Hecht R, Thielmann H W
Eur J Biochem. 1978 Sep 1;89(2):607-18. doi: 10.1111/j.1432-1033.1978.tb12565.x.
An endonuclease which is active with regard to depurinated, alkylated, arylated, and arylamidated DNA has been purified 500-fold from Micrococcus luteus. In this purification, separation from the pyrimidine-dimer-specific ultraviolet-endonuclease has been achieved. The enzyme has a molecular weight of 30000 on the basis of gel filtration; its activity is not absolutely dependent upon the presence of Mg2+, but 5--30 mM Mg2+ produces a five-fold stimulation. Potassium chloride concentrations of less than 100 mM are optimal, while concentrations exceeding 100 mM inhibit. The enzyme has no effect on native DNA, but introduces single-strand breaks into DNA containing apurinic/apyrimidinic sites produced by heating at an acidic pH. DNA treated with such carcinogens as N-alkyl-N-nitrosoureas, alkyl methanesulfonates, alkyl sulfates, nitrogen mustard, beta-propiolactone, 7-bromomethyl-benz[a]anthracene, N-acetoxy-2-acetylaminofluorene, and 7,12-dimethyl-benz[a]anthracene-5,6-oxide also becomes susceptible to enzymic action. The activity of the enzyme has been detected by making use of the difference in mobility between supercoiled closed-circular DNA of Pseudomonas phage PM2 and its nicked form in agarose gel elctrophoresis. Even depurinated or carcinogen-modified supercoiled PM2 DNA migrated faster than the respective relaxed nicked forms. A comparison of the number of enzyme-catalyzed single-strand breaks with the number of alkali-labile (i.e. apurinic) sites in carcinogen-modified PM2 DNA showed that the enzyme preparation introduced approximately twice as many breaks into the substrates as the number of apurinic sites present. We conclude that the enzyme preparation either recognizes both apurinic sites and DNA bases carrying carcinogenic residues or contains DNA glycosidase activity in addition to the endonuclease activity. Exposure of ultraviolet-irradiated PM2 DNA to the endonuclease preparation showed that pyrimidine dimers were not substrates. The yield of enzyme-catalyzed single-strand breaks found in ultraviolet-irradiated DNA was five times the number of alkali-labile sites present suggesting that minor photoproducts, possibly 5,6-saturated pyrimidine residues, were recognized in addition to apurinic sites.
一种对脱嘌呤、烷基化、芳基化和芳酰胺化DNA具有活性的核酸内切酶已从藤黄微球菌中纯化了500倍。在该纯化过程中，已实现了与嘧啶二聚体特异性紫外线核酸内切酶的分离。基于凝胶过滤，该酶的分子量为30000；其活性并非绝对依赖于Mg2+的存在，但5-30 mM的Mg2+会产生五倍的刺激作用。氯化钾浓度低于100 mM时最为适宜，而超过100 mM则会产生抑制作用。该酶对天然DNA无作用，但会在酸性pH下加热产生的含有脱嘌呤/脱嘧啶位点的DNA中引入单链断裂。用N-烷基-N-亚硝基脲、烷基甲烷磺酸盐、烷基硫酸盐、氮芥、β-丙内酯、7-溴甲基苯并[a]蒽、N-乙酰氧基-2-乙酰氨基芴和7,12-二甲基苯并[a]蒽-5,6-氧化物等致癌物处理的DNA也变得易受酶作用的影响。该酶的活性已通过利用假单胞菌噬菌体PM2的超螺旋闭环DNA与其在琼脂糖凝胶电泳中的切口形式之间迁移率的差异进行检测。即使是脱嘌呤或致癌物修饰的超螺旋PM2 DNA迁移速度也比相应的松弛切口形式快。对致癌物修饰的PM2 DNA中酶催化的单链断裂数量与碱不稳定（即脱嘌呤）位点数量的比较表明，酶制剂在底物中引入的断裂数量约为存在的脱嘌呤位点数量的两倍。我们得出结论，酶制剂要么识别脱嘌呤位点和携带致癌残基的DNA碱基，要么除了核酸内切酶活性外还含有DNA糖苷酶活性。将紫外线照射的PM2 DNA暴露于核酸内切酶制剂中表明，嘧啶二聚体不是底物。在紫外线照射的DNA中发现的酶催化单链断裂产量是存在的碱不稳定位点数量的五倍，这表明除了脱嘌呤位点外，还识别了可能是5,6-饱和嘧啶残基的次要光产物。
