Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence
Binding of EcoP15I DNA methyltransferase to DNA reveals a large structural distortion within the recognition sequence
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DOI：
10.1006/jmbi.2000.3673
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发表时间：
2000-05-12
影响因子：
5.6
通讯作者：
Rao, DN
中科院分区：
文献类型：
--
作者：
Reddy, YVR;Rao, DN
EcoP 15 I DNA甲基转移酶是III型限制-修饰系统的成员，与序列5 '-CAGCAG-3'结合，将甲基从S-腺苷-L-甲硫氨酸转移到第二个腺嘌呤碱基。我们用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，该酶具有较高的亲和力，在识别序列内的目标碱基含有错配的DNA。高锰酸钾足迹法研究表明，有一个高反应性的高锰酸盐切割位点与腺嘌呤一致，腺嘌呤是甲基化的靶碱基。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测定法。当EcoP 15 I DNA甲基转移酶结合到同源序列内含有2-氨基嘌呤取代的DNA时，观察到由靶腺嘌呤碱基的酶促翻转引起的8至10倍荧光增强。此外，荧光光谱分析表明，可归因于结构畸变的变化仅对识别序列内的碱基具有特异性。更重要的是，我们观察到，在识别位点的腺嘌呤碱基似乎在结构上扭曲到相同的程度。虽然靶腺嘌呤碱基可能从DNA双链体中翻转出来，但我们的研究结果也表明，荧光增强可能来自于蛋白质-DNA相互作用而不是碱基翻转。两者合计，我们的研究结果支持建议的碱基翻转腺嘌呤甲基转移酶的机制。(C)北京大学出版社.
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. (C) 2000 Academic Press.