Common kinetic mechanism of abasic site recognition by structurally different apurinic/apyrimidinic endonucleases

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

Research output: Contribution to journalArticlepeer-review

Abstract

Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site–containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes’ intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.

Original languageEnglish
Article number8874
JournalInternational Journal of Molecular Sciences
Volume22
Issue number16
DOIs
Publication statusPublished - 2 Aug 2021

Keywords

  • Abasic site
  • Apurinic/apyrimidinic endonuclease
  • Conformational dynamics
  • Damaged DNA
  • DNA repair
  • Stopped-flow enzyme kinetics
  • Catalytic Domain
  • Escherichia coli
  • Humans
  • Substrate Specificity
  • Molecular Dynamics Simulation
  • Nucleotides/chemistry
  • DNA Repair
  • Protein Conformation
  • DNA Damage
  • Kinetics
  • Nucleic Acid Conformation
  • Binding Sites
  • DNA Cleavage
  • DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry

OECD FOS+WOS

  • 1.02 COMPUTER AND INFORMATION SCIENCES
  • 2.04 CHEMICAL ENGINEERING
  • 1.04 CHEMICAL SCIENCES
  • 1.06 BIOLOGICAL SCIENCES

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