**2. Computational methods**

**1. Introduction**

32 Mitochondrial DNA - New Insights

of living cells.

High-fidelity DNA replication is a central issue in molecular biology [1]. During DNA replication, spontaneous point mutations [2–4] arise with frequencies 10−9 ÷ 10−11 [5–8] in functioning

Nowadays, it is reliably known that the root cause of the origin of the spontaneous point mutations is the formation in the very tight, slightly deformable base pair recognition pocket of the high-fidelity DNA polymerase in its close state of the "wrong" DNA base pairs (i.e., mismatches) able to acquire in the process of thermal fluctuations the conformation of the correct Watson-Crick DNA base pair (i.e., enzymatically competent conformation), which guarantees their incorporation into the chemical structure of the synthesized DNA double helix [4].

In the literature, two approaches are currently presented, according to physico-chemical principles of the occurrence of the mispairs leading to spontaneous point mutations in DNA. One of them is the "tautomeric hypothesis" suggested by J. Watson and F. Crick [9], which consists in the spontaneous tautomeric transition of the DNA bases from canonical to mutagenic tautomeric forms leading to the formation of the adenine·cytosine (A·C\*)/A\*·C and guanine·thymine (G\*·T)/G·T\* (here and below, mutagenic tautomers are marked with asterisk) Watson-Cricklike mispairs with correct enzymatically competent conformation [10] containing mutagenic tautomers [11–13]. Despite great advances in experimental, in particular X-ray analysis [14, 15], NMR, in particular relaxation dispersion measurements [11–13, 16–18], and theoretical [19–21] investigations, there is no unique approach to the physico-chemical mechanisms enabling DNA bases in the canonical tautomeric form to acquire rare or mutagenic tautomeric form before the dissociation of the Watson-Crick nucleobase pairs into the monomers by the replication machinery in order to produce mispairs resulting in further misincorporations and as a result the spontaneous point mutations at the DNA replication. It is generally accepted in the literature that mutagenic tautomers of the DNA bases can arise *via* the double proton transfer (DPT) along intermolecular H-bonds in the Watson-Crick [22–25] and wobble [26] base pairs, and also in the protein-DNA complexes [27]. However, some authors also consider as the source of the

origin of the spontaneous transitions the formation of the ionized DNA base pairs [28].

polymerase remain unclear [30, 31].

plinary challenge with fundamental and applied consequences.

On contrary, according to second approach, other researchers believe that spontaneous point mutations arise due to the formation of the incorrect base pairs involving only DNA bases in the main, canonical tautomeric form—so-called wobble or shifted A·C and G·T base pairs [29, 30]. However, the mechanisms of their adaptation to the enzymatically competent sizes in the very tight, slightly deformable base pair recognition pocket of the high-fidelity DNA

The common feature of these approaches is the absence of the general physico-chemical theory according the nature of these mispairs causing spontaneous point mutations, and the emergence of each of them is considered as a unique phenomenon. In the literature, there are no attempts or ideas aimed at combining these approaches into a unique, internally noncontradictory conception. Nevertheless, creation of such a microstructural theory is an interdisciAll geometric, energetic and vibrational calculations of the considered base mispairs and transition states (TSs) of their conversion have been performed by Gaussian'09 package [42] using B3LYP [43, 44] and MP2 [45] levels of quantum-mechanical (QM) theory combined with a wide variety of basis sets followed by the intrinsic reaction coordinate (IRC) calculations in the forward and reverse directions from each ТS using Hessian-based predictor-corrector integration algorithm [46] in vacuum and in the continuum with ε = 4, which is characteristic for the active center of the DNA polymerase [47, 48]. Bader's quantum theory of Atoms in Molecules (QTAIM) was applied to analyze the electron density distribution [49]. Physico-chemical parameters have been estimated by the known formulas of physico-chemical kinetics [50].
