**3.3. Novel mechanisms of the wobble (w)**↔**Watson-Crick (WC) tautomeric interconversions in the canonical and incorrect DNA base pairs as a key to understand origins of spontaneous transitions and transversions**

For the first time, a novel theoretical approach to elucidate microstructural mechanisms of incorporation and replication point errors arising at the DNA replication was proposed. We show for the first time that pairs of nucleotide bases with Watson-Crick architecture of the H-bonding—classical, long, short, in which one or both bases are in the main or rare tautomeric forms, are in a slow tautomeric equilibrium with the corresponding wobble base pairs in comparison with the time, in which high-fidelity DNA polymerase spends on the incorporation of one nucleotide into the DNA double helix (~8.3 × 10−4 s [67]). In fact, a novel pathway of the chemical reaction was discovered—tautomerization with significant changes of the geometry of the base pair—from Watson-Crick to wobble and *vice versa*.

We have discovered novel structural hypostases of the classical A·T(WC) and G·C(WC) Watson-Crick DNA base pairs arising due to their ability to switch into the wobble А\*·Т↑(w), А·Т\*O2↑(w), А·Т\*↓(w) and G·C\*↑(w), G\*·C↓(w), G·C\*↓(w), G\*·C↑(w) H-bonded mismatches containing rare tautomers (**Figure 2**) [68]. Estimated populations of the tautomerized states of the А·Т(WC) (6.1 × 10−9–1.5 × 10−7) and G·C(WC) (4.2 × 10−11–1.4 × 10−9) base pairs in the continuum with ε = 4 correspond to the interface of the protein-nucleic acid interactions. This evidences their involvement in nucleation of spontaneous point replication errors in DNA arising with frequencies ~10−11–10−9 errors *per* replicated nucleotide.

We found for the first time the intrinsic ability of the purine·pyrimidine (A·C [69, 70] and G·T [69, 71]), purine·purine (A·A [72], G·G [72] and A·G [73]) and pyrimidine·pyrimidine (С·С [74], Т·Т [74] and С·T [73]) DNA base mispairs to perform wobble↔Watson-Crick tautomeric transitions *via* the sequential intrapair DPT and subsequent shifting of the bases relative to each other (**Figure 3**, **Table 2**). These nondissociative tautomerizations *via* the sequential PT are controlled by the highly stable (ΔEint > 100 kcal·mol−1), highly polar and zwitterionic transition states of the type (protonated base)·(deprotonated base). These interconversions are accompanied by a significant rebuilding of the base mispairs with Watson-Crick architecture into the mismatches wobbled toward both DNA minor and major grooves and *vice versa*.

**Figure 2.** Energetic profiles of the mutagenic tautomerization *via* the wobbling of the (**a**) A·T(WC) and (**b**) G·C(WC) DNA base pairs to the H-bonded mismatches containing rare tautomers (B3LYP/6–311++G(d,p) level of theory, ε = 1) [68].

Notably, each of the discussed tautomerizations is realized precisely through four different topological and energetic pathways. The number of mobile protons (two in each pair) and number of wobbling directions of WC base pairs (two by the number of the grooves in DNA minor and major) determines the number of tautomerization pathways. Characteristically, in

**Figure 3.** Energetic profiles and stationary structures on the potential energy hypersurface of the biologically important transformations *via* the PT, accompanied by the shifting of the bases relative to each other within a base pair into the sides of the DNA minor or major grooves, leading to the occurrence of the spontaneous transitions and transversions—

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37

incorporation and replication errors (B3LYP/6–311++G(d,p) level theory, ε = 1).

Renaissance of the Tautomeric Hypothesis of the Spontaneous Point Mutations in DNA: New Ideas… http://dx.doi.org/10.5772/intechopen.77366 37

**3.3. Novel mechanisms of the wobble (w)**↔**Watson-Crick (WC) tautomeric interconversions in the canonical and incorrect DNA base pairs as a key to** 

of the geometry of the base pair—from Watson-Crick to wobble and *vice versa*.

arising with frequencies ~10−11–10−9 errors *per* replicated nucleotide.

For the first time, a novel theoretical approach to elucidate microstructural mechanisms of incorporation and replication point errors arising at the DNA replication was proposed. We show for the first time that pairs of nucleotide bases with Watson-Crick architecture of the H-bonding—classical, long, short, in which one or both bases are in the main or rare tautomeric forms, are in a slow tautomeric equilibrium with the corresponding wobble base pairs in comparison with the time, in which high-fidelity DNA polymerase spends on the incorporation of one nucleotide into the DNA double helix (~8.3 × 10−4 s [67]). In fact, a novel pathway of the chemical reaction was discovered—tautomerization with significant changes

We have discovered novel structural hypostases of the classical A·T(WC) and G·C(WC) Watson-Crick DNA base pairs arising due to their ability to switch into the wobble А\*·Т↑(w), А·Т\*O2↑(w), А·Т\*↓(w) and G·C\*↑(w), G\*·C↓(w), G·C\*↓(w), G\*·C↑(w) H-bonded mismatches containing rare tautomers (**Figure 2**) [68]. Estimated populations of the tautomerized states of the А·Т(WC) (6.1 × 10−9–1.5 × 10−7) and G·C(WC) (4.2 × 10−11–1.4 × 10−9) base pairs in the continuum with ε = 4 correspond to the interface of the protein-nucleic acid interactions. This evidences their involvement in nucleation of spontaneous point replication errors in DNA

We found for the first time the intrinsic ability of the purine·pyrimidine (A·C [69, 70] and G·T [69, 71]), purine·purine (A·A [72], G·G [72] and A·G [73]) and pyrimidine·pyrimidine (С·С [74], Т·Т [74] and С·T [73]) DNA base mispairs to perform wobble↔Watson-Crick tautomeric transitions *via* the sequential intrapair DPT and subsequent shifting of the bases relative to each other (**Figure 3**, **Table 2**). These nondissociative tautomerizations *via* the sequential PT are controlled by the highly stable (ΔEint > 100 kcal·mol−1), highly polar and zwitterionic transition states of the type (protonated base)·(deprotonated base). These interconversions are accompanied by a significant rebuilding of the base mispairs with Watson-Crick architecture into the mismatches wobbled toward both DNA minor and major grooves and *vice versa*.

**Figure 2.** Energetic profiles of the mutagenic tautomerization *via* the wobbling of the (**a**) A·T(WC) and (**b**) G·C(WC) DNA base pairs to the H-bonded mismatches containing rare tautomers (B3LYP/6–311++G(d,p) level of theory, ε = 1) [68].

**understand origins of spontaneous transitions and transversions**

36 Mitochondrial DNA - New Insights

**Figure 3.** Energetic profiles and stationary structures on the potential energy hypersurface of the biologically important transformations *via* the PT, accompanied by the shifting of the bases relative to each other within a base pair into the sides of the DNA minor or major grooves, leading to the occurrence of the spontaneous transitions and transversions incorporation and replication errors (B3LYP/6–311++G(d,p) level theory, ε = 1).

Notably, each of the discussed tautomerizations is realized precisely through four different topological and energetic pathways. The number of mobile protons (two in each pair) and number of wobbling directions of WC base pairs (two by the number of the grooves in DNA minor and major) determines the number of tautomerization pathways. Characteristically, in


Mutagenic pressure of the analogues of DNA bases could be explained within the framework of the proposed model of the w↔WC mutagenic tautomerization. In particular, mutagenic action of the analogue of C-6H,8H-3,4-dihydropyrimido[4,5-c] [1, 2]oxazin-7-one [11, 12] increases the population of the G·P\*↑ (4.5 × 10−3) and G·P\*↓ (1.4 × 10−4) base mispairs by its participation in comparison with the analogical values for the canonical C DNA base. Mutagenic activity of the halogen derivatives of the uracil base is associated with the decreas-

induced mutations (35 [71]), which is in good accordance with experimental data (from 20

All long purine·purine DNA base mispairs can acquire enzymatically competent conformations—А\*·Аsyn(TF), G·Аsyn, A\*·G\*syn and G·G\*syn—through the A\*·A(WC)↔A\*·Asyn(TF), G·A(WC)↔G·Asyn, A\*·G\*(WC)↔A\*·G\*syn and G·G\*(WC)↔G·G\*syn conformational transitions [77], eventually guaranteeing their chemical incorporation into the newly synthesized structure of the DNA double helix (TF-Topal-Fresco nucleobase pair [10]; *syn*-*syn*-orientation of the base according the sugar-phosphate moiety) (**Figure 4**). Characteristic time of these nondissociative conformational transitions (~10−7 s) is much less than the period of time the high-fidelity DNA polymerase spends on incorporating one nucleotide into the DNA double helix (~8.3 × 10−4 s [67]). So-called long A\*·A(WC), G·A(WC), A\*·G\*(WC) and G·G\*(WC) DNA base mispairs have been outlined as "node stations" on the way of the formation of the enzymatically competent conformations arising in the recognition pocket of the high-fidelity DNA

**3.5. Physico-chemical scenarios of the origin of the replication and incorporation** 

for three other cases, when G\*, T\* and C\* belong to the template strand of DNA.

In the framework of such qualitatively new model conceptions, we were able to shed light on the microstructural mechanisms of the occurrence of point mutations—replication and

Thus, the spontaneous mutagenic tautomerization of the Watson-Crick pairs of nucleotide bases into the wobble base mispairs, which includes the A\*, T\*, G\* and C\* mutagenic tautomers, has been established to be the source of the generation of the mutagenic tautomers of the DNA bases arising at the separation of DNA strands. At this juncture, *replication errors* would arise in the following way (as an example, we would consider the case, when A\* belongs to the template strand of DNA): A\* + C → A\*·C → A·C\*, A\* + A → A\*·A → A\*·Asyn, A\* + G → A \*·G → A·G → A\*·G\* → A\*·G\*syn. Similar schemes of structural transformations, which occur directly in the recognition pocket of the high-fidelity DNA polymerase, would take place also

*Incorporation errors* would occur according to the following scenario: in the recognition pocket of the high-fidelity DNA polymerase, it would form the appropriate wobble base mispair

XU\*(WC) mispairs with Watson-Crick geometry, thus inducing higher frequency

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XU(w) (X = H, CH<sup>3</sup>

, Br, Cl, F) mispairs

39

BrU-calculated frequency of the

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ing of the transformation barriers of the wobble G·5

of the transitions. The maximal effect is observed for the <sup>5</sup>

polymerase at its transition from the open to closed state.

**3.4.** *Anti*↔*syn* **conformational transitions of the long purine-purine DNA** 

into the G·5

[75] to 29 [76]).

**mismatches**

**point errors in DNA**

incorporation point errors.

Note: for designations see **Table 1**.

a The time necessary to reach 99.9% of the equilibrium concentration between the reactant and the product of the tautomerization reaction, s.

b Populations of the wobble mispairs containing mutagenic tautomers.

**Table 2.** Energetic and kinetic characteristics of the tautomeric transformations of the classical Watson-Crick or wobble DNA base pairs, which are involved into the processes of the spontaneous point mutagenesis, *via* the DPT accompanied by the substantial changes of their geometry in the continuum with ε = 1.

each case mostly, one pathway is most probable at the origin of the spontaneous point mutations (**Figures 2** and **3**, **Table 2**).

Obtained results are crucial for understanding the microstructural mechanisms of spontaneous transitions and transversions, since they allow us to explain how incorrect purine·pyrimidine, purine·purine and pyrimidine·pyrimidine wobble pairs adapt to the enzymatically competent sizes in the recognition pocket of the high-fidelity DNA polymerase. In particular, established A·C(w) →A·C\*(WC) [70] and G·T(w) → G\*·T(WC) [71] transformations *via* the sequential PT allow us to interpret the X-ray [14, 15] and molecular dynamics simulations data [19] according the acquisition by the wobble A·C(w)/G·T(w) mispairs of the Watson-Crick geometry by their transformation to the A·C\*(WC)/G\*·T(WC) Watson-Crick-like base mispairs by the participation of the C\* and G\* mutagenic tautomers in the recognition pocket of the high-fidelity DNA polymerase. Moreover, we theoretically predicted the G·T(w) → G\*·T(WC) transformation for the wobble G·T(w) base mispair, which was confirmed by an NMR experiment of a DNA duplex [16–18].

Mutagenic pressure of the analogues of DNA bases could be explained within the framework of the proposed model of the w↔WC mutagenic tautomerization. In particular, mutagenic action of the analogue of C-6H,8H-3,4-dihydropyrimido[4,5-c] [1, 2]oxazin-7-one [11, 12] increases the population of the G·P\*↑ (4.5 × 10−3) and G·P\*↓ (1.4 × 10−4) base mispairs by its participation in comparison with the analogical values for the canonical C DNA base. Mutagenic activity of the halogen derivatives of the uracil base is associated with the decreasing of the transformation barriers of the wobble G·5 XU(w) (X = H, CH<sup>3</sup> , Br, Cl, F) mispairs into the G·5 XU\*(WC) mispairs with Watson-Crick geometry, thus inducing higher frequency of the transitions. The maximal effect is observed for the <sup>5</sup> BrU-calculated frequency of the induced mutations (35 [71]), which is in good accordance with experimental data (from 20 [75] to 29 [76]).
