Purification and characterization of alu I methylase

Abstract

Alu I methylase has been isolated from 300g (wet weight) cells of Arthrobacter luteus. After ammonium sulfate fractionation, the protein which has methylase activity was purified through phosphocellulose, DEAE-cellulose, Heparin agarose, and Hydroxylapatite column chromatography. The methylated DNA by the purified methylase was resistatnt against Alu I endonuclease. The purified Alu I methylase was essentially homogeneous as judged by 10% SDS-polyacrylamide gel electrophoresis, and the apparent subunit molecular weight was 56,000±1,000. The specific activity of the enzyme was 1.32 × 10 units per mg protein. Since the discovery by Smith (1970), Type II restriction and modification enzymes have been invaluable both as tools for the study of gene structure and as models for sequence specific DNA-protein interactions. In particular, comparison of a Type II endonuclease with its cognate methylase may reveal valuable information on two distinct proteins that interact with the same nucleotide sequence. In 1976, Roberts et al. (1976) reported that Alu I endonuclease from Arthrobacter luteus recognizes basesequence 5'-d (AG ↓ CT)-3' with the cleavage site indicated by the arrow, but there was no report about Alu I methylase activity. In this paper, a procedure for the purfication of Alu I methylase is described. The subunit molecular weight and the specificity of the enzyme are also presented. Materials and Methods