The Kinetic Mechanism of 3′-5′ Exonucleolytic Activity of AP Endonuclease Nfo from E. coli

Svetlana I. Senchurova, Aleksandra A. Kuznetsova, Alexander A. Ishchenko, Murat Saparbaev, Olga S. Fedorova, Nikita A. Kuznetsov

Результат исследования: Научные публикации в периодических изданияхстатьярецензирование

Аннотация

Escherichia coli apurinic/apyrimidinic (AP) endonuclease Nfo is one of the key participants in DNA repair. The principal biological role of this enzyme is the recognition and hydrolysis of AP sites, which arise in DNA either as a result of the spontaneous hydrolysis of an N-glycosidic bond with intact nitrogenous bases or under the action of DNA glycosylases, which eliminate various damaged bases during base excision repair. Nfo also removes 3′-terminal blocking groups resulting from AP lyase activity of DNA glycosylases. Additionally, Nfo can hydrolyze the phosphodiester linkage on the 5′ side of some damaged nucleotides on the nucleotide incision repair pathway. The function of 3′-5′-exonuclease activity of Nfo remains unclear and probably consists of participation (together with the nucleotide incision repair activity) in the repair of cluster lesions. In this work, using polyacrylamide gel electrophoresis and the stopped-flow method, we analyzed the kinetics of the interaction of Nfo with various model DNA substrates containing a 5′ single-stranded region. These data helped to describe the mechanism of nucleotide cleavage and to determine the rates of the corresponding stages. It was revealed that the rate-limiting stage of the enzymatic process is a dissociation of the reaction product from the enzyme active site. The stability of the terminal pair of nucleotides in the substrate did not affect the enzymatic-reaction rate. Finally, it was found that 2′-deoxynucleoside monophosphates can effectively inhibit the 3′-5′-exonuclease activity of Nfo.

Язык оригиналаанглийский
Номер статьи2998
ЖурналCells
Том11
Номер выпуска19
DOI
СостояниеОпубликовано - окт. 2022

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