SUBSTRATE RECOGNITION AND SELECTIVITY IN THE TYPE-IC DNA MODIFICATIONMETHYLASE M.ECORI124I

The type I DNA modification methylase M.EcoR124I binds sequence specif ically to DNA and protects a 25bp fragment containing its cognate reco gnition sequence from digestion by exonuclease III. Using modified syn thetic oligonucleotide duplexes we have investigated the catalytic pro perties of the methylase, and have established that a specific adenine on each strand of DNA is the site of methylation. We show that the ra te of methylation of each adenine is increased at least 100 fold by pr ior methylation at the other site. However, this is accompanied by a s ignificant decrease in the affinity of the methylase for these substra tes according to competitive gel retardation assays. In contrast, meth ylation of an adenine in the recognition site which is not a target fo r the enzyme results in only a small decrease in both DNA binding affi nity and rate of methylation by the enzyme.