Fluorescently labeled human apurinic/apyrimidinic endonuclease APE1 reveals effects of DNA polymerase β on the APE1–DNA interaction
Bakman A.S. Kuznetsova A.A. Yanshole L.V. Ishchenko A.A. Saparbaev M. Fedorova O.S. Kuznetsov N.A.
March 2023Elsevier B.V.
DNA Repair
2023#123
The base excision repair (BER) pathway involves sequential action of DNA glycosylases and apurinic/apyrimidinic (AP) endonucleases to incise damaged DNA and prepare DNA termini for incorporation of a correct nucleotide by DNA polymerases. It has been suggested that the enzymatic steps in BER include recognition of a product–enzyme complex by the next enzyme in the pathway, resulting in the “passing-the-baton” model of transfer of DNA intermediates between enzymes. To verify this model, in this work, we aimed to create a suitable experimental system. We prepared APE1 site-specifically labeled with a fluorescent reporter that is sensitive to stages of APE1–DNA binding, of formation of the catalytic complex, and of subsequent dissociation of the enzyme–product complex. Interactions of the labeled APE1 with various model DNA substrates (containing an abasic site) of varied lengths revealed that the enzyme remains mostly in complex with the DNA product. By means of the fluorescently labeled APE1 in combination with a stopped-flow fluorescence assay, it was found that Polβ stimulates both i) APE1 binding to an abasic-site–containing DNA duplex with the formation of a catalytically competent complex and ii) the dissociation of APE1 from its product. These findings confirm DNA-mediated coordination of APE1 and Polβ activities and suggest that Polβ is the key trigger of the DNA transfer between the enzymes participating in initial steps of BER.
Apurinic/apyrimidinic endonuclease , Conformational change , Damaged DNA transfer , DNA repair , DNA-protein interaction , Fluorescence , Pre-steady-state kinetics , Protein-protein interaction
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Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of Russian Academy of Sciences (SB RAS), 8 Prospekt Akad. Lavrentyeva, Novosibirsk, 630090, Russian Federation
International Tomography Center SB RAS, 3a Institutskaya Str, Novosibirsk, 630090, Russian Federation
Group Mechanisms of DNA Repair and Carcinogenesis, Gustave Roussy Cancer Campus, CNRS UMR9019, Université Paris-Saclay, Villejuif, 94805, France
NCJSC “Al-Farabi Kazakh National University”, Almaty, Kazakhstan
Department of Natural Sciences, Novosibirsk State University, 2 Pirogova Str, Novosibirsk, 630090, Russian Federation
Institute of Chemical Biology and Fundamental Medicine
International Tomography Center SB RAS
Group Mechanisms of DNA Repair and Carcinogenesis
NCJSC “Al-Farabi Kazakh National University”
Department of Natural Sciences
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