CLEAVAGE OF DEOXYRIBONUCLEIC-ACID BY THE 1,10-PHENANTHROLINE-CUPROUS COMPLEX - HYDROGEN-PEROXIDE REQUIREMENT AND PRIMARY AND SECONDARY STRUCTURE SPECIFICITY
CLEAVAGE OF DEOXYRIBONUCLEIC-ACID BY THE 1,10-PHENANTHROLINE-CUPROUS COMPLEX - HYDROGEN-PEROXIDE REQUIREMENT AND PRIMARY AND SECONDARY STRUCTURE SPECIFICITY
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DOI：
10.1021/bi00505a003
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发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
SIGMAN, DS
中科院分区：
文献类型：
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作者：
MARSHALL, LE;GRAHAM, DR;SIGMAN, DS
1，10-菲咯啉(OP)与铜离子配位，在需要过氧化氢和硫醇的反应中裂解DNA。交替共聚物聚([~3H]DA-[~3H]T)(10微克/毫升)在好氧和厌氧条件下，用20微米OP、2微米铜离子、7毫米3-巯基丙酸和7毫米H_2O_2使95%的酸溶于-30 S。在没有添加过氧化氢的情况下，1，10-二氮杂菲和铜离子在硫醇催化下断裂DNA需要氧气，因为它是2：1邻菲咯啉-铜络合物催化硫醇原位氧化生成过氧化氢的前体。在没有1，10-邻菲咯啉和铜离子存在的情况下，H_2O_2对聚(da-DT)没有明显的降解作用。在配体和金属离子存在的情况下，双氧水和硫醇同时存在时的反应比单独存在时要快得多。亚铜复合体的参与是从对还原剂的需求和亚铜特异性配体对反应的抑制中推断出来的。在反应过程中，1，10-二氮杂菲插入DNA是由于插层剂如溴化乙锭对DNA断裂的抑制，以及对双链[聚(Da).cntdot]的强烈(如果不是绝对的)偏好。Poly(T)]与作为底物的单链[Poly(Da)或Poly(T)]DNA进行切割比较。对大肠杆菌乳胶操纵子片段与配位络合物反应的电泳图显示，每个碱基的断裂强度几乎相等，这表明反应中的初级序列特异性很小。由于缺乏特异性，这种化学可能有助于研究其他试剂与DNA相互作用中的初级序列特异性。
1,10-Phenanthroline (OP) coordinated to Cu ion cleaves DNA in a reaction requiring H2O2 and thiol. The alternating copolymer poly([3H]dA-[3H]T) (10 .mu.g/ml) is made 95% acid soluble in < 30 s with 20 .mu.M OP, 2 .mu.M Cu ion, 7 mM 3-mercaptopropionic acid and 7 mM H2O2 under aerobic and anaerobic conditions. In the absence of added H2O2, oxygen is required for the thiol-mediated scission of DNA by 1,10-phenanthroline and Cu ion because it serves as a precursor for H2O2 formed as a result of the in situ oxidation of thiol catalyzed by the 2:1 phenanthroline-cupric complex. In the absence of 1,10-phenanthroline and Cu ion, H2O2 does not measurably degrade poly(dA-dT). In the presence of the ligand and metal ion, the reaction is much faster when both H2O2 and thiol are present than when either is present alone. The involvement of the cuprous complex is inferred from the requirement for reducing agent and from the inhibition of the reaction by cuprous specific ligands. Intercalation of 1,10-phenanthroline into the DNA during the course of the reaction is suggested by inhibition of DNA scission by intercalating agents such as ethidium bromide and a strong, if not absolute, preference for double-stranded [poly(dA) .cntdot. poly(T)] as compared to single-stranded [poly(dA) or poly(T)] DNA as a substrate for the cleavage. Electrophoresis of a fragment of Escherichia coli lac operon reacted with the coordination complex revealed scission of nearly equal intensity at every base, suggesting little, if any, primary sequence specificity in the reaction. Because of this lack of specificity, this chemistry may be useful in studying the primary sequence specificity of other agents in their interactions with DNA.