Kinetic Features of 3′–5′–Exonuclease Activity of Apurinic/Apyrimidinic Endonuclease Apn2 from Saccharomyces cerevisiae
Résumé
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.
Mots clés
Kuznetsova
A.A. Gavrilova
A.A. Ishchenko
A.A. Saparbaev
M. Fedorova
O.S. Kuznetsov
N.A. Kinetic Features of AP endonuclease DNA repair pre-steady-state kinetics fluorescence 3 -5 -exonuclease activity
A.A.
Gavrilova
Ishchenko
Saparbaev
M.
Fedorova
O.S.
Kuznetsov
N.A. Kinetic Features of AP endonuclease
DNA repair
pre-steady-state kinetics
fluorescence
3 -5 -exonuclease activity
Domaines
Sciences du Vivant [q-bio]
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Kuznetsova AA_Kinetic Features of 3ꞌ-5'-Exonuclease Activity of AP Endonuclease Apn2 from S. cerevisiae_IJMS2022_reprint.pdf (6.16 Mo)
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Licence : CC BY - Paternité
Licence : CC BY - Paternité