Preprint Article Version 1 Preserved in Portico This version is not peer-reviewed

Non-Covalent Interactions between dUTP C5-Substituents and DNA Polymerase Result in a Decrease in PCR Efficiency

Olga A. Zasedateleva
* ORCID logo ,
Sergey A. Surzhikov
,
Viktoriya E. Kuznetsova
,
Valeriy E. Shershov
,
Victor E. Barsky
,
Alexander S. Zasedatelev
,
Alexander V. Chudinov
Version 1 : Received: 31 July 2023 / Approved: 1 August 2023 / Online: 2 August 2023 (08:21:50 CEST)

A peer-reviewed article of this Preprint also exists.

Zasedateleva, O.A.; Surzhikov, S.A.; Kuznetsova, V.E.; Shershov, V.E.; Barsky, V.E.; Zasedatelev, A.S.; Chudinov, A.V. Non-Covalent Interactions between dUTP C5-Substituents and DNA Polymerase Decrease PCR Efficiency. Int. J. Mol. Sci. 2023, 24, 13643. Zasedateleva, O.A.; Surzhikov, S.A.; Kuznetsova, V.E.; Shershov, V.E.; Barsky, V.E.; Zasedatelev, A.S.; Chudinov, A.V. Non-Covalent Interactions between dUTP C5-Substituents and DNA Polymerase Decrease PCR Efficiency. Int. J. Mol. Sci. 2023, 24, 13643.

Abstract

The approach based on molecular modeling was developed to study dNTP derivatives characterized by new polymerase-specific properties. For this purpose, the relative efficiency of PCR amplification with modified dUTPs was studied using Taq, Tth, Pfu, Vent, Deep Vent, Vent (exo-), and Deep Vent (exo-) DNA polymerases. The efficiency of PCR amplification with modified dUTPs was compared with the results of molecular modeling using the known 3D structures of KlenTaq polymerase–DNA–dNTP complexes. The dUTPs were C5-modified with bulky functional groups (the Cy5 dye analogs) or lighter aromatic groups. Comparing the experimental data and the results of molecular modeling revealed the decrease of PCR efficiency in the presence of modified dUTPs with an increase in the number of non-covalent bonds between the substituents and the DNA polymerase (about 15% decrease per one extra non-covalent bond). Generalization of the revealed patterns to all the studied polymerases of the A and B families is discussed herein. The number of non-covalent bonds between the substituents and polymerase amino acid residues is proposed to be a potentially variable parameter for regulating enzyme activity.

Keywords

C5-modified dUTPs; A and B family DNA polymerases; PCR amplification; X-ray structure; molecular modeling; non-covalent interactions

Subject

Biology and Life Sciences, Biochemistry and Molecular Biology

Copyright: This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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