The SalGI restriction endonuclease. Mechanism of DNA cleavage

Abstract

The cleavage of supercoiled DNA of plasmid pMB9 by restriction endonuclease SalGI has been studied. Under the optimal conditions for this reaction, the only product is the linear form of the DNA, in which both strands of the duplex have been cleaved at the SalGI recognition site. DNA molecules cleaved in one strand at this site were found to be poor substrates for the SalGI enzyme. Thus, both strands of the DNA appear to be cleaved in a concerted reaction. However, under other conditions, the enzyme cleaves either one or both strands of the DNA; the supercoiled substrate is then converted to either open-circle or linear forms, the two being produced simultaneously rather than consecutively. We propose a mechanism for the SalGI restriction endonuclease which accounts for the reactions of this enzyme under both optimal and other conditions. These reactions were unaffected by the tertiary structure of the DNA.