Cloning and Characterization of a Wheat Homologue of Apurinic/Apyrimidinic Endonuclease Ape1L
Cloning and Characterization of a Wheat Homologue of Apurinic/Apyrimidinic Endonuclease Ape1L
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DOI：
10.1371/journal.pone.0092963
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发表时间：
2014-03-25
期刊：
影响因子：
3.7
通讯作者：
Saparbaev, Murat
中科院分区：
文献类型：
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作者：
Joldybayeva, Botagoz;Prorok, Paulina;Saparbaev, Murat
背景：脱氧核糖核酸内切酶是碱基切除修复(BER)途径中的关键DNA修复酶。在BER中，AP内切酶在AP位点切割DNA，并阻止DNA糖基酶和/或氧化损伤产生的39个部分。从小麦中分离到一个编码ExoIII家族AP内切酶同源序列的基因，该基因包括E.coliXth、人APE1和拟南芥AtApe1L，并对其蛋白产物进行了纯化和鉴定。方法/主要研究结果：我们报道了小麦AP内切酶TaApe1L具有AP内切酶、3‘-修复磷酸二酯酶、3’-磷酸酶和3‘-GT；5’外切核酸酶活性。令人惊讶的是，与细菌和人AP内切酶相反，在反应混合物中加入5-10 mM的镁和钙抑制了TaApe1L，而存在0.1-1.0 mM的Mn2+、Co2+和Fe2+则强烈刺激了TaApe1L的DNA修复活性。对反应条件的优化表明，TaApe1L具有较低的二价阳离子浓度(0.1 mM)、中等酸性(6-7)、低离子强度(20 MM KCl)，最适反应温度为20℃左右。稳态反应动力学参数表明，TaApe1L能有效去除39个封闭的糖基和3‘-磷酸基团(k(CAT)/K-M分别为630和485 mM-1.min(-1))，但与人APE1相比，其AP内切酶活性很弱。这些数据证实了小麦AP内切酶的DNA底物特异性，并暗示其可能在内源和环境因素造成的DNA损伤修复中发挥作用。
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, 3'-repair phosphodiesterase, 3'-phosphatase and 3'-> 5' exonuclease activities. Surprisingly, in contrast to bacterial and human AP endonucleases, addition of Mg2+ and Ca2+ (5-10 mM) to the reaction mixture inhibited TaApe1L whereas the presence of Mn2+, Co2+ and Fe2+ 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 degrees C. The steady-state kinetic parameters of enzymatic reactions indicate that TaApe1L removes 39-blocking sugar-phosphate and 3'-phosphate groups with good efficiency (k(cat)/K-M = 630 and 485 mu M-1.min(-1), 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.