ENDONUCLEASE SEGE OF PHAGE-T4 .1. CLONING, EXPRESSION, AND BIOCHEMICAL-CHARACTERIZATION OF ENDONUCLEASE ACTIVITY

Phage T4 gene segE (hoc.2) was cloned in translation vector pET-3a and expressed in Escherichia coli using bacteriophage CE6 as a source of T7 RNA polymerase. The SegE protein was purified by sequential chromat ography on phosphocellulose, phenyl-Sepharose, and DEAE-Sepharose. It proved to be a Mg-dependent site-specific endonuclease cleaving plasmi d and DNA as well as wild-type T4 DNA containing glucosylated oxymethy lcytosine. Some sites were preferred for cleavage but in all cases hyd rolysis was incomplete. An increase of enzyme concentration or replace ment of Mg2+ with Mn2+ decreased the specificity. Optimal conditions w ere determined for cleavage at one of the preferred sites.