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

• Published in: International journal of molecular sciences 2022-11, Vol.23 (22), p.14404
• Main Authors: Kuznetsova, Aleksandra A, Gavrilova, Anastasia A, Ishchenko, Alexander A, Saparbaev, Murat, Fedorova, Olga S, Kuznetsov, Nikita A
• Format: Article
• Language: English
• Subjects: 3′–5′–exonuclease activity; Adenosine; AP endonuclease; Base excision repair; DNA damage; DNA repair; DNA-(Apurinic or Apyrimidinic Site) Lyase - metabolism; Endonuclease; Endonucleases; Enzymes; Exonuclease; Exonucleases; fluorescence; Kinetics; Life Sciences; Mammals; Nucleotides; Phosphodiesterase; Polyacrylamide; pre–steady–state kinetics; Reaction products; Saccharomyces cerevisiae; Saccharomyces cerevisiae - metabolism; Saccharomyces cerevisiae Proteins; Yeast
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms232214404

In yeast 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.