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. 2020 Jun 19;5(25):15317-15324.
doi: 10.1021/acsomega.0c01345. eCollection 2020 Jun 30.

Preferential DNA Polymerase β Reverse Reaction with Imidodiphosphate

Affiliations

Preferential DNA Polymerase β Reverse Reaction with Imidodiphosphate

Lalith Perera et al. ACS Omega. .

Abstract

DNA replication and repair reactions involve the addition of a deoxynucleoside monophosphate onto a growing DNA strand with the loss of pyrophosphate. This chemical reaction is also reversible; the addition of pyrophosphate generates a deoxynucleoside triphosphate, thereby shortening the DNA by one nucleotide. The forward DNA synthesis and reverse pyrophosphorolysis reactions strictly require the presence of divalent metals, usually magnesium, at the reactive center as cofactors. The overall equilibrium enzymatic reaction strongly favors DNA synthesis over pyrophosphorolysis with natural substrates. The DNA polymerase β chemical reaction has been structurally and kinetically characterized, employing natural and chemically modified substrates. Substituting an imido-moiety (NH) for the bridging oxygen between Pβ and Pγ of dGTP dramatically decreased the overall enzymatic activity and resulted in a chemical equilibrium that strongly favors the reverse reaction (i.e., K ≪ 1). Using QM/MM calculations in conjunction with the utilization of parameters such as quantum mechanically derived atomic charges, we have examined the chemical foundation for the altered equilibrium with this central biological reaction. The calculations indicate that the rapid reverse reaction is likely due, in part, to the increased nucleophilicity of the reactive oxygen on the tautomeric form of imidodiphosphate.

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Conflict of interest statement

The authors declare no competing financial interest.

Figures

Figure 1
Figure 1
Energy profiles for the pol β-assisted pyrophosporolysis with various P–X–P substrates. X = O (black), X = NH (red), and the tautomeric form with P–N=P (green).
Figure 2
Figure 2
(a) Minimalist’s conformation of the reactive atoms as the largest system in the cluster calculation. All atoms with reported charges are marked. The hydrogens are in lighter gray, carbons in gray, oxygens in red, nitrogens in blue, and the two metal ions in light green. The pyrophosphate and the three residues of pol β involved in the catalytic scaffold are also marked. Water oxygens are marked with “W”. (b) Only two components in the quantum system undergo a chemical change: the phosphate group (from the terminal nucleotide) capped with two methyl groups and the pyrophosphate substrate that reacts with it during pyrophosphorolysis. (c) Tautomeric forms of the imidodiphosphate substrate used in the present study.

References

    1. Bebenek K.; Kunkel T. A.. Functions of DNA polymerases. In DNA Repair and Replication; Advances in Protein Chemistry; Yang W., Ed.; Academic Press: San Diago, 2004; pp 137-165. - PubMed
    1. Deutscher M. P.; Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. 28. Phyrophosphate exchange and phyrophosphorolysis reactions of deoxyribonucleic acid polymerase. J. Biol. Chem. 1969, 244, 3019–3028. - PubMed
    1. Torgovnick A.; Schumacher B. DNA repair mechanisms in cancer development and therapy. Front. Genet. 2015, 6, 157.10.3389/fgene.2015.00157. - DOI - PMC - PubMed
    1. Bessman M. J.; Lehman I. R.; Simms E. S.; Kornberg A. Enzymatic synthesis of deoxyribonucleic acid. II. General properties of the reaction. J. Biol. Chem. 1958, 233, 171–177. - PubMed
    1. Beese L. S.; Steitz T. A. Structural Basis for the 3’-5’ Exonuclease Activity of Escherichia-Coli DNA-Polymerase-I - a 2 Metal-Ion Mechanism. EMBO J. 1991, 10, 25–33. 10.1002/j.1460-2075.1991.tb07917.x. - DOI - PMC - PubMed