DNA duplexes containing photoactive derivatives of 2'-deoxyuridine as photocrosslinking probes for EcoRII DNA methyltransferase-substrate interaction

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.     

Affinity modification of EcoRII DNA methyltransferase by the dialdehyde-substituted DNA duplexes: mapping the enzyme region that interacts with DNA

Affinity modification of EcoRII DNA methyltransferase (M x EcoRII) by DNA duplexes containing oxidized 2'-O-beta-D-ribofuranosylcytidine (Crib*) or 1-(beta-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M x EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly268-Met391 of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M x EcoRII region. Our results support the theoretically predicted DNA binding region of M x EcoRII.     

DNA duplexes containing altered sugar residues as probes of EcoRII and MvaI endonuclease interactions with sugar-phosphate backbone

Oligonucleotides containing 1-(beta-D-2'-deoxy-threo-pentofuranosyl)cytosine (dCx) and/or 1-(beta-D-2'-deoxy-threo-pentofuranosyl)thymine (dTx) in place of dC and dT residues in the EcoRII and MvaI recognition site CC(A/T)GG were synthesized in order to investigate specific recognition of the DNA sugar-phosphate backbone by EcoRII and MvaI restriction endonucleases. In 2'-deoxyxylosyl moieties of dCx and dTx, 3'-hydroxyl groups were inverted, which perturbs the related individual phosphates. Introduction of a single 2'-deoxyxylosyl moiety into a dC x dG pair resulted in a minor destabilization of double-stranded DNA structure. In the case of a dA x dT pair the effect of a 2'-deoxyxylose incorporation was much more pronounced. Multiple dCx modifications and their combination with dTx did not enhance the destabilization effect. Hydrolysis of dCx-containing DNA duplexes by EcoRII endonuclease was blocked and binding affinity was strongly depended on the location of an altered sugar. A DNA duplex containing a dTx residue was cleaved by the enzyme, but kcat/K(M) was slightly reduced. In contrast, MvaI endonuclease efficiently cleaved both types of sugar-altered substrate analogs. However it did not cleave conformationally perturbed scissile bonds, when the corresponding unmodified bonds were perfectly hydrolyzed in the same DNA duplexes. Based on these data the possible contributions of individual phosphates in the recognition site to substrate recognition and catalysis by EcoRII were proposed. We observed strikingly non-equivalent inputs for different phosphates with respect to their effect on EcoRII-DNA complex formation.     

Probing of contacts between <Emphasis Type="Italic">Eco</Emphasis>RII DNA methyltransferase and DNA with the use of substrate analogs and molecular modeling

The molecular mechanisms of DNA recognition and modification by EcoRII DNA methyltransferase (M.EcoRII) were studied using 14-mer substrate analogs containing 2-aminopurine or 1′,2′-dideoxy-D-ribofuranose in the M.EcoRII recognition site. The efficiency of DNA binding and methylation depended on the position of a modified nucleoside residue in the recognition site. A structural model of M.EcoRII in complex with substrate DNA and the cofactor analog S-adenosyl-L-homocysteine (AdoHcy) was constructed using the available crystal structures of M.Hha and M.HaeIII and the recent Frankenstein’s monster approach. The amino acid residues interacting with DNA were predicted based on the model. In addition, theoretical and experimental findings made it possible to predict the groups of atoms of the heterocyclic bases of the M.EcoRII recognition site that are presumably involved in the interactions with the enzyme.     

Imaging DNA loops induced by restriction endonuclease EcoRII. A single amino acid substitution uncouples target recognition from cooperative DNA interaction and cleavage

EcoRII is a type IIE restriction endonuclease characterized by a highly cooperative reaction mechanism that depends on simultaneous binding of the dimeric enzyme molecule to two copies of its DNA recognition site. Transmission electron microscopy provided direct evidence that EcoRII mediates loop formation of linear DNA containing two EcoRII recognition sites. Specific DNA binding of EcoRII revealed a symmetrical DNase I footprint occupying 16-18 bases. Single amino acid replacement of Val(258) by Asn yielded a mutant enzyme that was unaffected in substrate affinity and DNase I footprinting properties, but exhibited a profound decrease in cooperative DNA binding and cleavage activity. Because the electrophoretic mobility of the mutant enzyme-DNA complexes was significantly higher than that of the wild-type, we investigated if mutant V258N binds as a monomer to the substrate DNA. Analysis of the molecular mass of mutant V258N showed a high percentage of protein monomers in solution. The dissociation constant of mutant V258N confirmed a 350-fold decrease of the enzyme dimerization capability. We conclude that Val(258) is located in a region of EcoRII involved in homodimerization. This is the first report of a specific amino acid replacement in a restriction endonuclease leading to the loss of dimerization and DNA cleavage while retaining specific DNA binding.     

Interaction of restriction and modification enzyme EcoRII with synthetic DNA fragments. VII. The study of complex-formation of endonuclease EcoRII with substrates containing natural and modified recognition sites

As shown by a nitrocellulose filter binding assay, in the absence of Mg2+ EcoRII restriction endonuclease binds specifically to a set of synthetic concatemer DNA duplexes of varying chain length, containing natural and modified recognition sites of this enzyme. The binding of the substrates with the central AT, TT or AA-pair in the recognition site decreases at AT greater than TT much greater than AA. Substitution of the pyrophosphate bond at the cleavage site for the phosphodiester or phosphoramide bond produces little influence on the stability of the complexes. The affinity of the enzyme for nonspecific sites is two orders of magnitude less than that for the specific EcoRII sequences. Equilibrium association constant for a substrate with one recognition site is 3.9 X 10(8) M-1. Addition of Mg2+ leads to the destabilization of the EcoRII endonuclease complex with DNA duplex, containing pyrophosphate bonds. The dissociation rate constants and the lifetime of the EcoRII endonuclease--synthetic substrates complexes have been determined.     

Activation of restriction endonuclease EcoRII does not depend on the cleavage of stimulator DNA.

The restriction endonuclease EcoRII is unable to cleave DNA molecules when recognition sites are very far apart. The enzyme, however can be activated in the presence of DNA molecules with a high frequency of EcoRII sites or by oligonucleotides containing recognition sites: Addition of the activator molecules stimulates cleavage of the refractory substrate. We now show that endonucleolysis of the stimulator molecules is not a necessary prerequisite of enzyme activation. A total EcoRII digest of pBR322 DNA or oligonucleotide duplexes with simulated EcoRII ends (containing the 5' phosphate group), as well as oligonucleotide duplexes containing modified bases within the EcoRII site, making them resistant to cleavage, are all capable of enzyme activation. For activation EcoRII requires the interaction with at least two recognition sites. The two sites may be on the same DNA molecule, on different oligonucleotide duplexes, or on one DNA molecule and one oligonucleotide duplex. The efficiency of functional intramolecular cooperation decreases with increasing distance between the sites. Intermolecular site interaction is inversely related to the size of the stimulator oligonucleotide duplex. The data are in agreement with a model whereby EcoRII simultaneously interacts with two recognition sites in the active complex, but cleavage of the site serving as an allosteric activator is not necessary.     

Effects of benzo[a]pyrene-deoxyguanosine lesions on DNA methylation catalyzed by EcoRII DNA methyltransferase and on DNA cleavage effected by EcoRII restriction endonuclease

DNA methylation is an important cellular mechanism for controlling gene expression. Whereas the mutagenic properties of many DNA adducts, e.g., those arising from polycyclic aromatic hydrocarbons, have been widely studied, little is known about their influence on DNA methylation. We have constructed site-specifically modified 18-mer oligodeoxynucleotide duplexes containing a pair of stereoisomeric adducts derived from a benzo[a]pyrene-derived diol epoxide [(+)- and (-)-r7,t8-dihydroxy-t9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, or B[a]PDE] bound to the exocyclic amino group of guanine. The adducts, either (+)- or (-)-trans-anti-B[a]P-N(2)-dG (G*), positioned either at the 5'-side or the 3'-side deoxyguanosine residue in the recognition sequence of EcoRII restriction-modification enzymes (5'-...CCA/TGG...) were incorporated into 18-mer oligodeoxynucleotide duplexes. The effects of these lesions on complex formation and the catalytic activity of the EcoRII DNA methyltransferase (M.EcoRII) and EcoRII restriction endonuclease (R.EcoRII) were investigated. The M.EcoRII catalyzes the transfer of a methyl group to the C5 position of the 3'-side cytosine of each strand of the recognition sequence, whereas R.EcoRII catalyzes cleavage of both strands. The binding of R.EcoRII to the oligodeoxynucleotide duplexes and the catalytic cleavage were completely abolished when G was positioned at the 3'-side dG position (5'-...CCTGG*...). When G* was at the 5'-side dG position, binding was moderately diminished, but cleavage was completely blocked. In the case of M.EcoRII, binding is diminished by factors of 5-30 but the catalytic activity was either abolished or reduced 4-80-fold when the adducts were located at either position. Somewhat smaller effects were observed with hemimethylated oligodeoxynucleotide duplexes. These findings suggest that epigenetic effects, in addition to genotoxic effects, need to be considered in chemical carcinogenesis initiated by B[a]PDE, since the inhibition of methylation may allow the expression of genes that promote tumor development.     

2-Pyrimidinone as a probe for studying the EcoRII DNA methyltransferase-substrate interaction.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the 5' em leader CC*T/AGG em leader 3' DNA sequence and catalyzes the transfer of the methyl group from S-adenosyl-l-methionine to the C5 position of the inner cytosine residue (C*). Here, we study the mechanism of inhibition of M.EcoRII by DNA containing 2-pyrimidinone, a cytosine analogue lacking an NH(2) group at the C4 position of the pyrimidine ring. Also, DNA containing 2-pyrimidinone was used for probing contacts of M.EcoRII with functional groups of pyrimidine bases of the recognition sequence. 2-Pyrimidinone was incorporated into the 5' em leader CCT/AGG em leader 3' sequence replacing the target and nontarget cytosine and central thymine residues. Study of the DNA stability using thermal denaturation of 2-pyrimidinone containing duplexes pointed to the influence of the bases adjacent to 2-pyrimidinone and to a greater destabilizing influence of 2-pyrimidinone substitution for thymine than that for cytosine. Binding of M.EcoRII to 2-pyrimidinone containing DNA and methylation of these DNA demonstrate that the amino group of the outer cytosine in the EcoRII recognition sequence is not involved in the DNA-M.EcoRII interaction. It is probable that there are contacts between the functional groups of the central thymine exposed in the major groove and M.EcoRII. 2-Pyrimidinone replacing the target cytosine in the EcoRII recognition sequence forms covalent adducts with M.EcoRII. In the absence of the cofactor S-adenosyl-l-methionine, proton transfer to the C5 position of 2-pyrimidinone occurs and in the presence of S-adenosyl-l-methionine, methyl transfer to the C5 position of 2-pyrimidinone occurs.     

Interaction of EcoRII restriction and modification enzyme with synthetic DNA fragments. IV. DNA duplexes with phosphoamide and pyrophosphate internucleotide bonds--the substrates for the study of single-strand breaks

A set of DNA duplexes with repeated EcoRII, EcoRI and AluI restriction endonuclease recognition sites in which EcoRII scissile phosphodiester bonds were replaced by phosphoramide or uncleavable pyrophosphate bonds have been synthesized. Endonuclease EcoRII was found not to cleave the substrate at the phosphoramide bond. The substrates containing non-nydrolysable pyrophosphate or phosphoramide bonds in one of the chains of EcoRII recognition sites were used to show that this enzyme is able to catalyze single-strand scissions. These scissions occur both in dA- and dT-containing chains of the recognition site. Endonuclease EcoRII interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of the other. Synthesized DNA-duplexes are cleaved specifically by EcoRI and AluI endonucleases, this cleavage being retarded if the modified bonds are in the recognition site (EcoRI) or flank it (AluI). For EcoRII and AluI this effect is more pronounced in the case of substrates with pyrophosphate bonds than with the phosphoramide ones.     

The cysteine conserved among DNA cytosine methylases is required for methyl transfer, but not for specific DNA binding.

All DNA (cytosine-5)-methyltransferases contain a single conserved cysteine. It has been proposed that this cysteine initiates catalysis by attacking the C6 of cytosine and thereby activating the normally inert C5 position. We show here that substitutions of this cysteine in the E. coli methylase M. EcoRII with either serine or tryptophan results in a complete loss of ability to transfer methyl groups to DNA. Interestingly, mutants with either serine or glycine substitution bind tightly to substrate DNA. These mutants resemble the wild-type enzyme in that their binding to substrate is not eliminated by the presence of non-specific DNA in the reaction, it is sensitive to methylation status of the substrate and is stimulated by an analog of the methyl donor. Hence the conserved cysteine is not essential for the specific stable binding of the enzyme to its substrate. However, substitution of the cysteine with the bulkier tryptophan does reduce DNA binding. We also report here a novel procedure for the synthesis of DNA containing 5-fluorocytosine. Further, we show that a DNA substrate for M. EcoRII in which the target cytosine is replaced by 5-fluorocytosine is a mechanism-based inhibitor of the enzyme and that it forms an irreversible complex with the enzyme. As expected, this modified substrate does not form irreversible complexes with the mutants.     

Simultaneous binding of three recognition sites is necessary for a concerted plasmid DNA cleavage by EcoRII restriction endonuclease

According to the current paradigm type IIE restriction endonucleases are homodimeric proteins that simultaneously bind to two recognition sites but cleave DNA at only one site per turnover: the other site acts as an allosteric locus, activating the enzyme to cleave DNA at the first. Structural and biochemical analysis of the archetypal type IIE restriction enzyme EcoRII suggests that it has three possible DNA binding interfaces enabling simultaneous binding of three recognition sites. To test if putative synapsis of three binding sites has any functional significance, we have studied EcoRII cleavage of plasmids containing a single, two and three recognition sites under both single turnover and steady state conditions. EcoRII displays distinct reaction patterns on different substrates: (i) it shows virtually no activity on a single site plasmid; (ii) it yields open-circular DNA form nicked at one strand as an obligatory intermediate acting on a two-site plasmid; (iii) it cleaves concertedly both DNA strands at a single site during a single turnover on a three site plasmid to yield linear DNA. Cognate oligonucleotide added in trans increases the reaction velocity and changes the reaction pattern for the EcoRII cleavage of one and two-site plasmids but has little effect on the three-site plasmid. Taken together the data indicate that EcoRII requires simultaneous binding of three rather than two recognition sites in cis to achieve concerted DNA cleavage at a single site. We show that the orthodox type IIP enzyme PspGI which is an isoschisomer of EcoRII, cleaves different plasmid substrates with equal rates. Data provided here indicate that type IIE restriction enzymes EcoRII and NaeI follow different mechanisms. We propose that other type IIE restriction enzymes may employ the mechanism suggested here for EcoRII.     

Interaction of EcoRII endonuclease with DNA substrates containing single recognition sites.

EcoRII is unusual among type II restriction enzymes in that, while it cleaves substrates such as pBR322 and bacteriophage lambda that contain several recognition sites for the enzyme efficiently, substrates such as the genomes of bacteriophages T3 and T7 which contain a small number of recognition sites are cut poorly by it. Interestingly, pBR322, or a short DNA duplex containing a single site for the enzyme, can activate the enzyme to cleave resistant substrates. We show here that, at low concentrations, activator short duplexes are themselves cleaved poorly by the enzyme. Further, the reaction shows substrate cooperativity, and at high concentrations, the duplexes are both activators and good substrates for the enzyme. This supports the model that the activation of EcoRII involves binding of more than one DNA molecule and provides a simple system to study the mechanism of activation. Using a gel mobility shift assay, we show that the enzyme forms sequence-specific, methylation-sensitive complexes with the duplexes in the absence of activating DNA. Therefore, resistance of the short duplexes to the enzyme at low concentrations cannot be due to an inability of the enzyme to bind the duplexes. Interestingly, these complexes are stable in the presence of Mg2+, the cofactor for the enzyme, and the complexes obtained in the presence of Mg2+ do not contain DNA that is cleaved by the enzyme. The inefficient step in the action of EcoRII on resistant substrates must occur subsequent to initial substrate binding and it is this step that the activating DNA must regulate.     

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. V. Study of single-strand cleavages.

Concatemer DNA duplexes which contain at the EcoRII restriction endonuclease cleavage sites (formula; see text) phosphodiester, phosphoamide or pyrophosphate internucleotide bonds have been synthesized. It has been shown that this enzyme did not cleave the substrate at phosphoamide bond. EcoRII endonuclease catalyzes single-strand cleavages both in dA- and dT-containing strands of the recognition site if the cleavage of the other strand has been blocked by modification of scissile bond or if the other strand has been cleaved. This enzyme interacts with both strands of the DNA recognition site, each of them being cleaved independently on the cleavage of another one. Nucleotide sequences flanking the EcoRII site on both sides are necessary for effective cleavage of the substrate.     

Interaction of EcoRII restriction and modification enzymes with synthetic DNA fragments. X. Hydrolysis of substrates with structural abnormalities

Interaction of the EcoRII restriction endonuclease with a set of 30-membered substrates having structural anomalies in the recognition site (decreases CCT/AGG) and in adjacent sequences has been studied. A nick in the centre of the EcoRII recognition site between dC and dA residues slows down hydrolysis of the nonmodified strand, whereas the modified one is not cleaved. Removal of the phosphate group from the nick in this substrate does not alter the rate of the cleavage. The absence of one of the phosphate groups in the flanking sequence at a two-base-pair "distance" from the recognition site slows down the enzymatic hydrolysis. Removal of dA or dT out of the EcoRII recognition site blocks the enzymatic reaction. It appears that EcoRII does not interact with the phosphate group between dC and dA residues in the recognition site. Suggestions are made concerning possible contacts of the EcoRII restriction endonuclease with dA- and dT-residues of the recognition site and with the sugar-phosphate backbone of the adjacent nucleotide sequences.     

Homology modeling of the CG-specific DNA methyltransferase SssI and its complexes with DNA and AdoHcy

Prokaryotic DNA methyltransferase M.SssI recognizes and methylates C5 position of the cytosine residue within the CG dinucleotides in DNA. It is an excellent model for studying the mechanism of interaction between CG-specific eukaryotic methyltransferases and DNA. We have built a structural model of M.SssI in complex with the substrate DNA and its analogues as well as the cofactor analogue S-adenosyl-L-homocysteine (AdoHcy) using the previously solved structures of M.HhaI and M.HaeIII as templates. The model was constructed according to the recently developed "FRankenstein's monster" approach. Based on the model, amino acid residues taking part in cofactor binding, target recognition and catalysis were predicted. We also modeled covalent modification of the DNA substrate and studied its influence on protein-DNA interactions.     

Peculiarities of recognition of CCA/TGG sequences in DNA by restriction endonucleases MvaI and EcoRII.

To elucidate the mechanism of action of restriction endonucleases MvaI and EcoRII a study was made of their interaction with a set of synthetic substrates in which the heterocyclic bases or the sugar-phosphate backbone had been modified; individual nucleotide residues had been removed or replaced with hydrocarbon bridges, and mismatched base pairs had been introduced. The groups of atoms in the heterocyclic bases and the phosphates in the recognition site that produce the most significant influence on the functioning of endonucleases MvaI and EcoRII were discerned. Profound differences were found in the functioning of the MvaI and EcoRII neoschizomers. The catalytic activity of EcoRII is significantly affected by any alteration in the recognition site structure and conformation, with a modification in one strand of the substrate causing the same decrease in the hydrolysis rate of both strands. Endonuclease MvaI is tolerant to a number of structural abnormalities; the latter sometimes affect only hydrolysis of one strand of the recognition site. The enzyme can preferentially cleave one of the substrate strands. Mismatched base pairs retard and sometimes block the hydrolysis. The effect depends on the particular enzyme, mismatch and its location.     

Mechanism of the interaction of EcoRII restriction endonuclease with two recognition sites. Probing of modified DNA duplexes as activators of the enzyme.

The efficiency of cleavage of DNA duplexes with single EcoRII recognition sites by the EcoRII restriction endonuclease decreases with increasing substrate length. DNA duplexes of more than 215 bp are not effectively cleaved by this enzyme. Acceleration of the hydrolysis of long single-site substrates by EcoRII is observed in the presence of 11-14-bp substrates. The stimulation of hydrolysis depends on the length and concentration of the second substrate. To study the mechanism of EcoRII endonuclease stimulation, DNA duplexes with base analogs and modified internucleotide phosphate groups in the EcoRII site have been investigated as activators. These modified duplexes are cleaved by EcoRII enzyme with different efficiencies or are not cleaved at all. It has been discovered that the resistance of some of them can be overcome by incubation with a susceptible canonical substrate. The acceleration of cleavage of long single-site substrates depends on the type of modification of the activator. The modified DNA duplexes can activate EcoRII catalyzed hydrolysis if they can be cleaved by EcoRII themselves or in the presence of the second canonical substrate. It has been demonstrated that EcoRII endonuclease interacts in a cooperative way with two recognition sites in DNA. The cleavage of one of the recognition sites depends on the cleavage of the other. We suggest that the activator is not an allosteric effector but acts as a second substrate.     

Cleavage by restriction endonucleases MvaI and EcoRII of substrates modified in amino groups of heterocyclic bases

14-membered DNA-duplexes containing modified nucleoside residues, viz 4-N-methyldeoxycytidine (m4dC), 6-N-methyldeoxyadenosine (m6dA) or deoxyinosine (dI), in only one strand of the recognition site (CCA/TGG) of MvaI and EcoRII endonucleases were synthesized. It was shown that MvaI and EcoRII endonucleases interact with the exocyclic amino groups of the external dC residues and of the central dA residue of the recognition site exposed into the DNA major groove. These endonucleases which are isochizomers were found to possess different mechanisms of substrate cleavage. The ability of MvaI endonuclease to hydrolyze only unmodified strand of methylated duplexes allows one to make site-directed single-strand nicks in double-stranded DNA. Elimination of the 2-NH2-group located in the minor groove of DNA by substituting dI for dG had little, if any, effect on the hydrolytic activity of EcoRII and MvaI endonucleases.     

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.