Результаты исследований: Научные публикации в периодических изданиях › статья › Рецензирование
Fluorescently labeled human apurinic/apyrimidinic endonuclease APE1 reveals effects of DNA polymerase β on the APE1-DNA interaction. / Bakman, Artemiy S; Kuznetsova, Aleksandra A; Yanshole, Lyudmila V и др.
в: DNA Repair, Том 123, 103450, 03.2023.Результаты исследований: Научные публикации в периодических изданиях › статья › Рецензирование
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TY - JOUR
T1 - Fluorescently labeled human apurinic/apyrimidinic endonuclease APE1 reveals effects of DNA polymerase β on the APE1-DNA interaction
AU - Bakman, Artemiy S
AU - Kuznetsova, Aleksandra A
AU - Yanshole, Lyudmila V
AU - Ishchenko, Alexander A
AU - Saparbaev, Murat
AU - Fedorova, Olga S
AU - Kuznetsov, Nikita A
N1 - Funding: This work was supported partially by the Russian Federal Ministry of Science and Higher Education (project No. 121031300041-4) to O.S.F. and N.A.K., by Electricit´e de France (RB 2020-02 and RB 2021-05) to M.S., a grant from the Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan (No. AP08856811) to M.S., and Fondation ARC (PJA-2021060003796) to A.A.I. The part of the work involving the stopped-flow analysis was specifically funded by Russian Science Foundation grant No. 21-64-00017. Copyright © 2023 Elsevier B.V. All rights reserved.
PY - 2023/3
Y1 - 2023/3
N2 - 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.
AB - 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.
KW - DNA Damage
KW - DNA Polymerase beta/metabolism
KW - DNA Repair
KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism
KW - DNA/metabolism
KW - Endonucleases/metabolism
KW - Humans
UR - https://www.scopus.com/record/display.uri?eid=2-s2.0-85149153961&origin=inward&txGid=8f2336a209b50fd543fd4de6b53170c6
UR - https://www.mendeley.com/catalogue/fe5fa699-ea8c-3ddc-9fde-d5aded2db088/
U2 - 10.1016/j.dnarep.2023.103450
DO - 10.1016/j.dnarep.2023.103450
M3 - Article
C2 - 36689867
VL - 123
JO - DNA Repair
JF - DNA Repair
SN - 1568-7864
M1 - 103450
ER -
ID: 43623912