Biochemical Characterization of the Restriction‐Modification System of Bacillus sphaericus

Abstract

A type II restriction endonuclease (endo R ·Bsp) has been purified from Bacillus sphaericus to electrophoretic homogeneity. The enzyme appears to be a single polypeptide chain with a molecular weight of 35000. Its pH optimum is around 8.2, it requires 20 mM Mg²⁺ for optimal activity and it is inhibited by Zn²⁺. The yield of the enzyme is higher than that of any type II restriction endonuclease so far reported. The enzyme also cleaves single-stranded DNA, albeit at a slower rate. It seems likely that single-stranded DNA is cleaved at the same sequences as double-stranded DNA. Bacillus sphaericus also contains a modification methylase (meth M ·Bsp) which completely protects the cell's own DNA against cleavage by its restriction endonuclease. The methylase activity has been partially purified, it copurifies with the nuclease until the next to the last step. The enzyme does not require ATP or Mg²⁺, it transfers the methyl group of S-adenosyl-methionine to cytosine residues of DNA. As the action of this methylase completely protects any DNA from endo R ·Bsp cleavage, it seems likely that the methylase recognizes and methylates the same sequence (dG-dG-dC-dC) as the nuclease.