The reactions of the EcoRi and other restriction endonucleases

The reaction of the EcoRI restriction endonuclease was studied with both the plasmid pMB9 and DNA from bacteriophage lambda as the substrates. With both circular and linear DNA molecules, the only reaction catalysed by the EcoRI restriction endonuclease was the hydrolysis of the phosphodiester bond within one strand of the recognition site on the DNA duplex. The cleavage of both strands of the duplex was achieved only after two independent reactions, each involving a single-strand scission. The reactivity of the enzyme for single-strand scissions was the same for both the first and the second cleavage within its recognition site. No differences were observed between the mechanism of action on supercoiled and linear DNA substrates. Other restriction endonucleases were tested against plasmid pMB9. The HindIII restriction endonuclease cleaved DNA in the same manner as the EcoRI enzyme. However, in contrast with EcoRI, the Sa/I and the BamHI restriction endonucleases appeared to cleave both strands of the DNA duplex almost simultaneously. The function of symmetrical DNA sequences and the conformation of the DNA involved in these DNA--protein interactions are discussed in the light of these observations. The fact that the same reactions were observed on both supercoiled and linear DNA substrates implies that these interactions do not involve the unwinding of the duplex before catalysis.