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Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae

In yeast Saccharomyces cerevisiae cells, apurinic/apyrimidinic (AP) sites are primarily repaired by base excision repair. Base excision repair is initiated by one of two AP endonucleases: Apn1 or Apn2. AP endonucleases catalyze hydrolytic cleavage of the phosphodiester backbone on the 5′ side of an...

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Autores principales: Kuznetsova, Aleksandra A., Gavrilova, Anastasia A., Ishchenko, Alexander A., Saparbaev, Murat, Fedorova, Olga S., Kuznetsov, Nikita A.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9697762/
https://www.ncbi.nlm.nih.gov/pubmed/36430884
http://dx.doi.org/10.3390/ijms232214404
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author Kuznetsova, Aleksandra A.
Gavrilova, Anastasia A.
Ishchenko, Alexander A.
Saparbaev, Murat
Fedorova, Olga S.
Kuznetsov, Nikita A.
author_facet Kuznetsova, Aleksandra A.
Gavrilova, Anastasia A.
Ishchenko, Alexander A.
Saparbaev, Murat
Fedorova, Olga S.
Kuznetsov, Nikita A.
author_sort Kuznetsova, Aleksandra A.
collection PubMed
description In yeast Saccharomyces cerevisiae cells, apurinic/apyrimidinic (AP) sites are primarily repaired by base excision repair. Base excision repair is initiated by one of two AP endonucleases: Apn1 or Apn2. AP endonucleases catalyze hydrolytic cleavage of the phosphodiester backbone on the 5′ side of an AP site, thereby forming a single–strand break containing 3′–OH and 5′–dRP ends. In addition, Apn2 has 3′–phosphodiesterase activity (removing 3′–blocking groups) and 3′ → 5′ exonuclease activity (both much stronger than its AP endonuclease activity). Nonetheless, the role of the 3′–5′–exonuclease activity of Apn2 remains unclear and presumably is involved in the repair of damage containing single–strand breaks. In this work, by separating reaction products in a polyacrylamide gel and by a stopped–flow assay, we performed a kinetic analysis of the interaction of Apn2 with various model DNA substrates containing a 5′ overhang. The results allowed us to propose a mechanism for the cleaving off of nucleotides and to determine the rate of the catalytic stage of the process. It was found that dissociation of a reaction product from the enzyme active site is not a rate–limiting step in the enzymatic reaction. We determined an influence of the nature of the 3′–terminal nucleotide that can be cleaved off on the course of the enzymatic reaction. Finally, it was found that the efficiency of the enzymatic reaction is context–specific.
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spelling pubmed-96977622022-11-26 Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae Kuznetsova, Aleksandra A. Gavrilova, Anastasia A. Ishchenko, Alexander A. Saparbaev, Murat Fedorova, Olga S. Kuznetsov, Nikita A. Int J Mol Sci Article In yeast Saccharomyces cerevisiae cells, apurinic/apyrimidinic (AP) sites are primarily repaired by base excision repair. Base excision repair is initiated by one of two AP endonucleases: Apn1 or Apn2. AP endonucleases catalyze hydrolytic cleavage of the phosphodiester backbone on the 5′ side of an AP site, thereby forming a single–strand break containing 3′–OH and 5′–dRP ends. In addition, Apn2 has 3′–phosphodiesterase activity (removing 3′–blocking groups) and 3′ → 5′ exonuclease activity (both much stronger than its AP endonuclease activity). Nonetheless, the role of the 3′–5′–exonuclease activity of Apn2 remains unclear and presumably is involved in the repair of damage containing single–strand breaks. In this work, by separating reaction products in a polyacrylamide gel and by a stopped–flow assay, we performed a kinetic analysis of the interaction of Apn2 with various model DNA substrates containing a 5′ overhang. The results allowed us to propose a mechanism for the cleaving off of nucleotides and to determine the rate of the catalytic stage of the process. It was found that dissociation of a reaction product from the enzyme active site is not a rate–limiting step in the enzymatic reaction. We determined an influence of the nature of the 3′–terminal nucleotide that can be cleaved off on the course of the enzymatic reaction. Finally, it was found that the efficiency of the enzymatic reaction is context–specific. MDPI 2022-11-19 /pmc/articles/PMC9697762/ /pubmed/36430884 http://dx.doi.org/10.3390/ijms232214404 Text en © 2022 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Kuznetsova, Aleksandra A.
Gavrilova, Anastasia A.
Ishchenko, Alexander A.
Saparbaev, Murat
Fedorova, Olga S.
Kuznetsov, Nikita A.
Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae
title Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae
title_full Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae
title_fullStr Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae
title_full_unstemmed Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae
title_short Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae
title_sort kinetic features of 3′–5′–exonuclease activity of apurinic/apyrimidinic endonuclease apn2 from saccharomyces cerevisiae
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9697762/
https://www.ncbi.nlm.nih.gov/pubmed/36430884
http://dx.doi.org/10.3390/ijms232214404
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