Direct analysis of enzyme-catalyzed DNA demethylation.
N/O-methylation of DNA can be cytotoxic and mutagenic; therefore, enzymes that reverse DNA methylation are essential for organism survival. Several 2-oxoglutarate-dependent oxygenases and methyltransferases that remove a methyl group from a methylated DNA base have been identified. Studies of their kinetics and search for their inhibitors have been retarded by the lack of an approach to directly quantitate DNA substrates and products that differ by a single methyl group. Here, we introduce such an approach, which is based on capillary electrophoresis with laser-induced fluorescence detection. We achieved baseline separation of a fluorescently labeled 15-nucleotide-long single-base methylated DNA substrate from its demethylated product, followed by its quantitative detection. We then used this approach to study the kinetics of AlkB-catalyzed DNA demethylation and screen a number of potential inhibitors of this reaction. Ten new inhibitors, which can be used as templates in developing therapies targeting AlkB-like enzymes, were identified. Our approach will be applicable for in vitro kinetic studies of known DNA demethylating and methylating enzymes and in the discovery of new ones.
Karkhanina
Anna A
AA
Department of Chemistry, York University, Toronto, Ontario M3J 1P3, Canada.
Mecinović
Jasmin
J
Musheev
Michael U
MU
Krylova
Svetlana M
SM
Petrov
Alexander P
AP
Hewitson
Kirsty S
KS
Flashman
Emily
E
Schofield
Christopher J
CJ
Krylov
Sergey N
SN
D001483:Base Sequence; D055162:Biocatalysis; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D019175:DNA Methylation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D029968:Escherichia coli Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D020128:Inhibitory Concentration 50; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D004791:Enzyme Inhibitors; D029968:Escherichia coli Proteins; D004247:DNA; D006899:Mixed Function Oxygenases; C466427:AlkB protein, E coli