)//B3LYP/6- 311++G(d,p) level of theory, ε = 1).

2AP—high-energy structural isomer of A nucleotide base [75–77, 80–82]. In the literature, a great amount of experimental and theoretical phenomenological data on 2AP has been collected [83–87] without proper justification and substantiation.

> We have shown for the first time that 2AP very effectively produces induced *incorporation errors* by binding with C DNA base and forming the wobble С·2АР(w) mispair, which is tautomerized *via* the С·2АР(w) → С\*·2АР(WC) tautomeric reaction into the Watson-Cricklike С\*·2АР(WC) base mispair, which quite easily in the process of the thermal fluctuations acquires enzymatically competent conformation (estimated ratio of probabilities РС·2АР/

> pVDZ//B3LYP/6–311++G(d,p) level of theory in vacuum at T = 298.15 К). The base, belonging to the template strand of

(cm−1) at the TSs of the interconversions are presented below them in brackets (MP2/aug-cc-

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**Figure 8.** Reaction pathways of the biologically important tautomerizations and conformational transitions of the structures containing canonical DNA bases and 2AP in the main and rare tautomeric forms leading to the replication (**a**) and incorporation (**b**, **c**, **d**) errors—transitions and transversions. Relative electronic ∆E and Gibbs free ∆G energies, electronic ΔEint and Gibbs free ΔGint energies of interaction, the deformation energies ΔEdef(A·T)/ΔEdef(G·C) necessary to apply to the mismatch to acquire the sizes of the A·T(WC)/G·C(WC) Watson-Crick DNA base pairs (in kcal·mol−1),

By estimating the probability ratio РА·2АР/РА·А = 40.5, we conclude that 2AP in the case of the А·2АР(w)→А\*·2АР(WC)→А\*·2АРsyn structural transformations (**Figure 8c**, **Table 4**) causes transversion, when a pyrimidine base (in this case T) is substituted by a purine, in particular—A [89].

РС·А = 1.92·10<sup>4</sup>

imaginary frequencies *ν<sup>i</sup>*

) (**Figure 8b**, **Table 4**) [89].

DNA, is situated on the left, while the base of the incoming nucleotide—on the right.

We have found for the first time that the microstructural mechanism of the mutagenic action of 2AP, causing induced *replication errors*, generates with higher probability the mutagenic tautomer T\* according to the 2AP·Т(WC) → 2AP·Т\*(w) tautomeric reaction, than for the Watson-Crick A·Т(WC) DNA base pair according to the A·Т(WC) → A·Т\*(w) tautomerization reaction [68, 88]. At this point, the ratio of probabilities determining *replication errors* consists Р2АР·T/РА·T = 1.8·10<sup>3</sup> . The mutagenic effect is achieved due to the greater stability of the 2AP·Т\*(w) complex by the participation of 2AP (ΔEint = −20.95 and ΔGint = −9.18 kcal·mol−1) in comparison with the analogical A·Т\*(w) base mispair by the participation of A (ΔEint = −13.44 and ΔGint = −1.61 kcal·mol−1) (**Figure 8a**, **Table 4**) [68, 88].

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**Figure 8.** Reaction pathways of the biologically important tautomerizations and conformational transitions of the structures containing canonical DNA bases and 2AP in the main and rare tautomeric forms leading to the replication (**a**) and incorporation (**b**, **c**, **d**) errors—transitions and transversions. Relative electronic ∆E and Gibbs free ∆G energies, electronic ΔEint and Gibbs free ΔGint energies of interaction, the deformation energies ΔEdef(A·T)/ΔEdef(G·C) necessary to apply to the mismatch to acquire the sizes of the A·T(WC)/G·C(WC) Watson-Crick DNA base pairs (in kcal·mol−1), imaginary frequencies *ν<sup>i</sup>* (cm−1) at the TSs of the interconversions are presented below them in brackets (MP2/aug-ccpVDZ//B3LYP/6–311++G(d,p) level of theory in vacuum at T = 298.15 К). The base, belonging to the template strand of DNA, is situated on the left, while the base of the incoming nucleotide—on the right.

2AP—high-energy structural isomer of A nucleotide base [75–77, 80–82]. In the literature, a great amount of experimental and theoretical phenomenological data on 2AP has been col-

**Table 3.** Selected structural and energetic (in kcal·mol−1) characteristics of the canonical and noncanonical DNA base pairs, responsible for the origin of the spontaneous transitions and transversions (MP2/6-311++G(2df,pd)//B3LYP/6-

The glycosidic angles for the bases situated on the left and right within the base pair, respectively, degree.

The glycosidic angles for the bases situated on the left and right within the base pair, respectively, degree.

The contribution of the total energy of the intermolecular H-bonds to the electronic energy of interaction, %.

<sup>d</sup>The electronic energy of deformation, necessary to apply to the mismatch to acquire the sizes of the A·T (in the left

We have found for the first time that the microstructural mechanism of the mutagenic action of 2AP, causing induced *replication errors*, generates with higher probability the mutagenic tautomer T\* according to the 2AP·Т(WC) → 2AP·Т\*(w) tautomeric reaction, than for the Watson-Crick A·Т(WC) DNA base pair according to the A·Т(WC) → A·Т\*(w) tautomerization reaction [68, 88]. At this point, the ratio of probabilities determining *replication errors* con-

2AP·Т\*(w) complex by the participation of 2AP (ΔEint = −20.95 and ΔGint = −9.18 kcal·mol−1) in comparison with the analogical A·Т\*(w) base mispair by the participation of A (ΔEint = −13.44

. The mutagenic effect is achieved due to the greater stability of the

lected [83–87] without proper justification and substantiation.

The distance between the glycosidic protons at the N1/N9 atoms, Å.

column) and G·C (in the right column) Watson-Crick DNA base pairs.

**Mispairs Geometrical parameters Energetic parameters**

**<sup>b</sup> α<sup>2</sup>**

**<sup>c</sup> ΔEdef**

А·C\* 9.996 55.3 58.2 0.10 0.29 15.73 91.8 2.27 A\*·С 10.059 55.3 57.2 0.29 0.53 23.50 65.9 10.76 G\*·T 10.291 51.5 51.1 0.14 0.40 19.79 87.7 7.09 G·T\* 10.202 50.6 52.2 0.45 0.90 33.40 61.3 20.66 G·Asyn 10.399 51.6 38.5 3.00 3.61 17.00 65.9 2.80 A\*·G\*syn 10.411 50.3 37.5 3.18 3.72 23.00 72.6 11.47 A\*·Asyn 10.322 53.9 41.2 2.18 2.72 16.73 74.8 3.83 G·G\*syn 10.425 48.7 36.1 4.04 4.66 19.82 69.5 7.28 C·T 8.215 59.7 57.0 8.67 8.87 13.86 85.4 1.54 C·C\* 8.086 60.3 59.5 8.57 8.76 14.75 91.2 2.34 T·T\* 8.385 53.6 58.1 10.97 10.91 16.67 84.0 4.69 A·T 10.130 54.3 54.8 0.00 0.25 14.92 86.9 1.43 G·C 10.209 52.9 55.3 0.11 0.00 29.28 60.8 15.97

**<sup>d</sup> -ΔEint**

**<sup>e</sup> ΣEHB/|ΔEint|f -ΔGint**

**g**

**R(HN1/N9-HN1/N9)<sup>a</sup> α<sup>1</sup>**

44 Mitochondrial DNA - New Insights

and ΔGint = −1.61 kcal·mol−1) (**Figure 8a**, **Table 4**) [68, 88].

sists Р2АР·T/РА·T = 1.8·10<sup>3</sup>

The electronic energy of interaction.

311++G(d,p) level of theory, ε = 1).

The Gibbs free energy of interaction (T = 298.15 K).

a

b

c

e

f

g

We have shown for the first time that 2AP very effectively produces induced *incorporation errors* by binding with C DNA base and forming the wobble С·2АР(w) mispair, which is tautomerized *via* the С·2АР(w) → С\*·2АР(WC) tautomeric reaction into the Watson-Cricklike С\*·2АР(WC) base mispair, which quite easily in the process of the thermal fluctuations acquires enzymatically competent conformation (estimated ratio of probabilities РС·2АР/ РС·А = 1.92·10<sup>4</sup> ) (**Figure 8b**, **Table 4**) [89].

By estimating the probability ratio РА·2АР/РА·А = 40.5, we conclude that 2AP in the case of the А·2АР(w)→А\*·2АР(WC)→А\*·2АРsyn structural transformations (**Figure 8c**, **Table 4**) causes transversion, when a pyrimidine base (in this case T) is substituted by a purine, in particular—A [89].


**4. Conclusions**

fidelity DNA polymerase.

> > G·G ≈ C·C (10−6) [93].

NMR [16–18] experiments.

still remain without proper theoretical justification:

quencies agree well with the experimental data.

processes determining the protein synthesis.

pairs by the quantum-chemical methods.

**Acknowledgements**

Reported results are crucial for understanding the microstructural mechanisms of the spontaneous transitions and transversions, since they allow us to explain, on one side, the origin of the mutagenic tautomers at the separation of the DNA strands before DNA replication and, on the other side, how incorrect purine·pyrimidine, purine·purine and pyrimidine·pyrimidine wobble mispairs adapt to enzymatically competent sizes in the recognition pocket of the high-

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Obtained results allow us to explain biological experiments available in the literature, which

• Numerical estimations of the frequencies of the mispair occurrence satisfactorily explain experimental data: (10−3÷10−4) G·T/T·G > > A·C/C·A > > C·T/T·C > A·A > G·A/A·G

• Established A·C(w)↔A·C\*(WC) and G·T(w)↔G\*·T(WC) wobble(w)↔Watson-Crick(WC) transformations *via* the sequential PT 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 also to interpret X-ray [14, 15] and

• Presented approach allows us to clarify the microstructural mechanisms of the mutations induced by the classical mutagens, in particular 2-aminopurine, for which induced fre-

• Ionization mechanism cannot entirely explain the nature of the spontaneous transitions [94]. These data clarify the nature of genome variability and reveals new facets of the Watson-Crick hypothesis of the spontaneous point mutagenesis arising during DNA replication and significantly expands the possibilities for rational design of chemical mutagens with targeted

Finally, authors believe that these principles could be extended without any constrains to the

In view of the prominent role, that play parallel and antiparallel Hoogsteen pairings in DNA:RNA helices, as it was reliably established by Prof. Seligmann [95, 96] for mitochondrial genomes, it is important to explore in future mutagenic tautomerization of these classical base

The authors gratefully appreciate technical support and computational facilities of joint computer cluster of SSI "Institute for Single Crystals" of the National Academy of Sciences of Ukraine (NASU) and Institute for Scintillation Materials of the NASU incorporated into Ukrainian National Grid. This work was partially supported by the Grant of the NASU for young scientists, Grant of the President of Ukraine to support the research of young scientists [project number F70] from the State Fund for Fundamental Research of Ukraine of the Ministry

action, which could be interesting for synthetic biology and biotechnology.

**Table 4.** Energetic and kinetic characteristics of the biologically important tautomeri*z*ations and conformational transitions of the structures containing canonical DNA bases and 2AP in the main or rare tautomeric forms leading to replication and incorporation errors*—*transitions and transversions (MP2/aug-cc-pVDZ//B3LYP/6-311++G(d,p) level of theory, ε = 1).

We prove 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 G·2AP\*(w) mispair converts according to the route of the sequential tautomeric and conformational transformations—G·2AP\*(w) → G\*·2AP(w) → G·2AP(WC) → G·2APsyn (**Figure 8d**, **Table 4**) [91]. Estimated ratio of probabilities РG·2АР\*/РG·А\* = 1.90·10<sup>7</sup> points that this route of the tautomerically conformational transformations is mutagenic, generating appropriate transversions, when pyrimidine bases (in this case C) are 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 DNA bases [91].

Our theoretical data are in good agreement with existing experimental results [80, 81, 83, 84] and also allow a unified physico-chemical interpretation of them.

By analyzing profiles of the physico-chemical characteristics for the tautomerization reactions *via* the DPT and PT involving 2AP, which are integral parts of the biologically important tautomerically conformational transformations, we have established that 2AP·Т(WC)↔2AP·Т\*(w) [79], 2AP·C\*(WC)↔2AP·C(w) [79], G\*·2AP(w)↔G·2AP(WC) [90] and А·2АР(w)↔А\*·2АР(WC) [90] tautomerization pathways proceed through the stepwise concerted mechanism *via* the sequential intrapair PT between the bases followed by the shifting of the 2AP relatively the T/C\*/G\*/A bases, accordingly, while the T·2AP\*(w)↔T\*·2AP(w) and G·2AP\*(w)↔G\*· 2AP(w) [92] DPT tautomerization reactions proceed through the asynchronous concerted mechanism.
