Determination of methylation specificity of sequence-specific DNA methyltransferases using matrix assisted laser desorption/ionization time-of-flight mass spectrometry.
We describe here a sensitive and straightforward method for characterizing the methylation specificity of type II DNA methyltransferase (MTase) using matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry. DNA substrate, prepared by ligation of a commercially available oligonucleotide, was modified by the subject MTase, and was derivatized to a mixture of single-stranded oligonucleotides through endonuclease treatment, heat-denaturation and limited digestion by 3'-terminus-specific phosphodiesterase I. MALDI-TOF mass spectrometry was used to determine the mass differences between the digestion products, and the methylated nucleotide was explicitly identified by the mass increase of 14 Da due to the base modification. The method was applicable to the three representative MTases M. Eco RI, M. Bam HI and M. Hae III.
Tamura
T
T
Department of Bioresources Chemistry, Faculty of Agriculture, Okayama University, 1-1-1 Tsushimanaka, Okayama 700, Japan.
Araki
Y
Y
Yamaoka
S
S
Inagaki
K
K
Tanaka
H
H
D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000378:metabolism*; D015257:DNA-Cytosine Methylases / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000378:metabolism; D013379:Substrate Specificity
D004247:DNA; D015254:DNA Modification Methylases; C042525:DNA modification methylase BamHI; C020098:DNA modification methylase EcoRI; D015257:DNA-Cytosine Methylases; C027823:Cytosine 5-methyltransferase; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific)