Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity. II. Catalytic properties, substrate specificity, and mode of action.
The properties of two DNA methyltransferases, termed M. BsuRIa and M. BsuRIb, whose isolation was described in the preceding paper (Günthert, U., Freund, M., and Trautner, T. A. (1981) J. Biol. Chem. 256, 9340-9345) were compared. Both enzymes recognize the same target sequence in double-stranded DNA, leading to methylation of the internal cytosine: 5'GGCC. The enzymes have identical reaction constants with their substrates, DNA (km = 2.7 nM for the 5' GGCC sequence), and S-adenosyl-L-methionine (km = 0.7 microM). Initial rates of methyl group transfer were proportional to enzyme concentration over a range of 50-fold, indicating absence of aggregation. The enzymes are different in their ionic strength requirements using Tris-HCl, pH 8.4. M. BsuRIa is most active at 100 mM, M. BsuRIb at 440 mM. As measured by incorporation kinetics and heat inactivation, M. BsuRIa is the more stable enzyme of the two. Equilibrium dialysis was used to study the mode of methyl group transfer to the DNA with either enzyme. The data indicate that initially S-adenosyl-L-methionine binds to methyltransferase. This complex attaches to either modified or nonmodified DNA. The methyl group will then be transferred to a nonmodified target sequence, leading to the dissociation of enzyme and S-adenosyl-L-homocysteine from the DNA.
D001412:Bacillus subtilis / Q000201:enzymology*; D004248:DNA (Cytosine-5-)-Methyltransferases / Q000378:metabolism*; D004262:DNA Restriction Enzymes / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000378:metabolism; D007700:Kinetics; D008780:Methyltransferases / Q000378:metabolism*; D009994:Osmolar Concentration; D013379:Substrate Specificity; D013696:Temperature
D007527:Isoenzymes; D008780:Methyltransferases; D004248:DNA (Cytosine-5-)-Methyltransferases; D004262:DNA Restriction Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific; C024403:GGCC-specific type II deoxyribonucleases