title: A kinetic mechanism of repair of DNA containing α-anomeric deoxyadenosine by human apurinic/apyrimidinic endonuclease 1.
abstract: α-Anomers of 2'-deoxyadenosine (αdA) are major products of deoxyadenosine damage when DNA is γ-irradiated under anoxic conditions. Such lesions are a threat to genomic stability and are known to be processed by human apurinic/apyrimidinic endonuclease 1 (APE1). The aim of this study was to determine whether the α-anomeric structure enhances enzyme recognition. For this purpose, we analyzed the kinetic mechanism of αdA conversion by APE1 using a stopped-flow fluorescence technique. Our data reveals that the initial formation of the complex of APE1 with an αdA-containing substrate is followed by at least three conformational transitions in this complex that correspond to the induced fit leading to the formation of a catalytically competent complex. A local perturbation around the αdA lesion in the DNA duplex allows APE1 to avoid the initial conformational changes observed earlier in the case of the enzyme binding to an undamaged ligand, abasic-site-, tetrahydrofuran-, or 5,6-dihydrouridine-containing substrates. The αdA structure promotes recognition by the enzyme but dramatically impedes formation of the catalytically competent complex and hydrolysis of the 5'-phosphodiester bond. A step following the chemical reaction, possibly a release of the αdA-containing product, is rate-limiting for the overall enzymatic process, though an α-anomeric nucleotide at the 5' terminus of the DNA nick accelerates dissociation of the enzyme-product complex. Our results show that the efficiency of αdA lesion conversion by APE1 is very low. Nonetheless, αdA repair by APE1 is probably a biologically relevant process.
authors.affiliation: Siberian Branch of the Russian Academy of Sciences, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia. fedorova@niboch.nsc.ru na_timof@niboch.nsc.ru.
authors.forename: N A
authors.initials: NA
authors.lastname: Timofeyeva
authors.affiliation: Siberian Branch of the Russian Academy of Sciences, Institute of Chemical Biology and Fundamental Medicine, Novosibirsk 630090, Russia. fedorova@niboch.nsc.ru na_timof@niboch.nsc.ru.
authors.forename: O S
authors.identifier: 0000-0002-4001-7187
authors.initials: OS
authors.lastname: Fedorova
mesh_terms: D016880:Anisotropy; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000378:metabolism*; D003839:Deoxyadenosines / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity
chemical_list: D003839:Deoxyadenosines; D004247:DNA; C085108:APEX1 protein, human; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; C058118:2'-deoxyadenosine