DNA methyltransferases genes of the BspACI restriction-modification system from Bacillus psychrodurans AC have been cloned in E. coli cells. Analysis of amino acid sequences of the proteins showed that both of these genes belong to C5 DNA methyltransferases.
Gene M1.BspACI has been subcloned in pJW2 vector. A high-purity recombinant enzyme has been obtained using chromatography on different carriers.
It has been shown that M1.BspACI modifies the first cytosine residue in the sequence 5′-CCGC-3′. Kinetic parameters of DNA
methylation by the enzyme have been determined. Catalytic constant appears to be 0.095 ± 0.002 min−1. K
mphage is λ DNA—0.053 ± 0.007 μM, and K
mSAM is 5.1 ± 0.3 μM.
KeywordsDNA methyltrasferase–gene cloning–
Moscow University Biological Sciences Bulletin 06/2011; 66(2):76-78. DOI:10.3103/S0096392511020106
A restriction-modification system from Bacillus psychrodurans AC (recognition sequence 5'-CCGC-3') comprises two DNA methyltransferases: M1.BspACI and M2.BspACI. The bspACIM1 gene was cloned in the pJW2 vector and expressed in Escherichia coli cells. High-purity M1.BspACI preparation has been obtained by chromatography on different carriers. M1.BspACI has a temperature optimum of 30°C and demonstrates maximum activity at pH 8.0. M1.BspACI modifies the first cytosine in the recognition sequence 5'-CCGC-3'. The kinetic parameters of M1.BspACI DNA methylation are as follows: K(m) for phage λ DNA is 0.053 µM and K(m) for S-adenosyl-L-methionine is 5.1 µM. The catalytic constant (k(cat)) is 0.095 min(-1).
Biochemistry (Moscow) 12/2010; 75(12):1484-90. DOI:10.1134/S0006297910120096 · 1.35 Impact Factor
The M1.Bst19I DNA-methyltransferase gene from restriction-modification system Bst19I (recognition sequence 5′-GCATC-3′) in
Bacillus stearothermophilus 19 has been cloned in the expressing vector pJW that carries a tandem of thermo inducible promoters P
from phage λ. Highly purified enzyme has been isolated by chromatography on various resins from Escherichia coli cells where it is accumulated in a soluble form. The study of M1.Bst19I properties has revealed that the enzyme has a temperature
optimum at 50°C and demonstrates maximal activity at pH 8.0. M1.Bst19I modifies adenine in sequence 5′-GCATC-3′. Kinetic parameters
of M1.Bst19I DNA methylation reaction have been determined as follows: Km for λ DNA is 0.68 ± 0.07 μM, Km for S-adenosyl-L-methionine is 2.02 ± 0.31 μM. Catalytical constant (k
cat) is 1.8 ± 0.05 min−1. Comparative analysis of Target Recognition Domain amino acid sequences for M1.Bst19I and other α-N6-DNA-methyltransferases
has allowed us to suggest the presence of two types of the enzymes containing ATG or ATC triplets in the recognition sequence.
-enzyme kinetics-amino acid sequence
Molecular Biology 08/2010; 44(4):606-615. DOI:10.1134/S0026893310040163 · 0.74 Impact Factor
Optimal conditions for DNA methylation by the M3.BstF5I enzyme from Bacillus stearothermophilus and kinetic parameters of lambda phage DNA modification and that of a number of oligonucleotide substrates are established. Comparison of M1.BstF5I and M3.BstF5I kinetic parameters revealed that with similar temperature optima and affinity for DNA, M3.BstF5I has nearly fourfold lower turnover number (0.24 min(-1)) and modifies the hemimethylated recognition site with lower efficiency under optimal conditions than the unmethylated one. In contrast to another three methylases of the BstF5I restriction-modification system, the M3.BstF5I enzyme is able to optionally modify the noncanonical 5'-GGATC-3' DNA sequence with a rate more than one order of magnitude lower than the methylation rate of the canonical 5'-GGATG-3' recognition site.
Biochemistry (Moscow) 01/2010; 75(1):63-71. DOI:10.1134/S0006297910010086 · 1.35 Impact Factor
Mammalian DNA methyltransferase 1 (Dnmt1) is responsible for copying the DNA methylation pattern during cell division. Since
Dnmt1 plays an important role in carcinogenesis, it is of particular interest to search for its specific inhibitors. To design
oligonucleotide inhibitors of human Dnmt1, a number of singlestranded, double-stranded, and hairpin DNA structures containing
a canonical or a modified Dnmt1 recognition site (5′-CG) were constructed on the basis of a 22-nt sequence. Structural features
such as a C:A mismatch, phosphorothioates, and hairpins proved capable of incrementally increasing the oligonucleotide affinity
for Dnmt1. An improvement of inhibitory properties was also achieved by replacing the target cytosine with 5,6-dihydro-5-azacytosine,
5-methyl-2-pyrimidinone, or 6-methyl-pyrrolo-[2,3-d]-2-pyrimidinone. The concentration that caused 50% inhibition of methylation
of 1 μM poly(dI-dC) · poly(dI-dC), a conventional DNA substrate, was approximately 10−7 M for the most efficient oligonucleotides. Under the same in vitro conditions, these oligonucleotide inhibitors demonstrated
a substantially stronger effect compared to known Dnmt1 inhibitors, which were used as controls.
Molecular Biology 06/2009; 43(3):418-425. DOI:10.1134/S0026893309030108 · 0.74 Impact Factor