An essential role for sodium in the bicarbonate transporting system of the cyanobacterium Anabaena variabilis

The Eco dam methylase is active on denatured DNA and single-stranded synthetic oligonucleotides containing GATC sites. The results suggest that on interaction with single-stranded oligonucleotides the Eco dam methylase is able to form a duplex structure within the GATC site, and that this duplex site is a substrate for enzyme.     

DNA-methylase Sau 3A: isolation and various properties

DNA-methylase Sau 3A has been isolated for the first time from Staphylococcus aureus 3A cells and purified by column chromatography on phosphocellulose PII, heparin-Sepharose and blue Sepharose. The purified enzyme methylates the GATC sequence with the formation of GATm5C as can be evidenced from the protection of DNA from digestion with restrictases Sau 3A and Bam HI, the lack of the C3H3-group incorporation into Sau 3A DNA-restricts and the formation of a single methylated base m5C. Sau 3A methylase modifies only a two-filament (but not one-filament) DNA. Thus, methylase Sau 3A modifies the both DNA chains in the recognition site during a single binding act. The 5-azacytidine-containing DNA inhibits by 95% the activity of methylase Sau 3A. Ado-met is the single methyl group donor for methylase Sau 3A. The presence of m6A in the recognition site does not affect the activity of methylase Sau 3A. The practical recommendations for the use of M. Sau 3A, alongside with M. Eco dam, for the study of dam methylation by additional methylation of the DNA in vitro in the presence of [methyl-3H]-S-adenosyl-methionine are given.     

Chemical synthesis and properties of oligonucleotide substrates for restriction endonuclease BamHI and methyltransferase Eco dam

Oligodeoxyribonucleotides which form a number of duplexes, containing the recognition sequences for endonuclease BamHI and DNA methylase Eco dam, were synthesised by the phosphotriester approach. Furthermore, synthesis of 3'-phosphorylated oligodeoxyribonucleotides from corresponding S-methyl phosphorothioate triester oligomers is described. The synthetic duplexes are characterized by some defects in the recognition sequences for endonuclease BamHI and methylase Eco dam, viz. nick, absence of an internucleotide phosphate, modifications (including partial single-strandedness) of the recognition site. Interaction of the enzymes with these synthetic substrates was investigated.     

Effect of Ecodam DNA-methylase on single-stranded sequences and synthetic oligonucleotides

Interaction of the Ecodam methylase with different substrates were investigated among them the double- and single-stranded DNAs and synthetic oligonucleotides containing some defects in the GATC sequence. These defects were:nick, the absence of one internucleotide phosphate of nucleotide; partially single-stranded form on the recognition site etc. It was demonstrated that the presence of both G . A-dinucleotides in the recognition site is necessary for productive enzyme-substrate interaction. The absence of T and/or C residues is less dramatic for methylase activity. The Ecodam methylase is capable to modify the single-stranded oligonucleotides by forming the double-stranded structure in the symmetric recognition sequences GATC.     

Application of the small-angle x-ray scattering technique for the study of two-step equilibrium enzyme-substrate interactions

The small-angle x-ray scattering (SAXS) technique is used for the investigation of two-stage equilibrium macromolecular interactions of the enzyme-substrate type in solution. Experimental procedures and methods of analyzing the data obtained from SAXS have been elaborated. The algorithm for the data analysis allows one to determine the stoichiometric, equilibrium, and structural parameters of the enzyme-substrate complexes obtained. The thermodynamic characteristics for the formation of complexes of double-stranded oligonucleotide with Eco dam methyltransferase (MTase) have been determined and demonstrate a high cooperativity of MTase binding when the ternary complex containing the dimeric enzyme is formed. The structural parameters (R_g, R_c, semiaxes) have been determined for free enzyme and polynucleotides and of enzyme-substrate complexes, indicating structural rearrangements of the enzyme in the interaction with substrates. © 1996 John Wiley & Sons, Inc.     

Relaxed specificity of the EcoRV restriction endonuclease

The EcoRV restriction endonuclease normally shows a high specificity for its recognition site on DNA, GATATC. In standard reactions, it cleaves DNA at this site several orders of magnitude more readily than at any alternative sequence. But in the presence of dimethyl sulphoxide and at high pH, the EcoRV enzyme cleaves DNA at several sites that differ from its recognition site by one nucleotide. Of the 18 (3 X 6) possible sequences that differ from GATATC by one base, all were cleaved readily except for the following 4 sites: TATATC, CATATC, GATATA and GATATG. However, two of the sites that could be cleaved by EcoRV in the presence of dimethyl sulphoxide, GAGATC and GATCTC, were only cleaved on DNA that lacked dam methylation: both contain the sequence GATC, the recognition site for the dam methylase of Escherichia coli.     

An analysis of methyltransferase SsoII-DNA contacts in the enzyme-substrate complex

The functional groups of the DNA methylation site that are involved in the DNA interaction with methyltransferase SsoII at the recognition stage were identified. The contacts in the enzyme-substrate complex were analyzed in the presence of S-adenosyl-L-homocysteine using the interference footprinting assay with formic acid, hydrazine, dimethyl sulfate, or N-ethyl-N-nitrosourea as a modifying reagent. It was shown that the replacement of the central A.T by the G.C pair in the methylation site did not affect the enzyme-DNA interaction, whereas the use of a substrate with one chain methylated (monomethylated substrate) instead of the unmethylated substrate dramatically changes the DNA contacts. The binding constants of unmethylated and monomethylated substrates with methyltransferase SsoII in the presence of S-adenosyl-L-homocysteine were calculated.     

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.     

Recognition sequence of the dam methylase of Escherichia coli K12 and mode of cleavage of Dpn I endonuclease.

The recognition sequence for the dam methylase of Escherichia coli K12 has been determined directly by use of in vivo methylated ColE1 DNA or DNA methylated in vitro with purified enzyme. The methylase recognizes the symmetric tetranucleotide d(pG-A-T-C) and introduces two methyl groups per site in duplex DNA with the product of methylation being 6-methylaminopurine. This work has also demonstrated that Dpn I restriction endonuclease cleaves on the 3' side of the modified adenine within the methylated sequence to yield DNA fragments possessing fully base-paired termini. All sequences in ColE1 DNA methylated by the dam enzyme are subject to double strand cleavage by Dpn I endonuclease. Therefore, this restriction enzyme can be employed for mapping the location of sequences possessing the dam modification.     

Single turnover kinetics of phage T4 DNA-(N6-adenine)methyltransferase

Interaction of T4 DNA-(N6-adenine)-methyltransferase [EC 2.1.1] was studied with a variety of synthetic oligonucleotide substrates containing the native recognition site GATC or its modified variants. The data obtained in the decisecond and second intervals of the reaction course allowed for the first time the substrate methylation rates to be compared with the parameters of the steady-state reaction. It was established that the substrate reaction proceeds in two stages. Because it is shown that in steady-state conditions T4 MTase forms a dimeric structure, the following sequence of events is assumed. Upon collision of a T4 MTase monomer with an oligonucleotide duplex, an asymmetrical complex forms in which the enzyme randomly oriented relative to one of the strands of the specific recognition site catalyzes a fast transfer of the methyl group from S-adenosylmethionine to the adenosine residue (k1 = 0.21 s-1). Simultaneously, a second T4 MTase subunit is added to the complex, providing for the continuation of the reaction. In the course of a second stage, which is by an order of magnitude slower (k2 = 0.023 s-1 for duplex with the native site), the dimeric T4 MTase switches over to the second strand and the methylation of the second residue, target. The rate of the methyl group transfer from donor, S-adenosylmethionine, to DNA is much higher than the overall rate of the T4 MTase-catalyzed steady-state reaction, although this difference is considerably less than that shown for EcoRI Mtase. Substitutions of bases and deletions in the recognition site affect the substrate parameters in different fashions. When the GAT sequence is disrupted, the proportion of the initial productive enzyme-substrate complexes is usually sharply reduced. The flipping of the adenosine residue, a target for the modification in the recognition site, revealed by fluorescence titration, upon interaction with the enzyme supports the existing notions about the involvement of such a DNA deformation in reactions catalyzed by various DNA-MTases.     

Effect of 5-azacytidine on E. coli cells with different DNA-methylases

A correlation was found between the bacteriocide effect of 5-aza-C and the amount of cytosine DNA-methylases in E. coli cells. 5-Aza-C-DNA induced partial or complete inhibition of bacterial DNA-methylases with different site specificity; cytosine DNA-methylases were inhibited by the DNA more effectively than adenine DNA-methylase Eco dam. The inhibitory influence of 5-aza-C-DNA on cytosine DNA-methylases was due to the formation of stable inactive complexes between the enzyme and the non-methylating cytosine analog in the recognition sites. Cytosine DNA-methylase Eco RII formed a relatively firm bond with 5-aza-C-DNA, which could be disrupted by 1 M KCl; this disruption restores the DNA-methylase activity and the inhibiting capacity of 5-aza-C-DNA. Thus, the binding of cytosine DNA-methylase to 5-aza-C in DNA is noncovalent; the inhibition of the enzyme by 5-aza-C-DNA is reversible.     

Sequence and substrate specificity of isolated DNA methylases from Escherichia coli C

Two DNA methylase activities of Escherichia coli C, the mec (designates DNA-cytosine-methylase gene, which is also designated dcm) and dam gene products, were physically separated by DEAE-cellulose column chromatography. The sequence and substrate specificity of the two enzymes were studied in vitro. The experiments revealed that both enzymes show their expected sequence specificity under in vitro conditions, methylating symmetrically on both DNA strands. The mec enzyme methylates exclusively the internal cytosine residue of CCATGG sequences, and the dam enzyme methylates adenine residues at GATC sites. Substrate specificity experiments revealed that both enzymes methylate in vitro unmethylated duplex DNA as efficiently as hemimethylated DNA. The results of these experiments suggest that the methylation at a specific site takes place by two independent events. A methyl group in a site on one strand of the DNA does not facilitate the methylation of the same site on the opposite strand. With the dam methylase it was found that the enzyme is incapable of methylating GATC sites located at the ends of DNA molecules.     

Study of the substrate-induced changes in the state of Eco dam methylase using a method of small-angle x-ray scattering

Interaction of Ecodam methylase (E.C. 2.1.1) with synthetic oligonucleotide substrates of various primary structure was studied by the small angle X-ray scattering method. Complex formation between the enzyme and substrates occurs after addition of double-stranded oligonucleotides to the methylase. In the presence of 1 M NaC1 (when the enzyme is inactive) addition of the synthetic substrates does not result in complex formation. Comparison of the experimental scattering parameters with the calculated ones has been made. The best coincidence of these data is obtained for the model which proposed Ecodam methylase dimer formation in the course of its interaction with the substrates.     

Purification and properties of the Hpa I methylase.

The purification and catalytic properties of the homogeneous Hpa I methylase is described. The enzyme exists as a single polypeptide chain with a molecular weight of 37,000 +/- 2,000 was shown by sedimentation equilibrium and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The Hpa I methylase transfers methyl groups of S-adenosylmethionine to adenine present in the recognition sequence d(G-T-T-A-A*-C), A* is the N6 methyl adenosine. An average of 2.1 methyl groups per recognition site are transferred by the Hpa I methylase.     

Substrate recognition and selectivity in the type IC DNA modification methylase M.EcoR124I.

The type I DNA modification methylase M.EcoR124I binds sequence specifically to DNA and protects a 25bp fragment containing its cognate recognition sequence from digestion by exonuclease III. Using modified synthetic oligonucleotide duplexes we have investigated the catalytic properties of the methylase, and have established that a specific adenine on each strand of DNA is the site of methylation. We show that the rate of methylation of each adenine is increased at least 100 fold by prior methylation at the other site. However, this is accompanied by a significant decrease in the affinity of the methylase for these substrates according to competitive gel retardation assays. In contrast, methylation of an adenine in the recognition site which is not a target for the enzyme results in only a small decrease in both DNA binding affinity and rate of methylation by the enzyme.     

Equilibrium kinetics of substrate-enzyme interactions in single crystals of cytoplasmic aspartate aminotransferase

Orthorhombic single crystals of cytoplasmic aspartate aminotransferase were examined alone or in the presence of substrates or inhibitors to quantitatively compare the interaction of ligands with the active-site chromophore between soluble and crystalline enzyme. As in enzyme solutions, equilibrium kinetic measurements can be made between substrates and single crystals of cytoplasmic aspartate aminotransferase. The absorption spectra of ligand-free enzyme forms and of enzyme-substrate or-inhibitor complexes are as distinctive as when the enzyme is in solution. The dissociation constants for glutamate with the pyridoxal form of the enzyme are identical to those in solution. The substrate analog erythro--hydroxyaspartate also binds with equal affinity to the active site in enzyme crystals as in solution; and the affinity of -ketoglutarate to bind in nonproductive complexes with the pyridoxal form of the enzyme is also unimpaired in the crystal (K _d =2 mM). In contrast to the affinity constants, the stoichiometry of the interactions does not appear to correlate to those in solution. In the presence of an amino acid plus keto acid substrates pair, the absorbance values of the enzyme-substrate complex(es) could be interpreted as for occupany of only half the available sites in the crystals. Yet an amino acid, cysteine sulfinate, and -keto acids such as , -difluorooxalacetate convert all active sites in the crystal to the pyridoxamine or pyridoxal form when added to the pyridoxal or pyridoxamine forms, respectively. This ability to completely undergo substrate-induced half-transamination and the apparently conflicting results in trapping half the sites in enzyme-substrate complexes are incorporated into a proposed reciprocating mechanism applicable only to the crystalline state of the enzyme and dictated by crystal packing forces rather than an intrinsic property of the enzyme. Active-site bound pyridoxal phosphate continues to behave as a pH indicator; nevertheless, the pK value of the single crystals is a pH unit (pK=7.15) higher than that in solution. This variation is interpreted as indication of a difference in the environment of the chromophore between the crystal and solution states. While the environmental difference does not significantly alter the affinity for substrates, it could account for the reduced rates in transformation of the enzyme-substrate complexes in half-transamination reactions in the crystalline state.     

Purification and properties of the Eco57I restriction endonuclease and methylase--prototypes of a new class (type IV).

The Eco57I restriction endonuclease and methylase were purified to homogeneity from the E.coli RR1 strain carrying the eco57IRM genes on a recombinant plasmid. The molecular weight of the denaturated methylase is 63 kDa. The restriction endonuclease exists in a monomeric form with an apparent molecular weight of 104-108 kDa. R.Eco57I also possesses methylase activity. The methylation activities of both enzymes modify the outer A residue in the target sequence 5'CTGAAG yielding N6-methyladenine. M.Eco57I modifies both strands of the substrate while R.Eco57I modifies only one. Only the methylase enzyme is stimulated by Ca2+. The restriction endonuclease shows an absolute requirement for Mg2+ and is stimulated by AdoMet. ATP has no influence on either activity of the enzymes. The subunit structure and enzymatic properties of the Eco57I enzymes distinguish them from all other restriction-modification enzymes that have been described previously. Therefore, RM.Eco57I may be regarded as a representative of a novel class of restriction-modification systems, and we propose to classify it as type IV.     

Involvement of E. coli dcm methylase in Tn3 transposition

The effects of DNA methyltransferases on Tn3 transposition were investigated. The E. coli dam (deoxyadenosine methylase) gene was found to have no effect on Tn3 transposition. In contrast, Tn3 was found to transpose more frequently in dcm+ (deoxycytosine methylase) cells than in dcm- mutants. When the EcoRII methylase gene was introduced into dcm- cells (E. coli strain GM208), the frequency of Tn3 transposition in GM208 was dramatically increased. The EcoRII methylase recognizes and methylates the same sequence as does the dcm methylase. These results suggest that deoxycytosine methylase modified DNA may be a preferred target for Tn3 transposition. Experiments were also performed to determine whether the Tn3 transposase was involved in DNA modification. Plasmid DNA isolated from dcm- E. coli containing the Tn3 transposase gene was susceptible to ApyI digestion but resistant to EcoRI digestion, suggesting that Tn3 transposase modified the dcm recognition sequence. In addition, restriction enzymes TaqI, AvaII, BglI and HpaII did not digest this DNA completely, suggesting that the recognition sequences of TaqI, AvaII, BglI and HpaII were modified by Tn3 transposase to a certain degree. The type(s), the extent and mechanism(s) of this modification remain to be investigated.     

Determination of the recognition sites of cytosine DNA-methylases from Escherichia coli SK.

Two different cytosine DNA-methylases, NI and GII, are present in Escherichia coli SK. The GII methylase recognizes the five-member symmetric sequence: 5'...NpCpCpApGpGpN...3'. This sequence is identical with the recognition site of the hsp II type determined by RII plasmid but, in contrast to RII methylase, the GII enzyme methylates cytosine located on the 5' side of the site. By analogy with the isoshizomery of the restricting endonucleases, RII and GII DNA methylaeses may be called isomethymers which recognize the same site but methylate different bases. Since the phage of the SK and hsp II phenotypes is effectively restricted in respective cells it may be assumed that the isomethymeric modification does not provide any protection against the corresponding restrictases. NI methylase recognizes the five-member symmetric site which represents an inverted sequence of the GII site: 5'...NpGpGpApCpCpN...3'. In this case cytosine at the 3'-end of the recognition site is methylated.