McClelland M, Nelson M
Department of Biochemistry and Molecular Biology, University of Chicago, IL 60637.
Gene. 1988 Dec 25;74(1):169-76. doi: 10.1016/0378-1119(88)90278-8.
Site-specific DNA methylation is known to block cleavage by a number of restriction endonucleases. We show that methylation at 'non-canonical' DNA modification sites can also block methylation by five of 13 DNA methyltransferases (MTases) tested. Furthermore, MTases and endonucleases that recognize the same nucleotide sequence can differ in their sensitivity to non-canonical methylation. In particular, BamHI endonuclease can cut 5'-GGATCm5C efficiently, whereas M.BamHI cannot methylate this modified sequence. Methyltransferase/endonuclease pairs which differ in their sensitivity to non-canonical methylation can be exploited to generate rare DNA cleavage sites. For example, we show that M.HpaII, M.BamHI, and BamHI can be used sequentially in a three-step procedure to specifically cleave DNA at the 10-bp sequence 5'-CCGGATCCGG. Several highly selective DNA cutting strategies are made possible by these sequential double methylation-blocking reactions.
已知位点特异性DNA甲基化会阻碍多种限制性内切酶的切割。我们发现，在“非典型”DNA修饰位点的甲基化也会阻碍所测试的13种DNA甲基转移酶（MTases）中5种的甲基化作用。此外，识别相同核苷酸序列的甲基转移酶和内切酶对非典型甲基化的敏感性可能不同。特别地，BamHI内切酶可以高效切割5'-GGATCm5C，而M.BamHI不能甲基化这个修饰序列。对非典型甲基化敏感性不同的甲基转移酶/内切酶对可用于产生罕见的DNA切割位点。例如，我们表明M.HpaII、M.BamHI和BamHI可按三步程序依次使用，以在10个碱基对序列5'-CCGGATCCGG处特异性切割DNA。这些连续的双甲基化阻断反应使几种高度选择性的DNA切割策略成为可能。
