Properties of a human lymphoblast AP-endonuclease associated with activity for DNA damaged by ultraviolet light, gamma-rays, or osmium tetroxide.
Properties of a human lymphoblast AP-endonuclease associated with activity for DNA damaged by ultraviolet light, gamma-rays, or osmium tetroxide.
复制标题
人淋巴母细胞 AP-核酸内切酶的特性与紫外线、伽马射线或四氧化锇损伤的 DNA 的活性相关。
DOI：
10.1021/bi00288a024
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发表时间：
1983
期刊：
影响因子：
2.9
通讯作者：
Brent,TP
中科院分区：
文献类型：
--
作者：
Brent,TP
材料与方法DNA底物的制备。已经描述了噬菌体PM 2的生长、用[3 H]胸苷标记和分离放射性标记的DNA的方法（Teebor和布伦特，1981）。通过在0.1M柠檬酸钠缓冲液中在70 ℃下加热制备每个分子含有一至两个AP位点的部分脱嘌呤的DNA（pH5.5）20-40分钟。紫外线照射的DNA含有约一个碱稳定的，每个分子的核酸内切酶敏感位点通过在254-nm光下曝光来制备（1500 J/m2）从杀菌灯以500 J m-2 min-1的剂量率（通过使用Blak-Ray紫外线计测定）照射。在空气中用137 Cs辐照器以800拉德/分钟的剂量率辐照在20 mM Tris-HCl 1（pH 7.5）、1 mM EDTA和0.1 M NaCl（缓冲液A）中的PM2 DNA。总共5000拉德产生大约一个碱稳定的，内切核酸酶敏感的网站每个分子。通过将缓冲液A中的PM2 DNA预热至70 ℃ 2分钟，加入OsO 4至0.02%的浓度，并在70 ℃继续孵育5分钟，制备每个分子含有约一个内切核酸酶敏感位点的OsO 4处理的DNA。冷却至室温后，溶液用2体积的乙醚萃取3次，最后用缓冲液A充分透析。氧化DNA-Sepharose 4 B亲和柱的制备。小牛胸腺DNA（Sigma I型），500 mg在50 mL H2O中，通过煮沸和快速冷却变性。用2L的0.3MnH4Cl缓冲液（pH8.6）透析后，加入KMnO 4至浓度为1.5mM，并将混合物在4 ℃下搅拌30分钟。通过加入1 M焦亚硫酸钠终止反应，直至颜色消失。该步骤之后是用2L的10
Materials and MethodsPreparation of DNA Substrates. Procedures for the growth of bacteriophage PM2, labeling with [3H] thymidine, and isolation of the radiolabeled DNA have been described (Teebor & Brent, 1981). Partially depurinated DNA containing one to two AP sites per molecule was prepared by heating at 70 C in 0.1 M sodium citrate buffer (pH 5.5) for 20-40 min. Ultraviolet-irradiated DNA containing about one alkali-stable, endonuclease sensitive site per molecule was prepared by ex-posure to 254-nm light (1500 J/m2) from a germicidal lamp at a dose rate of 500 J m “2 min'1 (determined by using a Blak-Ray ultraviolet meter). PM2 DNA in 20 mM Tris-HCl1 (pH 7.5), 1 mM EDTA, and 0.1 M NaCl (buffer A) was irradiated under air at a dose rate of 800 rads/min with a 137Cs irradiator. A total of 5000 rads produced approximately one alkali-stable, endonuclease-sensitive site per molecule. Os04-treated DNA containingabout one endonuclease-sensitive site per molecule was prepared by preheating PM2 DNA in buffer A to 70 C for 2 min, adding Os04 to a concentration of 0.02%, and continuing incubation at 70 C for 5 min. After cooling to room temperature, the solution was extracted 3 times with 2 volumes of diethyl ether and finally dialyzed extensively against buffer A.Preparation of Oxidized DNA-Sepharose 4B Affinity Column. Calf thymus DNA (Sigma type I), 500 mg in 50 mL of H20, was denatured by boiling and rapid cooling. After dialysis against 2 L of 0.3 Mnh4c1 buffer, pH 8.6, KMn04 was added to a concentration of 1.5 mM and the mixture stirred at 4 C for 30 min. The reaction was stopped by addition of 1 M sodium metabisulfite until the color disappeared. This step was followed by dialysis against 2 L of 10