Probing a rate-limiting step by mutational perturbation of AdoMet binding in the HhaI methyltransferase

DNA methylation plays important roles via regulation of numerous cellular mechanisms in diverse organisms, including humans. The paradigm bacterial methyltransferase (MTase) HhaI (M.HhaI) catalyzes the transfer of a methyl group from the cofactor S-adenosyl-L-methionine (AdoMet) onto the target cytosine in DNA, yielding 5-methylcytosine and S-adenosyl-L-homocysteine (AdoHcy). The turnover rate (k cat) of M.HhaI, and the other two cytosine-5 MTases examined, is limited by a step subsequent to methyl transfer; however, no such step has so far been identified. To elucidate the role of cofactor interactions during catalysis, eight mutants of Trp41, which is located in the cofactor binding pocket, were constructed and characterized. The mutants show full proficiency in DNA binding and base-flipping, and little variation is observed in the apparent methyl transfer rate k chem as determined by rapid-quench experiments using immobilized fluorescent-labeled DNA. However, the Trp41 replacements with short side chains substantially perturb cofactor binding (100-fold higher K(AdoMet)D and K(AdoMet)M) leading to a faster turnover of the enzyme (10-fold higher k cat). Our analysis indicates that the rate-limiting breakdown of a long-lived ternary product complex is initiated by the dissociation of AdoHcy or the opening of the catalytic loop in the enzyme.     

5-Fluorocytosine in DNA is a mechanism-based inhibitor of HhaI methylase

5-Fluorodeoxycytidine (FdCyd) was incorporated into a synthetic DNA polymer containing the GCGC recognition sequence of HhaI methylase to give a polymer with about 80% FdCyd. In the absence of AdoMet, poly(FdC-dG) bound competitively with respect to poly(dG-dC) (Ki = 3 nM). In the presence of AdoMet, the analogue caused a time-dependent, first-order (k = 0.05 min-1) inactivation of the enzyme. There is an ordered mechanism of binding in which enzyme first binds to poly(FdC-dG), then binds to AdoMet, and subsequently forms stable, inactive complexes. The complexes did not dissociate over the course of 3 days and were stable to heat (95 degrees C) in the presence of 1% SDS. Gel filtration of a complex formed with HhaI methylase, poly(FdC-dG), and [methyl-3H] AdoMet gave a peak of radioactivity eluting near the void volume. Digestion of the DNA in the complex resulted in a reduction of the molecular weight to the size of the methylase, and the radioactivity in this peak was shown to be associated with protein. These data indicate that the complexes contain covalently bound HhaI methylase, poly(FdC-dG), and methyl groups and that 5-fluorodeoxycytidine is a mechanism-based inactivator of the methylase. By analogy with other pyrimidine-modifying enzymes and recent studies on the mechanism of HhaI methylase (Wu & Santi, 1987), these results suggest that an enzyme nucleophile attacks FdCyd residues at C-6, activating the 5-position for one-carbon transfer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Water-assisted dual mode cofactor recognition by HhaI DNA methyltransferase.

Energetically competent binary recognition of the cofactor S-adenosyl-L-methionine (AdoMet) and the product S-adenosyl-L-homocysteine (AdoHcy) by the DNA (cytosine C-5) methyltransferase (M.HhaI) is demonstrated herein. Titration calorimetry reveals a dual mode, involving a primary dominant exothermic reaction followed by a weaker endothermic one, for the recognition of AdoMet and AdoHcy by M.HhaI. Conservation of the bimodal recognition in W41I and W41Y mutants of M.HhaI excludes the cation-pi interaction between the methylsulfonium group of AdoMet and the pi face of the Trp(41) indole ring from a role in its origin. Small magnitude of temperature-independent heat capacity changes upon AdoMet or AdoHcy binding by M.HhaI preclude appreciable conformational alterations in the reacting species. Coupled osmotic-calorimetric analyses of AdoMet and AdoHcy binding by M.HhaI indicate that a net uptake of nearly eight and 10 water molecules, respectively, assists their primary recognition. A change in water activity at constant temperature and pH is sufficient to engender and conserve enthalpy-entropy compensation, consistent with a true osmotic effect. The results implicate solvent reorganization in providing the major contribution to the origin of this isoequilibrium phenomenon in AdoMet and AdoHcy recognition by M.HhaI. The observations provide unequivocal evidence for the binding of AdoMet as well as AdoHcy to M.HhaI in solution state. Isotope partitioning analysis and preincubation studies favor a random mechanism for M.HhaI-catalyzed reaction. Taken together, the results clearly resolve the issue of cofactor recognition by free M.HhaI, specifically in the absence of DNA, leading to the formation of an energetically and catalytically competent binary complex.     

Inhibition of HhaI DNA (Cytosine-C5) methyltransferase by oligodeoxyribonucleotides containing 5-aza-2'-deoxycytidine: examination of the intertwined roles of co-factor,target, transition state structure and enzyme conformation

The presence of 5-azacytosine (ZCyt) residues in DNA leads to potent inhibition of DNA (cytosine-C5) methyltranferases (C5-MTases) in vivo and in vitro. Enzymatic methylation of cytosine in mammalian DNA is an epigenetic modification that can alter gene activity and chromosomal stability, influencing both differentiation and tumorigenesis. Thus, it is important to understand the critical mechanistic determinants of ZCyt's inhibitory action. Although several DNA C5-MTases have been reported to undergo essentially irreversible binding to ZCyt in DNA, there is little agreement as to the role of AdoMet and/or methyl transfer in stabilizing enzyme interactions with ZCyt. Our results demonstrate that formation of stable complexes between HhaI methyltransferase (M.HhaI) and oligodeoxyribonucleotides containing ZCyt at the target position for methylation (ZCyt-ODNs) occurs in both the absence and presence of co-factors, AdoMet and AdoHcy. Both binary and ternary complexes survive SDS-PAGE under reducing conditions and take on a compact conformation that increases their electrophoretic mobility in comparison to free M.HhaI. Since methyl transfer can occur only in the presence of AdoMet, these results suggest (1) that the inhibitory capacity of ZCyt in DNA is based on its ability to induce a stable, tightly closed conformation of M.HhaI that prevents DNA and co-factor release and (2) that methylation of ZCyt in DNA is not required for inhibition of M.HhaI.     

S-adenosyl-L-methionine-dependent methyl transfer: observable precatalytic intermediates during DNA cytosine methylation

S-adenosyl-L-methionine- (AdoMet-) dependent methyltransferases are widespread, play critical roles in diverse biological pathways, and are antibiotic and cancer drug targets. Presently missing from our understanding of any AdoMet-dependent methyl-transfer reaction is a high-resolution structure of a precatalytic enzyme/AdoMet/DNA complex. The catalytic mechanism of DNA cytosine methylation was studied by structurally and functionally characterizing several active site mutants of the bacterial enzyme M.HhaI. The 2.64 A resolution protein/DNA/AdoMet structure of the inactive C81A M.HhaI mutant suggests that active site water, an approximately 13 degree tilt of the target base toward the active site nucleophile, and the presence or absence of the cofactor methylsulfonium are coupled via a hydrogen-bonding network involving Tyr167. The active site in the mutant complex is assembled to optimally align the pyrimidine for nucleophilic attack and subsequent methyl transfer, consistent with previous molecular dynamics ab initio and quantum mechanics/molecular mechanics calculations. The mutant/DNA/AdoHcy structure (2.88 A resolution) provides a direct comparison to the postcatalytic complex. A third C81A ternary structure (2.22 A resolution) reveals hydrolysis of AdoMet to adenosine in the active site, further validating the coupling between the methionine portion of AdoMet and ultimately validating the structural observation of a prechemistry/postchemistry water network. Disruption of this hydrogen-bonding network by a Tyr167 to Phe167 mutation does not alter the kinetics of nucleophilic attack or methyl transfer. However, the Y167F mutant shows detectable changes in kcat, caused by the perturbed kinetics of AdoHcy release. These results provide a basis for including an extensive hydrogen-bonding network in controlling the rate-limiting product release steps during cytosine methylation.     

Kinetic and catalytic mechanism of HhaI methyltransferase

Kinetic and catalytic properties of the DNA (cytosine-5)-methyltransferase HhaI are described. With poly(dG-dC) as substrate, the reaction proceeds by an equilibrium (or processive) ordered Bi-Bi mechanism in which DNA binds to the enzyme first, followed by S-adenosylmethionine (AdoMet). After methyl transfer, S-adenosylhomocysteine (AdoHcy) dissociates followed by methylated DNA. AdoHcy is a potent competitive inhibitor with respect to AdoMet (Ki = 2.0 microM) and its generation during reactions results in non-linear kinetics. AdoMet and AdoHcy significantly interact with only the substrate enzyme-DNA complex; they do not bind to free enzyme and bind poorly to the methylated enzyme-DNA complex. In the absence of AdoMet, HhaI methylase catalyzes exchange of the 5-H of substrate cytosines for protons of water at about 7-fold the rate of methylation. The 5-H exchange reaction is inhibited by AdoMet or AdoHcy. In the enzyme-DNA-AdoHcy complex, AdoHcy also suppresses dissociation of DNA and reassociation of the enzyme with other substrate sequences. Our studies reveal that the catalytic mechanism of DNA (cytosine-5)-methyltransferases involves attack of the C6 of substrate cytosines by an enzyme nucleophile and formation of a transient covalent adduct. Based on precedents of other enzymes which catalyze similar reactions and the susceptibility of HhaI to inactivation by N-ethylmaleimide, we propose that the sulfhydryl group of a cysteine residue is the nucleophilic catalyst. Furthermore, we propose that Cys-81 is the active-site catalyst in HhaI. This residue is found in a Pro-Cys doublet which is conserved in all DNA (cytosine-5)-methyltransferases whose sequences have been determined to date and is found in related enzymes. Finally, we discuss the possibility that covalent adducts between C6 of pyrimidines and nucleophiles of proteins may be important general components of protein-nucleic acid interactions.     

The role of Arg165 towards base flipping, base stabilization and catalysis in M.HhaI

Arg165 forms part of a previously identified base flipping motif in the bacterial DNA cytosine methyltransferase, M.HhaI. Replacement of Arg165 with Ala has no detectable effect on either DNA or AdoMet affinity, yet causes the base flipping and restacking transitions to be decreased approximately 16 and 190-fold respectively, thus confirming the importance of this motif. However, these kinetic changes cannot account for the mutant's observed 10(5)-fold decreased catalytic rate. The mutant enzyme/cognate DNA cocrystal structure (2.79 A resolution) shows the target cytosine to be positioned approximately 30 degrees into the major groove, which is consistent with a major groove pathway for nucleotide flipping. The pyrimidine-sugar chi angle is rotated to approximately +171 degrees, from a range of -95 degrees to -120 degrees in B DNA, and -77 degrees in the WT M.HhaI complex. Thus, Arg165 is important for maintaining the cytosine positioned for nucleophilic attack by Cys81. The cytosine sugar pucker is in the C2'-endo-C3'-exo (South conformation), in contrast to the previously reported C3'-endo (North conformation) described for the original 2.70 A resolution cocrystal structure of the WT M.HhaI/DNA complex. We determined a high resolution structure of the WT M.HhaI/DNA complex (1.96 A) to better determine the sugar pucker. This new structure is similar to the original, lower resolution WT M.HhaI complex, but shows that the sugar pucker is O4'-endo (East conformation), intermediate between the South and North conformers. In summary, Arg165 plays significant roles in base flipping, cytosine positioning, and catalysis. Furthermore, the previously proposed M.HhaI-mediated changes in sugar pucker may not be an important contributor to the base flipping mechanism. These results provide insights into the base flipping and catalytic mechanisms for bacterial and eukaryotic DNA methyltransferases.     

The mechanism of DNA cytosine-5 methylation. Kinetic and mutational dissection of Hhai methyltransferase

Kinetic and binding studies involving a model DNA cytosine-5-methyltransferase, M.HhaI, and a 37-mer DNA duplex containing a single hemimethylated target site were applied to characterize intermediates on the reaction pathway. Stopped-flow fluorescence studies reveal that cofactor S-adenosyl-l-methionine (AdoMet) and product S-adenosyl-l-homocysteine (AdoHcy) form similar rapidly reversible binary complexes with the enzyme in solution. The M.HhaI.AdoMet complex (k(off) = 22 s(-)1, K(D) = 6 microm) is partially converted into products during isotope-partitioning experiments, suggesting that it is catalytically competent. Chemical formation of the product M.HhaI.(Me)DNA.AdoHcy (k(chem) = 0.26 s(-)1) is followed by a slower decay step (k(off) = 0.045 s(-)1), which is the rate-limiting step in the catalytic cycle (k(cat) = 0.04 s(-)1). Analysis of reaction products shows that the hemimethylated substrate undergoes complete (>95%) conversion into fully methylated product during the initial burst phase, indicating that M.HhaI exerts high binding selectivity toward the target strand. The T250N, T250D, and T250H mutations, which introduce moderate perturbation in the catalytic site, lead to substantially increased K(D)(DNA(ternary)), k(off)(DNA(ternary)), K(M)(AdoMet(ternary)) values but small changes in K(D)(DNA(binary)), K(D)(AdoMet(binary)), k(chem), and k(cat). When the target cytosine is replaced with 5-fluorocytosine, the chemistry step leading to an irreversible covalent M.HhaI.DNA complex is inhibited 400-fold (k(chem)(5FC) = 0.7 x 10(-)3 s(-)1), and the Thr-250 mutations confer further dramatic decrease of the rate of the covalent methylation k(chem). We suggest that activation of the pyrimidine ring via covalent addition at C-6 is a major contributor to the rate of the chemistry step (k(chem)) in the case of cytosine but not 5-fluorocytosine. In contrast to previous reports, our results imply a random substrate binding order mechanism for M.HhaI.     

DNA containing 4'-thio-2'-deoxycytidine inhibits methylation by HhaI methyltransferase.

4'-Thio-2'-deoxycytidine was synthesized as a 5'- protected phosphoramidite compatible with solid phase DNA synthesis. When incorporated as the target cytosine (C*) in the GC*GC recognition sequence for the DNA methyltransferase M. HhaI, methyl transfer was strongly inhibited. In contrast, these same oligonucleotides were normal substrates for the cognate restriction endonuclease R. HhaI and its isoschizomer R. Hin P1I. M. HhaI was able to bind both 4'-thio-modified DNA and unmodified DNA to equivalent extents under equilibrium conditions. However, the presence of 4'-thio-2'-deoxycytidine decreased the half-life of the complex by >10-fold. The crystal structure of a ternary complex of M. HhaI, AdoMet and DNA containing 4'-thio-2'-deoxycytidine was solved at 2.05 A resolution with a crystallographic R-factor of 0.186 and R-free of 0.231. The structure is not grossly different from previously solved ternary complexes containing M. HhaI, DNA and AdoHcy. The difference electron density suggests partial methylation at C5 of the flipped target 4'-thio-2'-deoxycytidine. The inhibitory effect of the 4'sulfur atom on enzymatic activity may be traced to perturbation of a step in the methylation reaction after DNA binding but prior to methyl transfer. This inhibitory effect can be partially overcome after a considerably long time in the crystal environment where the packing prevents complex dissociation and the target is accurately positioned within the active site.     

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.     

Reconciling structure and function in HhaI DNA cytosine-C-5 methyltransferase

Pre-steady state partitioning analysis of the HhaI DNA methyltransferase directly demonstrates the catalytic competence of the enzyme.DNA complex and the lack of catalytic competence of the enzyme.S-adenosyl-L-methionine (AdoMet) complex. The enzyme.AdoMet complex does form, albeit with a 50-fold decrease in affinity compared with the ternary enzyme.AdoMet.DNA complex. These findings reconcile the distinct binding orientations previously observed within the binary enzyme.AdoMet and ternary enzyme. S-adenosyl-L-homocysteine.DNA crystal structures. The affinity of the enzyme for DNA is increased 900-fold in the presence of its cofactor, and the preference for hemimethylated DNA is increased to 12-fold over unmethylated DNA. We suggest that this preference is partially due to the energetic cost of retaining a cavity in place of the 5-methyl moiety in the ternary complex with the unmethylated DNA, as revealed by the corresponding crystal structures. The hemi- and unmethylated substrates alter the fates and lifetimes of discrete enzyme.substrate intermediates during the catalytic cycle. Hemimethylated substrates partition toward product formation versus dissociation significantly more than unmethylated substrates. The mammalian DNA cytosine-C-5 methyltransferase Dnmt1 shows an even more pronounced partitioning toward product formation.     

Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex.

The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity. The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography. The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste. M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor. The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet. From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined. The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique. Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 A and are suitable for structure determination.     

Engineering the DNA cytosine-5 methyltransferase reaction for sequence-specific labeling of DNA

DNA methyltransferases catalyse the transfer of a methyl group from the ubiquitous cofactor S-adenosyl-L-methionine (AdoMet) onto specific target sites on DNA and play important roles in organisms from bacteria to humans. AdoMet analogs with extended propargylic side chains have been chemically produced for methyltransferase-directed transfer of activated groups (mTAG) onto DNA, although the efficiency of reactions with synthetic analogs remained low. We performed steric engineering of the cofactor pocket in a model DNA cytosine-5 methyltransferase (C5-MTase), M.HhaI, by systematic replacement of three non-essential positions, located in two conserved sequence motifs and in a variable region, with smaller residues. We found that double and triple replacements lead to a substantial improvement of the transalkylation activity, which manifests itself in a mild increase of cofactor binding affinity and a larger increase of the rate of alkyl transfer. These effects are accompanied with reduction of both the stability of the product DNA–M.HhaI–AdoHcy complex and the rate of methylation, permitting competitive mTAG labeling in the presence of AdoMet. Analogous replacements of two conserved residues in M.HpaII and M2.Eco31I also resulted in improved transalkylation activity attesting a general applicability of the homology-guided engineering to the C5-MTase family and expanding the repertoire of sequence-specific tools for covalent in vitro and ex vivo labeling of DNA.     

Mutational analysis of conserved residues in HhaI DNA methyltransferase

HhaI DNA methyltransferase belongs to the C5-cytosine methyltransferase family, which is characterized by the presence of a set of highly conserved amino acids and motifs present in an invariant order. HhaI DNA methyltransferase has been subjected to a lot of biochemical and crystallographic studies. A number of issues, especially the role of the conserved amino acids in the methyltransferase activity, have not been addressed. Using sequence comparison and structural data, a structure-guided mutagenesis approach was undertaken, to assess the role of conserved amino acids in catalysis. Site-directed mutagenesis was performed on amino acids involved in cofactor S-adenosyl-L-methionine (AdoMet) binding (Phe18, Trp41, Asp60 and Leu100). Characterization of these mutants, by in vitro /in vivo restriction assays and DNA/AdoMet binding studies, indicated that most of the residues present in the AdoMet-binding pocket were not absolutely essential. This study implies plasticity in the recognition of cofactor by HhaI DNA methyltransferase.     

Bacteriophage T4 Dam DNA-[N6-adenine]methyltransferase. Kinetic evidence for a catalytically essential conformational change in the ternary complex.

We carried out a steady state kinetic analysis of the bacteriophage T4 DNA-[N6-adenine]methyltransferase (T4 Dam) mediated methyl group transfer reaction from S-adenosyl-l-methionine (AdoMet) to Ade in the palindromic recognition sequence, GATC, of a 20-mer oligonucleotide duplex. Product inhibition patterns were consistent with a steady state-ordered bi-bi mechanism in which the order of substrate binding and product (methylated DNA, DNA(Me) and S-adenosyl-l-homocysteine, AdoHcy) release was AdoMet downward arrow DNA downward arrow DNA(Me) upward arrow AdoHcy upward arrow. A strong reduction in the rate of methylation was observed at high concentrations of the substrate 20-mer DNA duplex. In contrast, increasing substrate AdoMet concentration led to stimulation in the reaction rate with no evidence of saturation. We propose the following model. Free T4 Dam (initially in conformational form E) randomly interacts with substrates AdoMet and DNA to form a ternary T4 Dam-AdoMet-DNA complex in which T4 Dam has isomerized to conformational state F, which is specifically adapted for catalysis. After the chemical step of methyl group transfer from AdoMet to DNA, product DNA(Me) dissociates relatively rapidly (k(off) = 1.7 x s(-1)) from the complex. In contrast, dissociation of product AdoHcy proceeds relatively slowly (k(off) = 0.018 x s(-1)), indicating that its release is the rate-limiting step, consistent with kcat = 0.015 x s(-1). After AdoHcy release, the enzyme remains in the F conformational form and is able to preferentially bind AdoMet (unlike form E, which randomly binds AdoMet and DNA), and the AdoMet-F binary complex then binds DNA to start another methylation cycle. We also propose an alternative pathway in which the release of AdoHcy is coordinated with the binding of AdoMet in a single concerted event, while T4 Dam remains in the isomerized form F. The resulting AdoMet-F binary complex then binds DNA, and another methylation reaction ensues. This route is preferred at high AdoMet concentrations.     

Kinetic mechanism of the EcoRI DNA methyltransferase

We present a kinetic analysis of the EcoRI DNA N6-adenosine methyltransferase (Mtase). The enzyme catalyzes the S-adenosylmethionine (AdoMet)-dependent methylation of a short, synthetic 14 base pair DNA substrate and plasmid pBR322 DNA substrate with kcat/Km values of 0.51 X 10(8) and 4.1 X 10(8) s-1 M-1, respectively. The Mtase is thus one of the most efficient biocatalysts known. Our data are consistent with an ordered bi-bi steady-state mechanism in which AdoMet binds first, followed by DNA addition. One of the reaction products, S-adenosylhomocysteine (AdoHcy), is an uncompetitive inhibitor with respect to DNA and a competitive inhibitor with respect to AdoMet. Thus, initial DNA binding followed by AdoHcy binding leads to formation of a ternary dead-end complex (Mtase-DNA-AdoHcy). We suggest that the product inhibition patterns and apparent order of substrate binding can be reconciled by a mechanism in which the Mtase binds AdoMet and noncanonical DNA randomly but that recognition of the canonical site requires AdoMet to be bound. Pre-steady-state and isotope partition analyses starting with the binary Mtase-AdoMet complex confirm its catalytic competence. Moreover, the methyl transfer step is at least 10 times faster than catalytic turnover.     

Caught in the act: visualization of an intermediate in the DNA base-flipping pathway induced by HhaI methyltransferase

Rotation of a DNA or RNA nucleotide out of the double helix and into a protein pocket (‘base flipping’) is a mechanistic feature common to some DNA/RNA-binding proteins. Here, we report the structure of HhaI methyltransferase in complex with DNA containing a south-constrained abasic carbocyclic sugar at the target site in the presence of the methyl donor byproduct AdoHcy. Unexpectedly, the locked south pseudosugar appears to be trapped in the middle of the flipping pathway via the DNA major groove, held in place primarily through Van der Waals contacts with a set of invariant amino acids. Molecular dynamics simulations indicate that the structural stabilization observed with the south-constrained pseudosugar will not occur with a north-constrained pseudosugar, which explains its lowered binding affinity. Moreover, comparison of structural transitions of the sugar and phosphodiester backbone observed during computational studies of base flipping in the M.HhaI–DNA–AdoHcy ternary complex indicate that the south-constrained pseudosugar induces a conformation on the phosphodiester backbone that corresponds to that of a discrete intermediate of the base-flipping pathway. As previous crystal structures of M.HhaI ternary complex with DNA displayed the flipped sugar moiety in the antipodal north conformation, we suggest that conversion of the sugar pucker from south to north beyond the middle of the pathway is an essential part of the mechanism through which flipping must proceed to reach its final destination. We also discuss the possibility of the south-constrained pseudosugar mimicking a transition state in the phosphodiester and sugar moieties that occurs during DNA base flipping in the presence of M.HhaI.     

M.HhaI binds tightly to substrates containing mismatches at the target base.

The (cytosine-5) DNA methyltransferase M.HhaI causes its target cytosine base to be flipped completely out of the DNA helix upon binding. We have investigated the effects of replacing the target cytosine by other, mismatched bases, including adenine, guanine, thymine and uracil. We find that M.HhaI binds more tightly to such mismatched substrates and can even transfer a methyl group to uracil if a G:U mismatch is present. Other mismatched substrates in which the orphan guanine is changed exhibit similar behavior. Overall, the affinity of DNA binding correlates inversely with the stability of the target base pair, while the nature of the target base appears irrelevant for complex formation. The presence of a cofactor analog. S-adenosyl-L-homocysteine, greatly enhances the selectivity of the methyltransferase for cytosine at the target site. We propose that the DNA methyltransferases have evolved from mismatch binding proteins and that base flipping was, and still is, a key element in many DNA-enzyme interactions.     

Binding of MmeI Restriction–modification Enzyme to its Specific Recognition Sequence is Stimulated by <Emphasis Type="Italic">S</Emphasis>-adenosyl-<Emphasis Type="SmallCaps">l</Emphasis>-methionine

Restriction endonucleases serve as a very good model for studying specific protein–DNA interaction. MmeI is a very interesting restriction endonuclease, but although it is useful in Serial Analysis of Gene Expression, still very little is known about the mechanism of its interaction with DNA. MmeI is a unique enzyme as besides cleaving DNA it also methylates specific sequence. For endonucleolytic activity MmeI requires Mg(II) and S-adenosyl-l-methionine (AdoMet). AdoMet is a methyl donor in the methylation reaction, but its requirement for DNA cleavage remains unclear. In the present article we investigated MmeI interaction with DNA with the use of numerous methods. Our electrophoretic mobility shift assay revealed formation of two types of specific protein–DNA complexes. We speculate that faster migrating complex consists of one protein molecule and one DNA fragment whereas, slower migrating complex, which appears in the presence of AdoMet, may be a dimer or multimer form of MmeI interacting with specific DNA. Additionally, using spectrophotometric measurements we showed that in the presence of AdoMet, MmeI protein undergoes conformational changes. We think that such change in the enzyme structure, upon addition of AdoMet, may enhance its specific binding to DNA. In the absence of AdoMet MmeI binds DNA to the much lower extent.