DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens: mechanism of action derived from steady state kinetics

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the 5'-GGATCC recognition site catalyzed by the DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens [EC 2.1.1.113] has shown that the dependence of the rate of methylation of the 20-meric substrate duplex on SAM and DNA concentration are normally hyperbolic, and the maximal rate is attained upon enzyme saturation with both substrates. No substrate inhibition is observed even at concentrations many times higher than the Km values (0.107 microM for DNA and 1.45 microM for SAM), which means that no nonreactive enzyme-substrate complexes are formed during the reaction. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAM decreases DNA decreases metDNA increases SAH increases (S-adenosyl-L-homocysteine). However, more detailed numerical analysis of the aggregate experimental data admits an alternative order of substrate binding, DNA decreases SAM decreases, though this route is an order of magnitude slower.     

DNA-(N4-Cytosine)-Methyltransferase from Bacillus amyloliquefaciens: Kinetic and Substrate-binding Properties

Interaction of DNA-(N4-cytosine)-methyltransferase from the Bacillus amyloliquefaciens (BamHI MTase, 49 kDa) with a 20-mer duplex containing a palindromic recognition site GGATCC was studied by methods of steady-state and pre-steady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaraldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward the 20-mer substrate. A linear dependence was observed for the reaction rate on the enzyme concentration and a Michaelis dependence of the reaction rate on the concentration of both substrates: S-adenosyl-L-methionine (SAM), the methyl group donor, and DNA, the methyl group acceptor. In independent experiments, the concentration of the 20-mer duplex or SAM was changed, the enzyme concentration being substantially lower than the concentrations of substrates. The k _cat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s^–1. The K _M values for the duplex and SAM are 0.35 and 1.6 M, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer duplex) revealed the following characteristics of the BamHI MTase-dependent methylation of DNA. The value of rate constant of the DNA methylation step at the enzyme saturating concentration is on average 0.085 s^–1, which is only 1.6 times higher than the value determined in steady-state conditions. Only one of two target cytidine residues was methylated in a single turnover of the enzyme, which coincides with the earlier data on EcoRI MTase. Regardless of the order of enzyme preincubation with SAM and DNA, both curves for the single turnover methylation are comparable. These results are consistent with the model of the random order of the productive ternary enzyme–substrate complex formation. In contrast to the relatively simple kinetic behavior of BamHI MTase in the steady-state reaction are the data on the enzyme binding with DNA. In gel retardation experiments, there was no stoichiometrically simple complex with the oligonucleotide duplex even at low enzyme concentrations. The molecular mass of the complexes was so high that they did not enter 12% PAG. In experiments on crosslinking of the BamHI MTase subunits, it was shown that the enzyme in a free state exists as a dimer. Introduction of substoichiometric amounts of DNA into the reaction mixture results in pronounced multimerization of the enzyme. However, addition of SAM in saturating concentration at an excess of the oligonucleotide duplex over BamHI MTase converts most of the enzyme into a monomeric state.     

The kinetic mechanism of phage T4 DNA-[N6-adenine]-methyltransferase

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the GATC recognition site catalyzed by the phage T4 DNA-[N6-adenine]-methyltransferase (MTase) [EC 2.1.1.72] showed that the reverse reaction is at least 500 times slower than the direct one. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAM decreases DNA decreases metDNA increases SAH increases (S-adenosyl-L-homocysteine). Pronounced inhibition was observed at high concentrations of the 20-meric substrate duplex, which may be attributed to formation of a dead-end complex MTase-SAH-DNA. In contrast, high SAM concentrations proportionally accelerated the reaction. Thus, the reaction may include a stage whereby the binding of SAM and the release of SAH are united into one concerted event. Computer fitting of alternative kinetic schemes to the aggregate of experimental data revealed that the most plausible mechanism involves isomerization of the enzyme.     

DNA-[N4-Cytosine]-Methyltransferase from Bacillus amyloliquefaciens: Mechanism of Action Derived from Steady-State Kinetics

Kinetic analysis of methyl group transfer from S-adenosyl-L-methionine (SAM) to the 5"-GGATCC recognition site catalyzed by the DNA-[N4-cytosine]-methyltransferase from Bacillus amyloliquefaciens [EC 2.1.1.113] has shown that the dependence of the rate of methylation of the 20-meric substrate duplex on SAM and DNA concentration are normally hyperbolic, and the maximal rate is attained upon enzyme saturation with both substrates. No substrate inhibition is observed even at concentrations many times higher than the K _M values (0.107 M for DNA and 1.45 M for SAM), which means that no nonreactive enzyme–substrate complexes are formed during the reaction. The overall pattern of product inhibition corresponds to an ordered steady-state mechanism following the sequence SAMDNAmetDNA SAH (S-adenosyl-L-homocysteine). However, more detailed numerical analysis of the aggregate experimental data admits an alternative order of substrate binding, DNA SAM }, though this route is an order of magnitude slower.     

Effect of S-adenosyl-L-methionine and its analogs on site-specific binding DNA-(adenine-N6)-methyltransferase from T4 phage to the oligonucleotide substrate

Using fluorescence of 2-aminopurine-substituted oligonucleotide duplexes, "flipping" of the target base in the process of interaction of T4 DNA-(adenine-N6)-methyltransferase (EC 2.1.1.72) with the substrate double-stranded DNA was revealed. It was shown that S-adenosyl-L-methionine, the methyl group donor, induces the reorientation of the enzyme relative to the asymmetrically modified recognition site.     

Effect ofS-adenosyl-L-methionine and its analogues on site-specific binding of DNA-(adenine-N 6)-methyltransferase of T4 phage with the oligonucleotide substrate

Using fluorescence of 2-aminopurine-substituted oligonucleotide duplexes, “flipping” of the target base in the process of interaction of T4 DNA-(adenine-N ⁶)-methyltransferase (EC 2.1.1.72) with the substrate double-stranded DNA was revealed. It was shown thatS-adenosyl-L-methionine, the methyl group donor, induces the reorientation of the enzyme relative to the unsymmetrically modified recognition site.     

Bacteriophage T4 Dam DNA-(N6-adenine)-methyltransferase. Processivity and orientation to the methylation target

We carried out steady state and pre-steady state (burst) kinetic analyses of the bacteriophage T4 Dam DNA-(N(6)-adenine)-methyltransferase (MTase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to Ade in oligonucleotide duplexes containing one or two specific GATC sites with different combinations of methylated and unmodified targets. We compared the results for ligated 40-mer duplexes with those of the mixtures of the two unligated duplexes used to generate the 40-mers. The salient results are as follows: (i) T4 Dam MTase modifies 40-mer duplexes in a processive fashion. (ii) During processive movement, T4 Dam rapidly exchanges product S-adenosyl-l-homocysteine (AdoHcy) for substrate AdoMet without dissociating from the DNA duplex. (iii) T4 Dam processivity is consistent with an ordered bi-bi mechanism AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. However, in contrast to the steady state, here DNA(Me) upward arrow signifies departure from a methylated site GMTC upward arrow without physically dissociating from the DNA. (iv) Following methyl transfer at one site and linear diffusion to a hemimethylated site, a reconstituted T4 Dam-AdoMet complex rapidly reorients itself to the (productive) unmethylated strand. T4 Dam-AdoHcy cannot reorient at an enzymatically created GMTC site. (v) The inhibition potential of fully methylated sites 5'-GMTC/5'-GMTC is much lower for a long DNA molecule compared with short single-site duplexes.     

A dual role for substrate S-adenosyl-L-methionine in the methylation reaction with bacteriophage T4 Dam DNA-[N6-adenine]-methyltransferase

The fluorescence of 2-aminopurine ((2)A)-substituted duplexes (contained in the GATC target site) was investigated by titration with T4 Dam DNA-(N6-adenine)-methyltransferase. With an unmethylated target ((2)A/A duplex) or its methylated derivative ((2)A/(m)A duplex), T4 Dam produced up to a 50-fold increase in fluorescence, consistent with (2)A being flipped out of the DNA helix. Though neither S-adenosyl-L-homocysteine nor sinefungin had any significant effect, addition of substrate S-adenosyl-L-methionine (AdoMet) sharply reduced the Dam-induced fluorescence with these complexes. In contrast, AdoMet had no effect on the fluorescence increase produced with an (2)A/(2)A double-substituted duplex. Since the (2)A/(m)A duplex cannot be methylated, the AdoMet-induced decrease in fluorescence cannot be due to methylation per se. We propose that T4 Dam alone randomly binds to the asymmetric (2)A/A and (2)A/(m)A duplexes, and that AdoMet induces an allosteric T4 Dam conformational change that promotes reorientation of the enzyme to the strand containing the native base. Thus, AdoMet increases enzyme binding-specificity, in addition to serving as the methyl donor. The results of pre-steady-state methylation kinetics are consistent with this model.     

Properties of DNA-(cytosine-5-)-methyltransferase in the brain

Beef brain DNA-(cytosine-5-)-methyltransferase was partially purified by chromatography on Ultrogel AcA34 and Dyematrex Blue A. The purification was of 360 times and the recovery of 75%. The pH optimum of the reaction is 7.6 NaCl inhibits double stranded DNA methylation, but stimulates single stranded DNA methylation up to 50 mM, before inhibiting. EDTA (1 mM) and MgCl2 (4 mM) stimulate DNA methylation. Polyamines inhibit the reaction.     

Oligomerization of DNA-(adenine-N6)-methyltransferase from phage T4 and its effect on the catalytic characteristics of the enzyme

The structural and catalytic properties of the phage T4 DNA-(adenine-N6)-methyltransferase (EC 2.1.1.72) were studied at different enzyme-substrate concentration ratios by chemical cross-linking of the protein subunits and by measuring the presteady state kinetics of the reactions. Various structural states of the methyltransferase were correlated with its catalytic activity, and it was shown that the oligomeric forms of the enzyme are catalytically active but are characterized by the reaction parameters different from those of the monomer.     

Sequence, internal homology and high-level expression of the gene for a DNA-(cytosine N4)-methyltransferase, M.Pvu II.

The base sequence of the pvuIIM gene has been determined. This gene codes for a DNA-(cytosine N4)-methyltransferase, M.Pvu II. The base sequence contains a single large open reading frame that predicts a 38.3kDa polypeptide, consistent with experimental data. The pvuIIM gene contains some sequences common to DNA methyltransferases in general, but includes none of the sequences specifically conserved among DNA-(cytosine 5)-methyltransferases. The pvuIIM sequence also reveals an internal homology at the amino acid level, each half of which spans over 100 amino acids and is itself homologous to the sequences of some DNA-(adenine N6)-methyltransferases. A derivative of the pvuIIM plasmid was constructed to allow high-level production of M.Pvu II. Specifically, the composite Ptac promoter was inserted 5' to pvuIIM, intervening DNA was deleted, and the resulting construct was used to transform an mcrB laclq strain of Escherichia coli. When this transformant was induced with isopropyl-B-D-galactopyranoside (IPTG), growth rapidly ceased and M.Pvu II accumulated to the point of comprising over 10% of the total soluble protein.     

Molecular enzymology of the EcoRV DNA-(Adenine-N (6))-methyltransferase: kinetics of DNA binding and bending, kinetic mechanism and linear diffusion of the enzyme on DNA

The EcoRV DNA-(adenine-N(6))-methyltransferase recognizes GATATC sequences and modifies the first adenine residue within this site. We show here, that the enzyme binds to the DNA and the cofactor S-adenosylmethionine (AdoMet) in an ordered bi-bi fashion, with AdoMet being bound first. M.EcoRV binds DNA in a non-specific manner and the enzyme searches for its recognition site by linear diffusion with a range of approximately 1800 bp. During linear diffusion the enzyme continuously scans the DNA for the presence of recognition sites. Upon specific M.EcoRV-DNA complex formation a strong increase in the fluorescence of an oligonucleotide containing a 2-aminopurine base analogue at the GAT-2AP-TC position is observed which, most likely, is correlated with DNA bending. In contrast to the GAT-2AP-TC substrate, a G-2AP-TATC substrate in which the target base is replaced by 2-aminopurine does not show an increase in fluorescence upon M.EcoRV binding, demonstrating that 2-aminopurine is not a general tool to detect base flipping. Stopped-flow experiments show that DNA bending is a fast process with rate constants >10 s(-1). In the presence of cofactor, the specific complex adopts a second conformation, in which the target sequence is more tightly contacted by the enzyme. M.EcoRV exists in an open and in a closed state that are in slow equilibrium. Closing the open state is a slow process (rate constant approximately 0.7 min(-1)) that limits the rate of DNA methylation under single turnover conditions. Product release requires opening of the closed complex which is very slow (rate constant approximately 0.05-0.1 min(-1)) and limits the rate of DNA methylation under multiple turnover conditions. M.EcoRV methylates DNA sequences containing more than one recognition sites in a distributive manner. Since the dissociation rate from non-specific DNA does not depend on the length of the DNA fragment, DNA dissociation does not preferentially occur at the ends of the DNA.     

Bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase): evidence for two distinct stages of methylation under single turnover conditions

We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5'-GATC/5'-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA < 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-l-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages. First, two preformed T4Dam-AdoMet complexes bind opposite strands of the unmodified target site, and one enzyme molecule catalyzes the rapid transfer of the AdoMet-methyl group (kmeth1 = 0.21 s-1); this is 2.5-fold slower than the rate observed with monomeric T4Dam-AdoMet bound under pre-steady-state conditions for burst determination. In the second stage, methyl transfer to adenine in GATC on the complementary strand occurs at a rate that is 1 order of magnitude slower (kmeth2 = 0.023 s-1). We suggest that under single turnover conditions, methylation of the second strand is rate-limited by Dam-AdoHcy dissociation or its clearance from the methylated complementary strand. The hemimethylated duplex 5'-GATC/5'-GMTC also interacts with T4Dam-AdoMet complexes in two stages under single turnover reaction conditions. The first stage (kmeth1) reflects methylation by dimeric T4Dam-AdoMet productively oriented to the strand with the adenine residue capable of methylation. The slower second stage (kmeth2) reflects methylation by enzyme molecules non-productively oriented to the GMTC chain, which then have to re-orient to the opposite productive chain. Substitutions of bases and deletions in the recognition site affect the kinetic parameters in different fashions. When the GAT portion of GATC was disrupted, the proportion of the initial productive enzyme-substrate complexes was sharply reduced.     

Conserved sequence motif DPPY in region IV of the phage T4 Dam DNA-[N6-adenine]-methyltransferase is important for S-adenosyl-L-methionine binding.

Comparison of the deduced amino acid sequences of DNA-[N6-adenine]-methyltransferases has revealed several conserved regions. All of these enzymes contain a DPPY [or closely related] motif. By site-directed mutagenesis of a cloned T4 dam gene, we have altered the first proline residue in this motif [located in conserved region IV of the T4 Dam-MTase] to alanine or threonine. The mutant enzymic forms, P172A and P172T, were overproduced and purified. Kinetic studies showed that compared to the wild-type [wt] the two mutant enzymic forms had: (i) an increased [5 and 20-fold, respectively] Km for substrate, S-adenosyl-methionine [AdoMet]; (ii) a slightly reduced [2 and 4-fold lower] kcat; (iii) a strongly reduced kcat/KmAdoMet [10 and 100-fold]; and (iv) almost the same Km for substrate DNA. Equilibrium dialysis studies showed that the mutant enzymes had a reduced [4 and 9-fold lower] Ka for AdoMet. Taken together these data indicate that the P172A and P172T alterations resulted primarily in a reduced affinity for AdoMet. This suggests that the DPPY-motif is important for AdoMet-binding, and that region IV contains or is part of an AdoMet-binding site.     

The M.EcoRV DNA-(adenine N6)-methyltransferase uses DNA bending for recognition of an expanded EcoDam recognition site

The M.EcoRV DNA methyltransferase recognizes GATATC sites. It is related to EcoDam, which methylates GATC sites. The DNA binding domain of M.EcoRV is similar to that of EcoDam suggesting a similar mechanism of DNA recognition. We show that amino acid residue Lys11 of M.EcoRV is involved in recognition of Gua1 and Arg128 contacts the Gua in base pair 6. These residues correspond to Lys9 and Arg124 in EcoDam, which recognize the Gua residues in both strands of the Dam recognition sequence, indicating that M.EcoRV and EcoDam make similar contacts to outermost base pairs of their recognition sequences and M.EcoRV recognizes its target site as an expanded GATC site. In contrast to EcoDam, M.EcoRV considerably bends the DNA (59+/-4 degrees) suggesting indirect readout of the AT-rich inner sequence. Recognition of an expanded target site by DNA bending is a new principle for changing DNA recognition specificity of proteins during molecular evolution. R128A is inefficient in DNA bending and binding, whereas K11A bends DNA with relaxed sequence specificity. These results suggest a temporal order of the formation of protein-DNA contacts in which the Gua6-Arg128 contact forms early followed by DNA bending and, finally, the formation of the Lys11-Gua1 contact.