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An Assay for the Activity of Base Excision Repair Enzymes in Cellular Extracts Using Fluorescent DNA Probes

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Abstract

Damaged DNA bases are removed by the base excision repair (BER) mechanism. This enzymatic process begins with the action of one of DNA glycosylases, which recognize damaged DNA bases and remove them by hydrolyzing N-glycosidic bonds with the formation of apurinic/apyrimidinic (AP) sites. Apurinic/apyrimidinic endonuclease 1 (APE1) hydrolyzes the phosphodiester bond on the 5′-side of the AP site with generation of the single-strand DNA break. A decrease in the functional activity of BER enzymes is associated with the increased risk of cardiovascular, neurodegenerative, and oncological diseases. In this work, we developed a fluorescence method for measuring the activity of key human DNA glycosylases and AP endonuclease in cell extracts. The efficacy of fluorescent DNA probes was tested using purified enzymes; the most efficient probes were tested in the enzymatic activity assays in the extracts of A549, MCF7, HeLa, WT-7, HEK293T, and HKC8 cells. The activity of enzymes responsible for the repair of AP sites and removal of uracil and 5,6-dihydrouracil residues was higher in cancer cell lines as compared to the normal HKC8 human kidney cell line.

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Abbreviations

AAG:

alkyladenine DNA glycosylase

APE1:

human AP endonuclease 1

AP site:

apurinic/apyrimidinic site

BHQ:

black hole quencher

DHU:

5,6-dihydrouridine

εA:

1,N6-ethenoadenosine

FAM:

6-carboxyfluorescein

FRET:

Förster resonance energy transfer

F:

(2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran residue

MBD4:

methyl-CpG-binding domain 4

NEIL1:

human endonuclease VIII

NTHL1:

human endonuclease III

OGG1:

8-oxoguanine DNA glycosylase

oxoG:

8-oxoguanosine

TDG:

thymine DNA glycosylase

UDG:

uracil-DNA glycosylase

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Funding

This work was supported by the Russian Science Foundation (project no. 18-14-00135, design of DNA probes and assays in cell lines; project no. 16-14-10038, measuring relative activities of purified MBD4 and TDG); Russian Ministry of Science and Education (project no. AAAA-A17-117020210022-4 for O.S.F. and N.A.K.); Ligue National Contre le Cancer “Equipe Labellisee”, Electricité de France (RB 2017 to M.S.); French National Research Agency (ANR-18-CE44-0008); and Foundation ARC (PJA-20181208015 to A.A.I.).

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Authors and Affiliations

  1. Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, 630090, Novosibirsk, Russia

    O. A. Kladova, D. A. Iakovlev, O. S. Fedorova & N. A. Kuznetsov

  2. Groupe “Réparation de l’AND”, Equipe Labellisée par la Ligue Nationale contre le Cancer, CNRS UMR 8200, Université Paris-Sud, Université Paris-Saclay, F-94805, Villejuif, France

    R. Groisman, A. A. Ishchenko & M. K. Saparbaev

  3. Gustave Roussy, Université Paris-Saclay, F-94805, Villejuif, France

    R. Groisman, A. A. Ishchenko & M. K. Saparbaev

  4. Novosibirsk State University, 630090, Novosibirsk, Russia

    O. S. Fedorova & N. A. Kuznetsov

Authors
  1. O. A. Kladova
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  2. D. A. Iakovlev
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  3. R. Groisman
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  4. A. A. Ishchenko
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  5. M. K. Saparbaev
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  6. O. S. Fedorova
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  7. N. A. Kuznetsov
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Correspondence to O. S. Fedorova or N. A. Kuznetsov.

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Kladova, O., Iakovlev, D., Groisman, R. et al. An Assay for the Activity of Base Excision Repair Enzymes in Cellular Extracts Using Fluorescent DNA Probes. Biochemistry Moscow 85, 480–489 (2020). https://doi.org/10.1134/S0006297920040082

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