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Global DNA dynamics of 8-oxoguanine repair by human OGG1 revealed by stopped-flow kinetics and molecular dynamic simulation

Abstract

Toxic action of different endogenous and exogenous agents leads to damages in the genomic DNA. 8-Oxoguanine is one of the mostly often generated and highly mutagenic oxidative damage in DNA. Normally in human cells it is promptly removed by 8-oxoguanine-DNA-glycosylase hOGG1, the key DNA-repair enzyme. An association between accumulation of oxidized guanine and increased risk of harmful processes in organism is already found. At the same time the detailed mechanism of damaged base recognition and removal is still full of questions. To clarify the role of the active site amino acids in the damage base coordination and to reveal the elementary steps in overall enzymatic process we have investigated hOGG1 mutant forms with substituted amino acid residues in the enzyme base-binding pocket. Replacing the functional groups of the enzyme active site allowed us to affect the rates of individual steps of the enzymatic reaction. To gain further insight into the mechanism of hOGG1 catalysis a detailed pre-steady state kinetic study of this enzymatic process was carried out using stopped-flow approach. The changes in the DNA structure after the mixing with enzymes were followed by recording of the FRET signal using Cy3/Cy5 labels in DNA substrates during time range from milliseconds to hundred seconds. DNA duplexes containing non-damaged DNA, 8-oxoG, AP-site or its unreactive synthetic analogue were used as DNA-substrates. The kinetic parameters of DNA binding and damage processing were obtained for mutant forms as well as for WT hOGG1. The analyses of fluorescence traces provided information about the DNA dynamics during damage recognition and removal. Kinetic study for mutant forms revealed that all introduced substitutions reduce the efficiency hOGG1 activity; however they play pivotal roles at certain elementary stages identified during the study. Taken together, our results gave the opportunity to restore the role of substituted amino acid and main “damaged base – amino acid” contacts, that provide an important link in the understanding the mechanism of the DNA repair process catalyzed by hOGG1.

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Publication details

The article was received on 07 Jun 2017, accepted on 10 Jul 2017 and first published on 14 Jul 2017


Article type: Paper
DOI: 10.1039/C7MB00343A
Citation: Mol. BioSyst., 2017, Accepted Manuscript
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    Global DNA dynamics of 8-oxoguanine repair by human OGG1 revealed by stopped-flow kinetics and molecular dynamic simulation

    M. V. Lukina, V. V. Koval, A. A. Lomzov, D. O. Zharkov and O. S. Fedorova, Mol. BioSyst., 2017, Accepted Manuscript , DOI: 10.1039/C7MB00343A

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