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首页> 外文期刊>Journal of Molecular Biology >Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence.
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Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence.

机译：EcoP15I DNA甲基转移酶与DNA的结合揭示了识别序列内的大结构扭曲。

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摘要

EcoP15I DNA methyltransferase, a member of the type III restriction-modification system, binds to the sequence 5'-CAGCAG-3' transferring a methyl group from S-adenosyl-l-methionine to the second adenine base. We have investigated protein-DNA interactions in the methylase-DNA complex by three methods. Determination of equilibrium dissociation constants indicated that the enzyme had higher affinity for DNA containing mismatches at the target base within the recognition sequence. Potassium permanganate footprinting studies revealed that there was a hyper-reactive permanganate cleavage site coincident with adenine that is the target base for methylation. More importantly, to detect DNA conformational alterations within the enzyme-DNA complexes, we have used a fluorescence-based assay. When EcoP15I DNA methyltransferase bound to DNA containing 2-aminopurine substitutions within the cognate sequence, an eight to tenfold fluorescent enhancement resulting from enzymatic flipping of the target adenine base was observed. Furthermore, fluorescence spectroscopy analysis showed that the changes attributable to structural distortion were specific for only the bases within the recognition sequence. More importantly, we observed that both the adenine bases in the recognition site appear to be structurally distorted to the same extent. While the target adenine base is probably flipped out of the DNA duplex, our results also suggest that fluorescent enhancements could be derived from protein-DNA interactions other than base flipping. Taken together, our results support the proposed base flipping mechanism for adenine methyltransferases. Copyright 2000 Academic Press.
机译：作为III型限制性修饰系统成员的EcoP15I DNA甲基转移酶与序列5'-CAGCAG-3'结合，将甲基从S-腺苷-1-蛋氨酸转移至第二个腺嘌呤碱基。我们通过三种方法研究了甲基化酶-DNA复合物中蛋白质-DNA的相互作用。平衡解离常数的测定表明该酶对识别序列内靶碱基处含有错配的DNA具有更高的亲和力。高锰酸钾足迹研究表明，高反应性高锰酸盐裂解位点与作为甲基化目标碱基的腺嘌呤相吻合。更重要的是，为了检测酶-DNA复合物中的DNA构象变化，我们使用了基于荧光的检测方法。当EcoP15I DNA甲基转移酶结合到同源序列中包含2-氨基嘌呤取代的DNA时，观察到目标腺嘌呤碱基的酶促翻转导致荧光增强了八到十倍。此外，荧光光谱分析表明，归因于结构变形的变化仅对识别序列内的碱基是特异性的。更重要的是，我们观察到识别位点的两个腺嘌呤碱基在结构上似乎都变形了相同程度。虽然目标腺嘌呤碱基可能会从DNA双链体中移出，但我们的研究结果还表明，荧光增强可能源自碱基翻转以外的蛋白质-DNA相互作用。两者合计，我们的结果支持腺嘌呤甲基转移酶的拟议的基础翻转机制。版权所有2000学术出版社。

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