A simple method for locating methylated bases in DNA, as applied to detect asymmetric methylation by M.FokIA.
Class-IIS restriction enzymes, which cut the DNA outside their recognition sequence, could be used for locating the bases methylated by a DNA-modification methylase. This is possible because methylation of the class-IIS cut sites does not interfere with the cleavage. The method consists of (i) selection of a nucleotide sequence with appropriate overlap between the methylase recognition site and the class-IIS enzyme cut site, (ii) methylation using S-adenosylmethionine as [3H]methyl donor, (iii) cleavage of the methylated sequence with the class-IIS enzyme, (iv) separation of the cleavage products and identification of the 3H-labelled fragment. Using this simple and straightforward method, we have shown that M.FokIA is an adenine methylase and methylates asymmetrically one strand of the FokI recognition site, resulting in the (Formula: see text) sequence. In addition, it was observed that another class-IIS restriction enzyme, SfaNI, is completely inhibited by methylation of its recognition site, (Formula: see text), by M.FokIA.
Pósfai
G
G
McArdle Laboratory for Cancer Research, University of Wisconsin, Madison 53706.
Szybalski
W
W
D001483:Base Sequence; D004247:DNA; D015252:Deoxyribonucleases, Type II Site-Specific; D008745:Methylation; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism*; D013379:Substrate Specificity
D004247:DNA; C058668:DNA modification methylase FokI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific