title: Cloning and characterization of a wheat homologue of apurinic/apyrimidinic endonuclease Ape1L.
abstract: BACKGROUND
  Apurinic/apyrimidinic (AP) endonucleases are key DNA repair enzymes involved in the base excision repair (BER) pathway. In BER, an AP endonuclease cleaves DNA at AP sites and 3'-blocking moieties generated by DNA glycosylases and/or oxidative damage. A Triticum aestivum cDNA encoding for a putative homologue of ExoIII family AP endonucleases which includes E. coli Xth, human APE1 and Arabidopsis thaliana AtApe1L has been isolated and its protein product purified and characterized.
  
  
  RESULTS
  We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 3'-repair phosphodiesterase, 3'-phosphatase and 3' → 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg(2+) and Ca(2+) (5-10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn(2+), Co(2+) and Fe(2+) cations (0.1-1.0 mM) strongly stimulated all its DNA repair activities. Optimization of the reaction conditions revealed that the wheat enzyme requires low divalent cation concentration (0.1 mM), mildly acidic pH (6-7), low ionic strength (20 mM KCl) and has a temperature optimum at around 20 °C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 3'-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (kcat/KM = 630 and 485 μM(-1) · min(-1), respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1.
  
  
  CONCLUSIONS
  Taken together, these data establish the DNA substrate specificity of the wheat AP endonuclease and suggest its possible role in the repair of DNA damage generated by endogenous and environmental factors.
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Biology, al-Farabi Kazakh National University, Almaty, Kazakhstan.
authors.forename: Botagoz
authors.initials: B
authors.lastname: Joldybayeva
authors.affiliation: Groupe «Réparation de l'ADN», CNRS UMR8200, Université Paris-Sud, Institut Gustave Roussy, Villejuif, France; Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland.
authors.forename: Paulina
authors.initials: P
authors.lastname: Prorok
authors.affiliation: Groupe «Réparation de l'ADN», CNRS UMR8200, Université Paris-Sud, Institut Gustave Roussy, Villejuif, France; SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia; Novosibirsk State University, Novosibirsk, Russia.
authors.forename: Inga R
authors.initials: IR
authors.lastname: Grin
authors.affiliation: SB RAS Institute of Chemical Biology and Fundamental Medicine, Novosibirsk, Russia; Novosibirsk State University, Novosibirsk, Russia.
authors.forename: Dmitry O
authors.initials: DO
authors.lastname: Zharkov
authors.affiliation: Groupe «Réparation de l'ADN», CNRS UMR8200, Université Paris-Sud, Institut Gustave Roussy, Villejuif, France.
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Ishenko
authors.affiliation: Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warsaw, Poland; Institute of Genetics and Biotechnology, University of Warsaw, Warsaw, Poland.
authors.forename: Barbara
authors.initials: B
authors.lastname: Tudek
authors.affiliation: Department of Molecular Biology and Genetics, Faculty of Biology, al-Farabi Kazakh National University, Almaty, Kazakhstan.
authors.forename: Amangeldy K
authors.initials: AK
authors.lastname: Bissenbaev
authors.affiliation: Groupe «Réparation de l'ADN», CNRS UMR8200, Université Paris-Sud, Institut Gustave Roussy, Villejuif, France.
authors.forename: Murat
authors.initials: M
authors.lastname: Saparbaev
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D003001:Cloning, Molecular; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010727:Phosphoric Diester Hydrolases / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011487:Protein Conformation; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity; D014908:Triticum / Q000201:enzymology* / Q000235:genetics*
chemical_list: D004247:DNA; D010744:Phosphoric Monoester Hydrolases; D010727:Phosphoric Diester Hydrolases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase