THE ECL18KI RESTRICTION-MODIFICATION SYSTEM - CLONING, EXPRESSION, PROPERTIES OF THE PURIFIED ENZYMES

Ecl18kI is a type II restriction-modification system isolated from Ent erobacter cloaceae 18kI strain. Genes encoding Ecl18kI methyltransfera se (M.Ecl18kI) and Ecl18kI restriction endonuclease (R.Ecl18kI) have b een cloned and expressed in Escherichia coli. These enzymes recognize the 5'...down arrow CCNGG...3' sequence in DNA; M.Ecl18kI methylates t he C5 carbon atom of the inner dC residue and R.Ecl18kI cuts DNA as sh own by the arrow, The restriction endonuclease and the methyltransfera se were purified from E. coli B834 [p18Ap1] cells to near homogeneity, The restriction endonuclease is present in the solution as a tetramer , while the methyltransferase is a monomer, The interactions of M.Ecl1 8kI and R.Ecl18kI with 1,2-dideoxy-D-ribofuranose containing DNA duple xes were investigated. The target base flipping-out mechanism is appli cable in the case of M.Ecl18kI. Correct cleavage of the abasic substra tes by R.Ecl18kI is accompanied by non-canonical hydrolysis of the mod ified strand. (C) 1998 Federation of European Biochemical Societies.