title: Direct and continuous fluorescence-based measurements of Pyrococcus horikoshii DNA N-6 adenine methyltransferase activity.
abstract: N-6 methylation of adenine destabilises duplex DNA and this can increase the proportion of DNA that dissociates into single strands. We have investigated utilising this property to measure the DNA adenine methyltransferase-catalyzed conversion of hemimethylated to fully methylated DNA through a simple, direct, fluorescence-based assay. The effects of methylation on the kinetics and thermodynamics of hybridisation were measured by comparing a fully methylated oligonucleotide product and a hemimethylated oligonucleotide substrate using a 13-bp duplex labeled on adjacent strands with a fluorophore (fluorescein) and quencher (dabcyl). Enzymatic methylation of the hemimethylated GATC site resulted in destabilisation of the duplex, increasing the proportion of dissociated DNA, and producing an observable increase in fluorescence. The assay provides a direct measurement of methylation rate in real time and is highly reproducible, with a coefficient of variance over 48 independent measurements of 3.6%. DNA methylation rates can be measured as low as 3.55 ± 1.84 fmols(-1) in a 96-well plate format, and the assay has been used to kinetically characterise the Pyrococcus horikoshii DNA adenine methyltransferase.
authors.affiliation: School of Chemistry, University of Southampton, Southampton, Hampshire SO17 1BJ, UK.
authors.forename: Michael D
authors.initials: MD
authors.lastname: Maynard-Smith
authors.forename: Jennifer C
authors.initials: JC
authors.lastname: McKelvie
authors.forename: Robert J
authors.initials: RJ
authors.lastname: Wood
authors.forename: Jenny E
authors.initials: JE
authors.lastname: Harmer
authors.forename: Rohan T
authors.initials: RT
authors.lastname: Ranasinghe
authors.forename: Claire L
authors.initials: CL
authors.lastname: Williams
authors.forename: Danielle M
authors.initials: DM
authors.lastname: Coomber
authors.forename: Alexandra F
authors.initials: AF
authors.lastname: Stares
authors.forename: Peter L
authors.initials: PL
authors.lastname: Roach
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D019175:DNA Methylation; D019793:Fluorescein / Q000032:analysis* / Q000737:chemistry; D007700:Kinetics; D043902:Pyrococcus horikoshii / Q000201:enzymology / Q000378:metabolism*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature; D013816:Thermodynamics; D004124:p-Dimethylaminoazobenzene / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry
chemical_list: C063127:4-(4-dimethylaminophenylazo)benzoic acid; D004247:DNA; D004124:p-Dimethylaminoazobenzene; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D019793:Fluorescein