Gopal J, Bhagwat A S
Department of Chemistry, Wayne State University, Detroit, MI 48202.
Nucleic Acids Res. 1995 Jan 11;23(1):29-35. doi: 10.1093/nar/23.1.29.
We have developed a simple new method that can identify the base methylated by a sequence-specific DNA methyltransferase and have used it to identify the cytosine that is methylated by DsaV methyltransferase (M. DsaV) within its recognition sequence 5'-CCNGG. The method utilizes the fact that exonuclease III of E. coli does not degrade DNA ends with 3' overhangs and cannot hydrolyze a phosphorothioate linkage. DNA duplexes containing phosphorothioate linkages at specific positions were methylated with M. DsaV in the presence of [methyl-3H] S-adenosylmethionine and were subjected to exonuclease III digestion. The pattern of [methyl-3H] dCMP release from the duplexes was consistent with the methylation of the internal cytosine in CCNGG, but not of the outer cytosine. To establish the accuracy of this method, we confirmed the known specificity of EcoRII methyltransferase by the method. We also confirmed the specificity of M. DsaV using an established biochemical method that involves the use of a type IIS restriction enzyme. Methylation of CCWGG (W = A or T) sequences at the internal cytosines is native to E. coli and is not restricted by the modified cytosine restriction (Mcr) systems. Surprisingly, the gene for M. DsaV was significantly restricted by the McrBC system. We interpret this to mean that M. DsaV may occasionally methylate at sequences other than CCNGG or may occasionally methylate the outer cytosine in its recognition sequence.
我们开发了一种简单的新方法，该方法可以识别由序列特异性DNA甲基转移酶甲基化的碱基，并已用它来识别在其识别序列5'-CCNGG内被DsaV甲基转移酶（M. DsaV）甲基化的胞嘧啶。该方法利用了这样一个事实，即大肠杆菌的核酸外切酶III不会降解具有3'突出端的DNA末端，并且不能水解硫代磷酸酯键。在[甲基-3H] S-腺苷甲硫氨酸存在下，用M. DsaV对在特定位置含有硫代磷酸酯键的DNA双链体进行甲基化，然后进行核酸外切酶III消化。双链体中[甲基-3H] dCMP释放的模式与CCNGG中内部胞嘧啶的甲基化一致，但与外部胞嘧啶的甲基化不一致。为了确定该方法的准确性，我们通过该方法证实了EcoRII甲基转移酶已知的特异性。我们还使用一种既定的生化方法（涉及使用IIS型限制酶）证实了M. DsaV的特异性。CCWGG（W = A或T）序列在内部胞嘧啶处的甲基化是大肠杆菌所固有的，不受修饰胞嘧啶限制（Mcr）系统的限制。令人惊讶的是，M. DsaV的基因受到McrBC系统的显著限制。我们将此解释为意味着M. DsaV可能偶尔会在CCNGG以外的序列处甲基化，或者可能偶尔会在其识别序列中的外部胞嘧啶处甲基化。
