Substrate binding in vitro and kinetics of RsrI [N6-adenine] DNA methyltransferase

Rsrl [N6-adenine] DNA methyltransferase (M . Rsrl), which recognizes GAATTC and is a member of a restriction-modification system in Rhodobacter sphaer oides, was purified to >95% homogeneity using a simplified procedure involv ing two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M . Rsrl were performed on unmethylated, hemimethylat ed, dimethylated or non-specific target DNA duplexes (25 bp) in the presenc e of sinefungin, a potent inhibitory analog of AdoMet, M . Rsrl binding was affected by the methylation status of the DNA substrate and was enhanced b y the presence of the cofactor analog. M . Rsrl bound DNA substrates in the presence of sinefungin with decreasing affinities: hemimethylated > unmeth ylated > dimethytated >> non-specific DNA, Gel retardation studies with DNA substrates containing an abasic site substituted for the target adenine DN A provided evidence consistent with M . Rsrl extruding the target base from the duplex. Consistent with such base flipping, an similar to1.7-fold fluo rescence intensity increase was observed upon stoichiometric addition of M . Rsrl to hemimethylated DNA containing the fluorescent analog P-aminopurin e in place of the target adenine, Pre-steady-state kinetic and isotope-part itioning experiments revealed that the enzyme displays burst kinetics, conf irmed the catalytic competence of the M . Rsrl-AdoMet complex and eliminate d the possibility of an ordered mechanism where DNA is required to bind fir st. The equilibrium dissociation constants for AdoMet, AdoHcy and sinefungi n were determined using an intrinsic tryptophan fluorescence-quenching assa y.