Common Kinetic Mechanism of Abasic Site Recognition by Structurally Different Apurinic/Apyrimidinic Endonucleases

Alexandra A Kuznetsova (1) , Svetlana I Senchurova (1) , Alexander Ishchenko (2) , Murat A Saparbaev (2) , Olga S Fedorova (1) , Nikita A Kuznetsov (1)

1 ICBFM SB RAS - Institute of Chemical Biology and Fundamental Medicine [Novosibirsk, Russia]
2 IGC - Intégrité du génome et cancers

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

Mots clés: stopped-flow enzyme kinetics; conformational dynamics; damaged DNA; abasic site; apurinic/apyrimidinic endonuclease; DNA repair; stopped-flow enzyme kinetics

Domaines: Biologie structurale [q-bio.BM]

Alexandra A Kuznetsova, Svetlana I Senchurova, Alexander Ishchenko, Murat A Saparbaev, Olga S Fedorova, et al.. Common Kinetic Mechanism of Abasic Site Recognition by Structurally Different Apurinic/Apyrimidinic Endonucleases. International Journal of Molecular Sciences, 2021, 22 (16), pp.8874. ⟨10.3390/ijms22168874⟩. ⟨hal-03432659⟩