Isolation and characterization of a restriction endonuclease, AtuS I, from agrobacterium tumefaciens SSI

Abstract

A new restriction endonuclease, AtuS i, was isolated from Agrobacterium tumefaciens SSI by the following steps: streptomycin sulfate fractionation, ammonium sulfate fractionation, phosphocellulose(Pll) column chromatography, CM-sepharose column chromatography, Heparin-agarose column chromatography, and Hydroxylapatite column chromatography. From the comparison of digestion patterns on various dnAs, it was demonstrated that AtuS I endonuclease recognizes the same sequence as BstN I (5``-CCAGG-3`` or 5``-CCTGG-3``). AtuS I enzyme cleaves lambda DNA at seventy one sites, Adeno2 DNA at one hundred thirty six sites. But Type II restriction enzyme, AtuS i, can not cleave dcm methylated DNA. AtuS I endoncuclease showes maximum activity at pH 8.0 and requires $Mg^{++}$ as the only cofactor. Also NaCl has no effect on enzyme activity at 37$^\circ$C.