2 DNA METHYLTRANSFERASES FROM MURINE ERYTHROLEUKEMIA-CELLS - PURIFICATION, SEQUENCE SPECIFICITY, AND MODE OF INTERACTION WITH DNA
2 DNA METHYLTRANSFERASES FROM MURINE ERYTHROLEUKEMIA-CELLS - PURIFICATION, SEQUENCE SPECIFICITY, AND MODE OF INTERACTION WITH DNA
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DOI：
10.1073/pnas.80.18.5559
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发表时间：
1983-01-01
期刊：
影响因子：
--
通讯作者：
INGRAM, VM
中科院分区：
文献类型：
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作者：
BESTOR, TH;INGRAM, VM
用Cibrron Blue F3GA-琼脂糖染色层析法从未诱导的小鼠红白血病细胞核提取液中拆分2种DNA(胞嘧啶-5-甲基转移酶)。每个物种都得到了高度纯化；第一峰和第二峰的活性分别与Mr[相对分子质量]150,000和175,000的多肽有关。用合成的DNA、vphi.X174复制型DNA和pBR322 DNA的限制性内切酶片段对底物的特异性分析表明，这两种酶都不依赖于CpG二核苷酸的序列背景；Poly(DG-DC)在所有被测试的未甲基化DNA底物中具有最大的甲基接受活性。与半甲基化位点的甲基化相比，这两种酶的去从头甲基化效率较低。接受甲基的活性强烈依赖于DNA链长。在DNA甲基转移酶定位其识别位点的机制中，与DNA的结合以及随后酶沿DNA分子的一维扩散是重要的。
Dye-ligand chromatography on Cibracron blue F3GA-agarose was used to resolve 2 species of DNA (cytosine-5-)-methyltransferase from nuclear extracts of uninduced Friend murine erythroleukemia cells. Each species has been highly purified; the activities in the 1st and 2nd peaks were associated with polypeptides of Mr [relative MW] 150,000 and 175,000, respectively. Analysis of substrate specificity with synthetic DNA and restriction fragments of .vphi.X174 replicative form DNA and pBR322 DNA showed that neither enzyme had dependence on the sequence context of CpG dinucleotides; poly(dG-dC) had the greatest methyl-accepting activity of any unmethylated DNA substrate tested. De-novo methylation by both enzymes was inefficient relative to methylation of hemimethylated sites. Methyl-accepting activity was strongly dependent on DNA chain length. Binding to DNA, followed by 1-dimensional diffusion of enzyme along the DNA molecule, is important in the mechanism by which DNA methyltransferase locates its recognition sites.