Bailly C, Gentle D, Hamy F, Purcell M, Waring M J
Department of Pharmacology, University of Cambridge, U.K.
Biochem J. 1994 May 15;300 ( Pt 1)(Pt 1):165-73. doi: 10.1042/bj3000165.
Four complementary footprinting and probing techniques utilizing DNAse I, methidiumpropyl EDTA (MPE).FeII, diethyl pyrocarbonate (DEPC) and KMnO4 as DNA-cleaving or DNA-modifying agents have been applied to investigate the sequence-specific binding to DNA of the antitumour antibiotic echinomycin. A 265 bp EcoRI-PvuII DNA restriction fragment excised from plasmid pBS was used as a substrate. Six regions of protection against DNAase I cleavage were located on the 265-mer: three sites encompass the sequences 5'-TCGA or 5'-GCGT and the three others contain 5'-GpG (CpC) dinucleotide sequences where the inhibition of DNAase I cutting by echinomycin is less pronounced. In contrast, MPE.FeII cleavage allows identification of only three echinomycin-binding sites on the 265-mer: two sites contain the sequence 5'-TCGA and one encompasses the sequence 5'-ACCA. Cleavage of DNA by MPE.FeII in the presence of echinomycin remains practically unaffected at the sequence 5'-GCGT, despite its identification by DNAase I as a strong site for binding the antibiotic, as well as at the two other sequences containing GpG steps. With both DNAase I and MPE.FeII, enhanced DNA cleavage is evident at AT-rich sequences in the presence of echinomycin. Enhanced reactivity towards KMnO4 and DEPC provides clear evidence for sequence-dependent conformational changes in DNA induced by the antibiotic. The experiments reveal that KMnO4 reacts most strongly with thymines located around, but not necessarily adjacent to, an echinomycin-binding site, whereas the carbethoxylation reactions caused by DEPC occur primarily at the adenine residues lying immediately 5' or 3' to the dinucleotide that denotes an echinomycin-binding site. The results reported here demonstrate that DEPC and KMnO4 serve as sensitive probes for different states of the DNA helix. It seems that the reaction with KMnO4 involves transient unstacking events, whereas the carbethoxylation reaction of DEPC requires larger-scale helix opening.
利用脱氧核糖核酸酶I（DNAse I）、甲磺酸丙酯乙二胺四乙酸（MPE）·FeII、焦碳酸二乙酯（DEPC）和高锰酸钾（KMnO4）作为DNA切割或DNA修饰剂的四种互补足迹和探测技术，已被用于研究抗肿瘤抗生素棘霉素与DNA的序列特异性结合。从质粒pBS切下的一个265bp的EcoRI - PvuII DNA限制片段用作底物。在这个265聚体上定位了六个抗DNAse I切割的保护区域：三个位点包含序列5'-TCGA或5'-GCGT，另外三个包含5'-GpG（CpC）二核苷酸序列，在这些序列处棘霉素对DNAse I切割的抑制作用不太明显。相比之下，MPE·FeII切割仅能在这个265聚体上鉴定出三个棘霉素结合位点：两个位点包含序列5'-TCGA，一个包含序列5'-ACCA。在棘霉素存在的情况下，MPE·FeII对DNA的切割在序列5'-GCGT处实际上不受影响，尽管DNAse I将其鉴定为抗生素结合的强位点，以及在另外两个包含GpG步移的序列处也是如此。对于DNAse I和MPE·FeII，在棘霉素存在的情况下，富含AT的序列处DNA切割增强是明显的。对抗生素诱导的DNA中序列依赖性构象变化而言，对KMnO4和DEPC的反应性增强提供了明确证据。实验表明，KMnO4与位于棘霉素结合位点周围但不一定相邻的胸腺嘧啶反应最强，而由DEPC引起的乙氧基化反应主要发生在表示棘霉素结合位点的二核苷酸紧邻5'或3'的腺嘌呤残基处。此处报道的结果表明，DEPC和KMnO4可作为DNA螺旋不同状态的敏感探针。似乎与KMnO4的反应涉及短暂的解堆积事件，而DEPC的乙氧基化反应需要更大规模的螺旋打开。
