Mechanism of action of rat liver DNA methylase. I. Interaction with double-stranded methyl-acceptor DNA

Rat liver DNA methylase forms two types of complexes with DNA which are distinguishable on the basis of their sensitivity to ionic strength. A weak complex which is dissociable by 0.2 m-NaCl is formed at 0 °C. A more tightly bound complex which is stable to 0.2 m-NaCl is formed at higher temperatures. On the basis of a comparison of the effects of salt upon tight complex formation and upon enzyme activity, it would appear that formation of the tightly bound complex is required for DNA methylation to occur. With tightly bound enzyme the methylation of high molecular weight DNA is nearly linear for 30 minutes in the presence of salt. Under the same conditions low molecular weight DNA's give non-linear kinetics which demonstrate a sharp reduction in the rate of methylation with time. The lower the molecular weight of the DNA fragments, the more marked is the deviation from linearity. On the basis of these data it is suggested that rat liver DNA cytosine-methylase which is tightly bound to DNA is able to accomplish several methyl group transfers without becoming detached from the DNA and that the enzyme must therefore walk along the DNA helix. We calculate that the walk is probably not random, but linear, and that the rate of walk may be about 1.5 to 3.5 base pairs per second.