CHROMOMYCIN, MITHRAMYCIN, AND OLIVOMYCIN BINDING-SITES ON HETEROGENEOUS DEOXYRIBONUCLEIC-ACID - FOOTPRINTING WITH (METHIDIUMPROPYL-EDTA)IRON(II)
CHROMOMYCIN, MITHRAMYCIN, AND OLIVOMYCIN BINDING-SITES ON HETEROGENEOUS DEOXYRIBONUCLEIC-ACID - FOOTPRINTING WITH (METHIDIUMPROPYL-EDTA)IRON(II)
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DOI：
10.1021/bi00279a011
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发表时间：
1983-01-01
期刊：
影响因子：
2.9
通讯作者：
DERVAN, PB
中科院分区：
文献类型：
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作者：
VANDYKE, MW;DERVAN, PB
Michael W.货车戴克和彼得B。Dervan* 摘要：用（甲基丙基-EDTA）铁（Ⅱ）[MPE-Fe（Ⅱ）] DNA切割抑制模式技术，测定了抗肿瘤、抗病毒、抗生素色霉素、光辉霉素和橄榄霉素在70个碱基对的异质DNA上的DNA结合位点。制备了两个含有乳糖操纵子启动子-操纵子区的DNA限制性片段，其长度分别为117和168个碱基对，互补链在3 '端用32 P标记。MPE-Fe（II）被允许部分切割在Mg 2+存在下用色霉素、光神霉素或橄榄霉素预平衡的限制片段。色霉素、光辉霉素和橄榄霉素的优选结合位点。一类在抗生素、抗病毒和抗肿瘤化学疗法中重要的小分子与双螺旋DNA结合（Gale等人，1981年）。药物与DNA结合的碱基序列偏好性通常是通过对合成的均聚物和共聚物DNA上药物结合的总亲和力和化学计量的分光光度分析来确定的。在亚铁离子和氧的存在下，（甲脒丙基-EDTA）铁（II）含有DNA嵌入剂甲脒（methidium），通过短的烃系链与金属螯合剂乙二胺四乙酸盐（EDTA）共价结合（图1），可有效地在双螺旋DNA中产生单链断裂（Hertzberg & Dervan，1982）。重要的是，MPE-Fe（II）是相对非序列特异性的DNA切割剂（货车Dyke等人，1982;货车Dyke & Der货车，1982）。实际上，MPE-Fe（II）是一种小型的DNA合成酶，模拟DNA裂解酶DNase I的行为。确定天然DNA上蛋白质结合位点的一种有用方法是DNA切割抑制模式技术，该技术结合了DNA酶I切割蛋白质保护的DNA片段和Maxam-Gilbert序列测定方法（Galas & Schmitz，1978; Schmitz & Galas，1982）。这种有用的“DNA酶I足迹”技术依赖于该酶在部分DNA切割反应中相对低的序列特异性和DNA结合蛋白阻止其覆盖的碱基对之间的DNA骨架切割的能力。蛋白质保护的DNA序列是
Michael W. Van Dyke and Peter B. Dervan* abstract: The DNA binding sites for the antitumor, antiviral, antibiotics chromomycin, mithramycin, and olivomycin on 70 base pairs of heterogeneous DNA have been determined by using the (methidiumpropyl-EDTA) iron (II)[MPE-Fe (II)] DNA cleavage inhibition patterntechnique. Two DNA re-striction fragments 117 and 168 base pairs in length containing the lactose operon promoter-operator region were prepared with complementary strands labeled with 32P at the 3'end. MPE-Fe (II) was allowed to partially cleave therestriction fragment preequilibrated with either chromomycin, mithra-mycin, or olivomycin in the presence of Mg2+. The preferred binding sites for chromomycin, mithramycin, and olivomycin. class of small molecules important in antibiotic, antiviral, and antitumor chemotherapy bind to double-helical DNA (Gale et al., 1981). The base sequence preferences of drugs binding to DNA have usually been determined from spec-trophotometric analyses of the overall affinity and stoichiometry of drug binding on synthetic homopolymer and copolymer DNAs.(Methidiumpropyl-EDTA) iron (II), whichcontains the DNA intercalator methidium covalently bound by a short hydrocarbon tether to the metal chelator ethylenediamine-tetraacetate (EDTA)(Figure 1) in the presence of ferrous ion and oxygen, efficiently produces single-strand breaks in dou-ble-helical DNA (Hertzberg & Dervan, 1982). Importantly, MPE-Fe (II) is a relatively non-sequence-specific DNA cleaving agent (Van Dyke et al., 1982; Van Dyke & Dervan, 1982). In effect, MPE-Fe (II) is a small synthetic scissor for DNA that mimics the behavior of the DNA cleaving enzyme DNase I. One useful method for determining protein binding sites on native DNA is the DNA cleavage inhibition pattern technique, which combines DNase I cleavage of protein-pro-tected DNA fragments and Maxam-Gilbert sequence deter-mination methods (Galas & Schmitz, 1978; Schmitz & Galas, 1982). This useful “DNase I footprinting” techniques relies on the relatively low sequence specificity of the enzyme in a partial DNA cleavage reaction and the ability of DNA-bound protein to prevent cleavage of the DNA backbone between the base pairs it covers. The protein-protected DNA sequence is