CLEAVAGE OF DNA WITH METHIDIUMPROPYL-EDTA-IRON(II) - REACTION CONDITIONS AND PRODUCT ANALYSES
CLEAVAGE OF DNA WITH METHIDIUMPROPYL-EDTA-IRON(II) - REACTION CONDITIONS AND PRODUCT ANALYSES
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DOI：
10.1021/bi00312a022
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发表时间：
1984-01-01
期刊：
影响因子：
2.9
通讯作者：
DERVAN, PB
中科院分区：
文献类型：
--
作者：
HERTZBERG, RP;DERVAN, PB
介绍了甲基丙基EDTA(MPE)的合成方法。MPE、MPE.cntdo.的结合亲和力N1(II)和MPE.cntdo.镁(II)对小牛胸腺DNA的抑制倍数为2.4倍。104M-1，1.5倍。105M-1和1.2倍。在50 mM的氯化钠、pH为7.4的条件下，分别为105M-1。结合位点大小为2个碱基对。Mpe.cntdo.镁(II)解离PM2 DNA 11.+-。3.学位。每个结合分子。Mpe.cntdo.在O2存在下，Fe(II)能有效地切割DNA，且序列特异性低。还原剂显著提高切割反应的效率，其顺序为抗坏血酸钠；二硫苏糖醇；NADPH。在0.1-0.01µm的MPE·cntdot中。在DNA碱基对中，抗坏血酸和二硫苏糖醇的最适切割浓度为1-5 mM。用MPE-cntdot有效地切割DNA(碱基对中的10µm)。Fe(II)(0.1-0.01µm)在pH为7-10的范围内存在，最适pH为7.4(Tris-HCl缓冲液)。最佳切割时间为3.5h(22.0h)。c)。在Na+离子浓度为5 mM~1 mM的范围内，DNA的切割效率最高，其中以5 mM的氯化钠的切割效果最好。每个MPE的超螺旋DNA上的单链切割数。在最佳条件下，Fe(II)为1.4。钴(II)、镁(II)、镍(II)和锌(II)等金属对MPE引起的断链有抑制作用。释放的DNA经MPE.cntdot裂解后的产物。Fe(II)是4个核苷酸碱基。裂解位点的DNA末端是5‘-磷酸，3’-磷酸和3‘-磷酸的比例大致相等。这些产物与DNA骨架脱氧核糖环的氧化降解是一致的，很可能是由羟基自由基引起的。
The synthesis of methidiumpropyl-EDTA (MPE) is described. The binding affinities of MPE, MPE .cntdot. N1(II) and MPE .cntdot. Mg(II) to calf thymus DNA are 2.4 .times. 104 M-1, 1.5 .times. 105 M-1 and 1.2 .times. 105 M-1, respectively, in 50 mM NaCl, pH 7.4. The binding site size is 2 base pairs. MPE .cntdot. Mg(II) unwinds PM2 DNA 11 .+-. 3.degree. per bound molecule. MPE .cntdot. Fe(II) in the presence of O2 efficiently cleaves DNA and with low sequence specificity. Reducing agents significantly enhance the efficiency of the cleavage reaction in the order sodium ascorbate > dithiothreitol > NADPH. At concentrations of 0.1-0.01 .mu.M in MPE .cntdot. Fe(II) and 10 .mu.M in DNA base pairs, optimum ascorbate and dithiothreitol concentrations for DNA cleavage are 1-5 mM. Efficient cleavage of DNA (10 .mu.M in base pairs) with MPE .cntdot. Fe(II) (0.1-0.01 .mu.M) occurs over a pH range of 7-10 with the optimum at 7.4 (Tris-HCl buffer). The optimum cleavage time is 3.5 h (22.degree. C). DNA cleavage is efficient in a Na+ ion concentration range of 5 mM to 1 mM, with the optimum at 5 mM NaCl. The number of single-strand scissions on supercoiled DNA per MPE .cntdot. Fe(II) under optimum conditions is 1.4. Metals such as Co(II), Mg(II), Ni(II) and Zn(II) inhibit strand scission by MPE. The released products from DNA cleavage by MPE .cntdot. Fe(II) are the 4 nucleotide bases. The DNA termini at the cleavage site are 5''-phosphate and roughly equal proportions of 3''-phosphate and 3''-(phosphoglycolic acid). The products are consistent with the oxidative degradation of the deoxyribose ring of the DNA backbone most likely by hydroxy radical.