Two subunits of EcoRII restriction endonuclease interact with two DNA recognition sites

The cleavage of a 14 base pair DNA duplex containing one EcoRII recognition site by EcoRII restriction endonuclease (R.EcoRII) was studied in single turnover experiments with varying enzyme concentrations in the micromolar range. The reaction rate increased with enzyme concentration until a ratio of one dimeric R.EcoRII enzyme to two double stranded oligonucleotide molecules. Excess of R.EcoRII lead to inhibition of cleavage. Maximum cleavage was also found with pBR322 DNA containing six EcoRII recognition sites at a ratio of one dimeric enzyme to two EcoRII recognition sites of the plasmid DNA. At higher ratios inhibition was observed. These observations indicate that the active enzyme complex is formed when two subunits of the enzyme interact with two R.EcoRII recognition sites.