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. 2000 Oct 15;28(20):3962-71.
doi: 10.1093/nar/28.20.3962.

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

S S Szegedi  1 , N O Reich, R I Gumport
Affiliations

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

S S Szegedi et al. Nucleic Acids Res. .

Abstract

RSR:I [N:6-adenine] DNA methyltransferase (M.RSR:I), which recognizes GAATTC and is a member of a restriction-modification system in Rhodobacter sphaeroides, was purified to >95% homogeneity using a simplified procedure involving two ion exchange chromatographic steps. Electrophoretic gel retardation assays with purified M.RSR:I were performed on unmethylated, hemimethylated, dimethylated or non-specific target DNA duplexes (25 bp) in the presence of sinefungin, a potent inhibitory analog of AdoMet. M. RSR:I binding was affected by the methylation status of the DNA substrate and was enhanced by the presence of the cofactor analog. M. RSR:I bound DNA substrates in the presence of sinefungin with decreasing affinities: hemimethylated > unmethylated > dimethylated >> non-specific DNA. Gel retardation studies with DNA substrates containing an abasic site substituted for the target adenine DNA provided evidence consistent with M.RSR:I extruding the target base from the duplex. Consistent with such base flipping, an approximately 1.7-fold fluorescence intensity increase was observed upon stoichiometric addition of M.RSR:I to hemimethylated DNA containing the fluorescent analog 2-aminopurine in place of the target adenine. Pre-steady-state kinetic and isotope- partitioning experiments revealed that the enzyme displays burst kinetics, confirmed the catalytic competence of the M.RSR:I-AdoMet complex and eliminated the possibility of an ordered mechanism where DNA is required to bind first. The equilibrium dissociation constants for AdoMet, AdoHcy and sinefungin were determined using an intrinsic tryptophan fluorescence-quenching assay.

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Figures

Figure 1
Figure 1
Binding isotherm of M·RsrI with unmethylated DNA and sinefungin. The DNA (5 nM) was sequence TB (Table 1). The curve was generated by fitting the average of three gel retardation assays as described in Materials and Methods. Error bars represent ± 1 SD. (Inset) Representative gel retardation image with [M·RsrI] increasing from 0 to 400 nM.
Figure 2
Figure 2
Stimulation of DNA binding by sinefungin. M·RsrI (15 nM) was incubated with increasing amounts of sinefungin. The data from three experiments were analyzed as described in Materials and Methods.
Figure 3
Figure 3
Steady-state fluorescence emission spectra of M·RsrI (I) and M·EcoRI (II) with 2ATmB DNA. (A) Buffer only, Raman peak at 348 nm; (B) enzyme only, ∼450 nM M·RsrI (λmax = 348 nm) or M·EcoRI (λmax = 348 nm); (C) 400 nM 2A-containing DNA duplex (λmax = ∼371 nm for both sets of spectra); (D) 400 nM single-strand 2A-containing DNA (λmax = 368 nm for both sets of spectra); (E) addition of ∼450 nM purified M·RsrI (λmax = 351nm and 364 nm) or M·EcoRI (λmax = 362 nm) to duplex 2-A-containing DNA.
Figure 4
Figure 4
Lehrer plot of quenching of M·RsrI fluorescence by sinefungin. Error bars represent ± 1 SD.
Figure 5
Figure 5
Kinetic analysis of M·RsrI. (A) Burst. M·RsrI was saturated with high specific activity AdoMet and diluted 50-fold into a solution containing the same specific activity AdoMet at the same concentration and DNA. (B) Isotope partition. Curve 1, re-plot of curve in (A); curve 2, enzyme was saturated with high specific activity AdoMet as in (A) but diluted into a solution containing the same concentration of unlabeled AdoMet and excess DNA; curve 3, control. Enzyme saturated with high specific activity AdoMet was diluted 50-fold into unlabeled AdoMet of the same concentration before addition of DNA. Error bars represent ± 1 SD.
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