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Issue 12, 2017
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Pre-steady-state kinetic analysis of damage recognition by human single-strand selective monofunctional uracil-DNA glycosylase SMUG1

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Abstract

In all organisms, DNA glycosylases initiate base excision repair pathways resulting in removal of aberrant bases from DNA. Human SMUG1 belongs to the superfamily of uracil-DNA glycosylases catalyzing the hydrolysis of the N-glycosidic bond of uridine and uridine lesions bearing oxidized groups at C5: 5-hydroxymethyluridine (5hmU), 5-formyluridine (5fU), and 5-hydroxyuridine (5hoU). An apurinic/apyrimidinic (AP) site formed as the product of an N-glycosylase reaction is tightly bound to hSMUG1, thus inhibiting the downstream action of AP-endonuclease APE1. The steady-state kinetic parameters (kcat and KM; obtained from the literature) correspond to the enzyme turnover process limited by the release of hSMUG1 from the complex with the AP-site. In the present study, our objective was to carry out a stopped-flow fluorescence analysis of the interaction of hSMUG1 with a DNA substrate containing a dU:dG base pair to follow the pre-steady-state kinetics of conformational changes in both molecules. A comparison of kinetic data obtained by means of Trp and 2-aminopurine fluorescence and Förster resonance energy transfer (FRET) detection allowed us to elucidate the stages of specific and nonspecific DNA binding, to propose the mechanism of damaged base recognition by hSMUG1, and to determine the true rate of the catalytic step. Our results shed light on the kinetic mechanism underlying the initiation of base excision repair by hSMUG1 using the “wedge” strategy for DNA lesion search.

Graphical abstract: Pre-steady-state kinetic analysis of damage recognition by human single-strand selective monofunctional uracil-DNA glycosylase SMUG1

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Publication details

The article was received on 25 Jul 2017, accepted on 11 Oct 2017 and first published on 11 Oct 2017


Article type: Paper
DOI: 10.1039/C7MB00457E
Citation: Mol. BioSyst., 2017,13, 2638-2649
  • Open access: Creative Commons BY license
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    Pre-steady-state kinetic analysis of damage recognition by human single-strand selective monofunctional uracil-DNA glycosylase SMUG1

    A. A. Kuznetsova, D. A. Iakovlev, I. V. Misovets, A. A. Ishchenko, M. K. Saparbaev, N. A. Kuznetsov and O. S. Fedorova, Mol. BioSyst., 2017, 13, 2638
    DOI: 10.1039/C7MB00457E

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