TY - JOUR

T1 - Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae

AU - Kuznetsova, Aleksandra A.

AU - Gavrilova, Anastasia A.

AU - Ishchenko, Alexander A.

AU - Saparbaev, Murat

AU - Fedorova, Olga S.

AU - Kuznetsov, Nikita A.

N1 - Funding Information: This work was supported by the Ministry of Science and Higher Education of the Russian Federation, agreement No. 075-15-2022-263. M.S. was supported by grant from the French National Research Agency (ANR-AAPG2022 MITOMUTREP), grants from the Electricité de France (RB 2020-02 and RB 2021-05) and grant from Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan No. AP08856811. A.A.I. was supported by grant from the Fondation ARC (PJA-2021060003796). Publisher Copyright: © 2022 by the authors.

PY - 2022/11

Y1 - 2022/11

N2 - 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.

AB - 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.

KW - 3′–5′–exonuclease activity

KW - AP endonuclease

KW - DNA repair

KW - fluorescence

KW - pre–steady–state kinetics

KW - DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism

KW - Saccharomyces cerevisiae/metabolism

KW - Endonucleases

KW - Saccharomyces cerevisiae Proteins

KW - Exonucleases

KW - Kinetics

UR - http://www.scopus.com/inward/record.url?scp=85142602565&partnerID=8YFLogxK

UR - https://www.mendeley.com/catalogue/491cb8fa-4aa8-31ed-b944-7d7ba510c6d2/

U2 - 10.3390/ijms232214404

DO - 10.3390/ijms232214404

M3 - Article

C2 - 36430884

AN - SCOPUS:85142602565

VL - 23

JO - International Journal of Molecular Sciences

JF - International Journal of Molecular Sciences

SN - 1661-6596

IS - 22

M1 - 14404

ER -