Effect of the structure of oligonucleotide substrates on interaction with methylase Eco dam

The interaction of Eco dam methylase with various synthetic oligonucleotide substrates was investigated. The "imperfect" duplexes contained a normal GATC recognition sequence in one chain of the enzyme recognition site and had some defects in the complementary chain, i.e., the absence of one or several nucleotide residues or the presence of S-methyl thiophosphate groups at the 3'-termini. The 3'-S-methyl thiophosphate residue has the same effect on the methylation of oligonucleotide complexes as does the absence of internucleotide phosphate in the analogous complexes. The presence of both GA dinucleotides in the recognition site is necessary for a productive enzyme-substrate interaction. The experimental data suggest that Eco dam methylase does form a symmetrical enzyme-substrate complex which is similar to that formed by type II restriction enzymes.     

Nucleotide sequences in DNA showing preferential binding with distamycin A and acridine derivatives

The specificity of DNA X dye binding was studied. Antibiotic distamycin A was bound most strongly to the DNA sequences composed of three or more neighboring A X T pairs. Acrichin and 7-aminoacrichin proved to be weak specific inhibitors binding predominantly within the A X T regions.     

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 oligonucleotide duplex containing the palindrome recognition site GGATCC was studied by methods of steady-state and presteady-state kinetics of the methyl group transfer, gel retardation, and crosslinking of the enzyme subunits with glutaric aldehyde. In steady-state conditions, BamHI MTase displays a simple kinetic behavior toward a 20-mer oligonucleotide 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 then the concentrations of substrates. The kcat values determined in these conditions are in good agreement with one another and approximately equal to 0.05 s-1. The Km values for the duplex and SAM are 0.35 and 1.6 microM, respectively. An analysis of single turnover kinetics (at limiting concentration of the 20-mer oligonucleotide 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 the course of the enzyme single turnover, which coincides with the earlier data on EcoRI MTase. Regardless of the order of the 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 of DNA. In gel retardation experiments, there was no stoichiometrically simple complexes 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.     

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.     

Molecular enzymology of phage T4 Dam DNA-methyltransferase

The review reflects results of studies on the molecular mechanism of phage T4 Dam DNA-methyltransferase action. The enzyme (T4Dam) catalyzes methyl group transfer from S-adenosyl-l-methionine (AdoMet) to N6-adenine position in the palindromic recognition sequence GATC (EC 2.1.1.72). The enzyme subunit structure, substrate-binding and kinetic parameters for a wide range of native and modified oligonucleotide duplexes, as well as steady-state reaction kinetic scheme, included T4Dam isomerization to catalytically active form, are considered. The found mechanisms of DNA induced T4Dam dimerization, target base flipping, enzyme reorientation in an asymmetrically modified recognition sequence, effector action of reaction substrates and processive methylation of DNA substrates, containing more than one specific site, are discussed. The results obtained with T4Dam may be useful for understanding mechanisms of action of other homologous enzymes, most of all for specimens of numerous family of Dam DNA-methyltransferases.     

Catalogue of Human Tissue Optical Properties at Terahertz Frequencies

Recently published studies suggest thatterahertz pulsed imaging will have applications inmedicine and biology, but there iscurrently very little information about the opticalproperties of human tissue at terahertzfrequencies. Such information would be useful forpredicting the feasibility of proposedapplications, optimising acquisition protocols,providing information about variability ofhealthy tissue and supplying data for studies of theinteraction mechanisms. Research ethicscommittee approval was obtained, andmeasurements made from samples of freshlyexcised human tissue, using a broadbandterahertz pulsed imaging system comprisingfrequencies approximately 0.5 to 2.5 THz.Refractive index and linear absorptioncoefficient were found. Reproducibility wasdetermined using blood from one volunteer,which was drawn and measured on consecutivedays. Skin, adipose tissue, striatedmuscle, vein and nerve were measured (to date, from oneindividual). Water had a higher refractiveindex (2.04 ± 0.07) than any tissue.The linear absorption coefficient was higher formuscle than adipose tissue, as expectedfrom the higher hydration of muscle. As these samples camefrom a single subject, there is currentlyinsufficient statistical power to draw firmconclusions, but results suggest that in vivo clinical imaging will be feasible in certainapplications.     

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.     

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.     

Aggregation of tryptophanyl-tRNA synthetase depending on temperature. Study by a low-angle scatter x-ray method

By means of small angle X-ray scattering, an aggregation of beef pancreas Trp-tRNA synthetase (EC 6.1.1.2) was observed at physiological temperatures. A Trp-tRNA synthetase preparation which is homogeneous after PAGE in beta-ME-SDS was found to be heterogeneous in particle sizes even at low (4-8 degrees C) temperature. At heating up to 30-45 degrees C, the oligomer sizes increased as well as its proportion depending on the incubation time and temperature; very large aggregates were observed 10 times exceeding the sizes of initial particles. Cooling to 20 degrees C caused no disaggregation due to disulphide bond formation between associated subunits of Trp-tRNA synthetase. A hypothesis is proposed that the aggregation of bovine Trp-tRNA synthetase evaluated in vitro and not observed earlier with any aminoacyl-tRNA synthetases of unicellular organisms might serve as one of the mechanisms of its compartmentation in pancreas.