Cloning and Characterization of a Wheat Homologue of Apurinic / Apyrimidinic Endonuclease Ape 1 L

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 39-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. Methodology/Principal Findings: We report that the putative wheat AP endonuclease, referred here as TaApe1L, contains AP endonuclease, 39-repair phosphodiesterase, 39-phosphatase and 39R59 exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg and Ca (5–10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn, Co and Fe 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 20uC. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 39-blocking sugar-phosphate and 39phosphate groups with good efficiency (kcat/KM = 630 and 485 mM ? min, respectively) but possesses a very weak AP endonuclease activity as compared to the human homologue, APE1. Conclusions/Significance: 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. Citation: Joldybayeva B, Prorok P, Grin IR, Zharkov DO, Ishenko AA, et al. (2014) Cloning and Characterization of a Wheat Homologue of Apurinic/Apyrimidinic Endonuclease Ape1L. PLoS ONE 9(3): e92963. doi:10.1371/journal.pone. 0092963 Editor: Robert W. Sobol, University of Pittsburgh, United States of America Received October 24, 2013; Accepted February 27, 2014; Published March 25, 2014 Copyright: 2014 Joldybayeva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from Science Committee of the Ministry of Education and Science of the Republic of Kazakhstan [grants No 0145/ GF and No 0738GF] to A.K. B. and from Agence Nationale pour la Recherche [ANR Blanc 2010 Projet ANR09-GENO-000 to M.S.] (http://www.agence-nationalerecherche.fr), Electricité de France (http://www.edf.fr) Contract Radioprotection [RB 2013–23 to M.S.]; Centre National de la Recherche Scientifique (http://www. cnrs.fr) [PICS N5479-Russie, and CNRS-INCA-MSHE Franco-Pologne #3037987 to M.S.] and by Fondation de France (http://www.fondationdefrance.org) [#2012 00029161 to A.A.I.] and by Polish-French collaborative grants Nu346/N-INCA/2008/0 and Nu303 819540 [to B.T. ] and by Russian Academy of Sciences [MKB 6.12 to D.O. Z. ]; Russian Foundation for Basic Research [12-04-33231-mol_a_ved to I.R. G. ]; P.P. was supported by the fellowship from International PhD Projects Programme of Foundation for Polish Science: ‘‘Studies of nucleic acids and proteins from basic to applied research’’. The project is co-financed by European Union Regional Development Fund. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have the following interests. This study was partially funded by a commercial source ‘‘Electricite de France, EDF (Scientific Commission Council of Radiation Protection)’’. There are no patents, products in development or marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials. * E-mail: smurat@igr.fr (MS); Amangeldy.Bisenbaev@kaznu.kz (AKB) . These authors contributed equally to this work.