title: Kinetic mechanism of human apurinic/apyrimidinic endonuclease action in nucleotide incision repair.
abstract: Human major apurinic/apyrimidinic endonuclease (APE1) is a multifunctional enzyme that plays a central role in DNA repair through the base excision repair (BER) pathway. Besides BER, APE1 is involved in an alternative nucleotide incision repair (NIR) pathway that bypasses glycosylases. We have analyzed the conformational dynamics and the kinetic mechanism of APE1 action in the NIR pathway. For this purpose we recorded changes in the intensity of fluorescence of 2-aminopurine located in two different positions in a substrate containing dihydrouridine (DHU) during the interaction of the substrate with the enzyme. The enzyme was found to change its conformation within the complex with substrate and also within the complex with the reaction product, and the release of the enzyme from the complex with the product seemed to be the limiting stage of the enzymatic process. The rate constants of the catalytic cleavage of DHU-containing substrates by APE1 were comparable with the appropriate rate constants for substrates containing apurinic/apyrimidinic site or tetrahydrofuran residue, which suggests that NIR is a biologically important process.
authors.affiliation: Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia.
authors.forename: N A
authors.initials: NA
authors.lastname: Timofeyeva
authors.forename: V V
authors.initials: VV
authors.lastname: Koval
authors.forename: A A
authors.initials: AA
authors.lastname: Ishchenko
authors.forename: M K
authors.initials: MK
authors.lastname: Saparbaev
authors.forename: O S
authors.initials: OS
authors.lastname: Fedorova
mesh_terms: D015075:2-Aminopurine / Q000737:chemistry; D004249:DNA Damage; D017931:DNA Primers / Q000737:chemistry; D004260:DNA Repair / Q000502:physiology*; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000378:metabolism*; D005470:Fluorometry; D006801:Humans; D007700:Kinetics; D008968:Molecular Conformation; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D015075:2-Aminopurine; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase