Isolation and properties of a new site-specific endonuclease Bmel42I from Bacillus megaterium 142

A new type II restriction endonuclease, Bmel42I, was partially purified and characterized from Bacillus megaterium 142. It has been seen in a crude extract, because this bacterial strain does not have significant amounts of contaminating nucleases. The enzyme was purified by chromatography on phosphocellulose Pll (elution buffer 10 mM K-phosphate, pH 7.0, 0.1 mM EDTA, 2 mM DTT, 0.2-1 M NaCl) and hydroxyapatite (elution buffer 0.01-0.5 M K-phosphate, pH 7.0, 1 mM EDTA, 2 mM DTT). Yield of enzyme is 2000 units per gram of wet cells. The enzyme is stable in elution buffer and may be stored at -20° after addition of glycerol to 50%. The recognition site was determined from the cleavage pattern of. pBR322 plasmid. The 5'end nucieotide is G. This has been shown by incubation of the 5'-end labelled DNA fragments with nuclease PI followed by thinlayer chromatography on a PEI cellulose plate (1). The points of cleavage of the recognition site were determined by the MaxamGilbert method. In contrast to its isoschisomer, the restriction endonuclease Haell, which cleaves the same recognition site to produce a 4 base 3' extension, the Smel42I produces blunt-ended DNA fragments: