RESUMO
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.
Assuntos
Metilases de Modificação do DNA/metabolismo , DNA/metabolismo , Geobacillus stearothermophilus/enzimologia , Sítios de Ligação , Metilação de DNA , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/isolamento & purificação , Cinética , DNA Metiltransferases Sítio Específica (Adenina-Específica)/química , DNA Metiltransferases Sítio Específica (Adenina-Específica)/metabolismo , Especificidade por SubstratoRESUMO
A gene encoding DNA methyltransferase (methylase) FauIA of the restriction-modification system FauI from Flavobacterium aquatile (recognizing sequence 5'-CCCGC-3') was cloned in pJW vector. The latter was used for transformation of E. coli RRI cells followed by subsequent thermoinduction and biomass elaboration. Highly purified DNA methyltransferase FauIA preparation was obtained using chromatography on different sorbents. The molecular mass of the isolated enzyme of about 39 kD corresponds to its theoretical value. The enzyme was characterized by temperature and pH optima of 33 degrees C and pH 7.5, respectively. Methylation of a synthetic oligonucleotide by FauIA methylase followed by its cleavage with various restrictases and analysis of the resultant restriction fragments revealed that FauIA methylase modified the second cytosine residue in the sequence 5'-CCCGC-3'. Kinetic analysis revealed Km and catalytic constant values of 0.16 microM and 0.05 min(-1), respectively.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Citosina/química , DNA (Citosina-5-)-Metiltransferases/isolamento & purificação , Metilases de Modificação do DNA/química , Metilases de Modificação do DNA/isolamento & purificação , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Clonagem Molecular , DNA (Citosina-5-)-Metiltransferases/química , DNA Bacteriano , Flavobacterium , Genes Bacterianos , Cinética , Metilação , Dados de Sequência Molecular , Peso Molecular , Especificidade por SubstratoRESUMO
The BstF5I restriction-modification system from Bacillus stearothermophilus F5 includes four site-specific DNA methyltransferases, thus differing from all known restriction-modification systems. Here we demonstrated for the first time that one bacterial cell can possess two pairs of methylases with identical substrate specificities (methylases BstF5I-1 and BstF5I-3 recognize GGATG, whereas methylases BstF5I-2 and BstF5I-4 recognize CATCC) that modify adenine residues on both DNA strands. Different chromatographic methods provide homogenous preparations of methylases BstF5I-2 and BstF5I-4. We estimated the principal kinetic parameters of the reaction of transfer of methyl group from the donor S-adenosyl-L-methionine to the recognition site 5;-CATCC-3; catalyzed by BstF5I-2 and BstF5I-4 DNA [N6-adenine]-methyltransferases from the BstF5I restriction-modification system.
