Detection and analysis of enzymatic DNA methylation of oligonucleotide substrates by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.
Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF) mass spectrometry was employed to analyze DNA methylation carried out by the Escherichia coli dam DNA methyltransferase using oligonucleotide substrates with molecular masses of 5000-10,000 Da per strand. The mass spectrometry assay offers several advantages: (i) it directly shows the methylation as the increase in the mass of the substrate DNA, (ii) it is nonradioactive, (iii) it is quantitative, and (iv) it can be automated for high-throughput applications. Since unmethylated and methylated DNA are detected, the ratio of methylation can be determined directly and accurately. Furthermore, the assay allows detection individually of the methylation of several substrates in competition, offering an ideal setup to analyze the specificity of DNA interacting with enzymes. We could not identify methylation at any noncanonical site, indicating that the dam MTase is a very specific enzyme. Finally, MALDI-TOF mass spectrometry permitted assessment of the number of methyl groups incorporated into each DNA strand, thereby, allowing study of mechanistic details such as the processivity of the methylation reaction. We provide evidence that the dam MTase modifies DNA in a processive reaction, confirming earlier findings.
Humeny
Andreas
A
Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander Universität Erlangen-Nürnberg, Fahrstrasse 17, 91054 Erlangen, Germany.
Beck
Carsten
C
Becker
Cord-Michael
CM
Jeltsch
Albert
A
D019175:DNA Methylation; D015254:DNA Modification Methylases / Q000032:analysis*; D004926:Escherichia coli / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D015254:DNA Modification Methylases