Kinetics of Methylation by EcoP1I DNA Methyltransferase
Figure 3
Preincubation analysis of M.EcoP1I and M.EcoP15I. (a) Preincubation analysis of M.EcoP1I. Methylation reactions were carried out in methylation buffer containing 400 nM M.EcoP1I, 1 M duplex I, or, alternatively, 1 M [3H-methyl]AdoMet. Methylation in the presence of a preformed M.EcoP1I-DNA binary complex (AdoMet added second) is indicated as filled circles (). Similarly, a preformed M.EcoP1I-AdoMet binary complex (DNA added second) is shown as empty circles (). 20 l aliquot of the reaction mixture was removed at a 15-second time interval, and the extent of methyl group incorporation was measured. (b) Preincubation analysis of M.EcoP15I (250 nM) with pUC19 DNA (1 M) and [3H-methyl]AdoMet (1 M). Methylation in the presence of a preformed M.EcoP15I-DNA binary complex (AdoMet added second) is indicated as filled circles (). Similarly, a preformed M.EcoP15I-AdoMet binary complex (DNA added second) is shown as empty circles (). (c) Isotope partitioning analysis of EcoP1I MTase. Methylation reactions were carried out in methylation buffer containing 400 nM M.EcoP1I, 1 M duplex I, and 1 M [3H-methyl]AdoMet. Curve 1 (, AdoMet*) shows product formation after enzyme was preincubated with 1 M [3H-methyl]AdoMet, and the reaction was started with addition of DNA and labeled [3H-methyl]AdoMet. Curve 2 (, AdoMet) shows product formation after the enzyme was preincubated with 1 M [3H-methyl]AdoMet and the reaction was started with addition of DNA and unlabeled AdoMet.
