• Overview

    Fidelity in this process refers to the ability of the polymerase to avoid or to correct errors in the newly synthesized DNA strand. It has been estimated that normal cells replicate their genome with a fidelity that translates to approximately a single error per cell generation (Loeb, 1991).

  • Our Focus

    The evolution and reproduction of life depends on the accurate replication of the genome by DNA polymerases. The synthesis of new DNA molecules would be impossible without these enzymes as they increase the rate of the phosphodiester bond formation by many orders of magnitude compared to the corresponding reaction in water. Furthermore, DNA polymerases are capable of selecting the right deoxyribonucleoside triphosphate (dNTP) substrate so that Watson-Crick base pairs are preferentially formed. The efforts to elucidate the source of such remarkable fidelity [1] have been greatly aided by the elucidation of the structures of ternary complexes of several DNA polymerases with DNA and dNTP substrates. Nevertheless, in order to convert the complex structural information into quantitative structure-function correlations it is essential to use some type of computer-based analysis.

    In principle, there are several major options that can account for the difference between the rate of enzymatic incorporation of the right (R) and wrong (W) incoming nucleotides. The options include rate determining conformational change during the dNTP binding step, conformational changes between the productive and non-productive binding configurations and a rate determining chemical step. In order to discriminate between them, we have been using a wide range of computational strategies, which have been developed, refined and validated over a long time. These strategies include EVB the PD and CDFT approach for evaluating reaction surfaces for the chemical steps. We also developed the linear response approximation (LRA), and advanced its LRA/α and LIE versions, which provide powerful accelerated ways for estimating binding free energies. The LRA approach is able to dissect the calculated free energy into its individual contributions in an additive way. This feature allows us to estimate the overall fidelity and the effect of different protein residues. The above methods have allowed us to move on several fronts, but most importantly, we have made major progress in quantifying the catalytic mechanism of DNA polymerases [2] [3] [4] and in elucidating the molecular origin of the fidelity [5] [6].