High-performance method for specific effect on nucleic acids in cells using TiO2~DNA nanocomposites.

Nanoparticles are used to solve the current drug delivery problem. We present a high-performance method for efficient and selective action on nucleic acid target in cells using unique TiO(2)·PL-DNA nanocomposites (polylysine-containing DNA fragments noncovalently immobilized onto TiO(2) nanoparticles capable of transferring DNA). These nanocomposites were used for inhibition of human influenza A (H3N2) virus replication in infected MDCK cells.

DNA methyltransferases: mechanistic models derived from kinetic analysis.

The sequence-specific transfer of methyl groups from donor S-adenosyl-L-methionine (AdoMet) to certain positions of DNA-adenine or -cytosine residues by DNA methyltransferases (MTases) is a major form of epigenetic modification. It is virtually ubiquitous, except for some notable exceptions. Site-specific methylation can be regarded as a means to increase DNA information capacity and is involved in a large spectrum of biological processes.

Dimeric/oligomeric DNA methyltransferases: an unfinished story.

DNA methyltransferases (MTases) are enzymes that carry out post-replicative sequence-specific modifications. The initial experimental data on the structure and kinetic characteristics of the EcoRI MTase led to the paradigm that type II systems comprise dimeric endonucleases and monomeric MTases. In retrospect, this was logical because, while the biological substrate of the restriction endonuclease is two-fold symmetrical, the in vivo substrate for the MTase is generally hemi-methylated and, hence, inherently asymmetric.

Differential methylation kinetics of individual target site strands by T4Dam DNA methyltransferase.

Prokaryote DNA methyltransferases (MTases) of the Dam family (including those of bacteriophages T2 and T4) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. Dam DNA MTases, as all site-specific enzymes interacting with polymeric DNA, require a mechanism of action that ensures a rapid search for specific targets for catalytic action, during both the initial and subsequent rounds of methylation. The results of pre-steady-state (reaction burst) and steady-state methylation analyses of individual targets permitted us to monitor the action of T4Dam, which has three degrees of freedom: sliding, reorientation and adaptation to the canonical GATC sequence.

Study of bacteriophage T4-encoded Dam DNA (adenine-N6)-methyltransferase binding with substrates by rapid laser UV cross-linking.

DNA methyltransferases of the Dam family (including bacteriophage T4-encoded Dam DNA (adenine-N(6))-methyltransferase (T4Dam)) catalyze methyl group transfer from S-adenosyl-L-methionine (AdoMet), producing S-adenosyl-L-homocysteine (AdoHcy) and methylated adenine residues in palindromic GATC sequences. In this study, we describe the application of direct (i.e.

A probabilistic approach to compact steady-state kinetic equations for enzymic reactions.

We describe a method for deriving kinetic equations based on the simplification of a complex graphical scheme of steady-state enzymic reactions to one that is comprised of an unbranched pathway. It entails compressing unbranched multi-step sequences into one step, and fusing some graph nodes into a single node. The final form of the equations is compact and well structured, and it simplifies the choice of independent kinetic parameters.

Bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase. Comparison of pre-steady state and single turnover methylation of 40-mer duplexes containing two (un)modified target sites.

We analyzed pre-steady state and single turnover kinetics of bacteriophage T4Dam DNA-(adenine-N(6))-methyltransferase-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to 40-mer duplexes containing native recognition sites (5'-GATC/5'-GATC) or some modified variant(s). The results extend a model from studies with single-site 20-mer duplexes. Under pre-steady state conditions, monomeric T4Dam methyltransferase-AdoMet complexes were capable of rapid methylation of adenine residues in 40-mer duplexes containing two sites.

Symmetry elements in DNA structure important for recognition/methylation by DNA [amino]-methyltransferases.

The phage T4Dam and EcoDam DNA-[adenine-N6] methyltransferases (MTases) methylate GATC palindromic sequences, while the BamHI DNA-[cytosine-N4] MTase methylates the GGATCC palindrome (which contains GATC) at the internal cytosine residue. We compared the ability of these enzymes to interact productively with defective duplexes in which individual elements were deleted on one chain. A sharp decrease in kcat was observed for all three enzymes if a particular element of structural symmetry was disrupted.

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

We compared the (pre)steady-state and single turnover methylation kinetics of bacteriophage T4Dam (DNA-(adenine-N6)-methyltransferase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to oligodeoxynucleotide duplexes containing a single recognition site (palindrome 5'-GATC/5'-GATC) or some modified variant. T4Dam-AdoMet functions as a monomer under steady-state conditions (enzyme/DNA << 1), whereas under single turnover conditions (enzyme/DNA > 1), a catalytically active complex containing two Dam-AdoMet molecules is formed initially, and two methyl groups are transferred per duplex (to produce a methylated duplex and S-adenosyl-l-homocysteine (AdoHcy)). We propose that the single turnover reaction proceeds in two stages.

DNA (cytosine-N4-)- and -(adenine-N6-)-methyltransferases have different kinetic mechanisms but the same reaction route. A comparison of M.BamHI and T4 Dam.

We studied the kinetics of methyl group transfer by the BamHI DNA-(cytosine-N(4)-)-methyltransferase (MTase) from Bacillus amyloliquefaciens to a 20-mer oligodeoxynucleotide duplex containing the palindromic recognition site GGATCC. Under steady state conditions the BamHI MTase displayed a simple kinetic behavior toward the 20-mer duplex. There was no apparent substrate inhibition at concentrations much higher than the K(m) for either DNA (100-fold higher) or S-adenosyl-l-methionine (AdoMet) (20-fold higher); this indicates that dead-end complexes did not form in the course of the methylation reaction.

Functional analysis of BamHI DNA cytosine-N4 methyltransferase.

We show that the kinetic mechanism of the DNA (cytosine-N(4)-)-methyltransferase M.BamHI, which modifies the underlined cytosine (GGATCC), differs from cytosine C(5) methyltransferases, and is similar to that observed with adenine N(6) methyltransferases. This suggests that the obligate order of ternary complex assembly and disassembly depends on the type of methylation reaction.

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

We carried out steady state and pre-steady state (burst) kinetic analyses of the bacteriophage T4 Dam DNA-(N(6)-adenine)-methyltransferase (MTase)-mediated methyl group transfer from S-adenosyl-l-methionine (AdoMet) to Ade in oligonucleotide duplexes containing one or two specific GATC sites with different combinations of methylated and unmodified targets. We compared the results for ligated 40-mer duplexes with those of the mixtures of the two unligated duplexes used to generate the 40-mers. The salient results are as follows: (i) T4 Dam MTase modifies 40-mer duplexes in a processive fashion.

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.