CLONING AND SEQUENCE-ANALYSIS OF THE PLASMID-BORNE GENES ENCODING THEECO29KI RESTRICTION AND MODIFICATION ENZYMES

The Eco29kI restriction-modification system (RMS2) has been found to b e localized on the plasmid pECO29 occurring naturally in the Escherich ia coli strain 29k (Pertzev, A.V., Ruban, N.M., Zakharova, M.V., Belet skaya, I.V., Petrov, S.I., Kravetz, A.N., Solonin, A.S., 1992. Eco29kI , a novel plasmid encoded restriction endonuclease from Escherichia co li. Nucleic Acids Res. 20, 1991). The genes coding for this RMS2, a Sa cII isoschizomer recognizing the sequence CCGCGG have been cloned in E scherichia coli K802 and sequenced. The DNA sequence predicts the rest riction endonuclease (ENase) of 214 amino acids (aa) (24 556 Dal and t he DNA-methyltransferase (MTase) of 382 aa (43 007 De) where the genes are separated by 2bp and arranged in tandem with eco29kIR preceding e co29kIM. The recombinant plasmid with Eco29kIR produces a protein of e xpected size. (M) over dot Eco29kI contains all the conserved aa seque nce motifs characteristic of m(5)C-MTases. Remarkably, its variable re gion exhibits a significant similarity to the part of the specific tar get-recognition domain (TRD) from (M) over dot BssHII-multispecific m( 5)C-MTase (Schumann, J.J., Waiter, J., Willert, J., Wild, C., Koch D., Trautner, T.A., 1996. (M) over dot BssHII: a multispecific cytosine-C 5-DNA-methyltransferase with unusual target recognizing properties. J. Mel. Biol. 257, 949-959), which recognizes five different sites on DN A (HaeII, MluI, Cfr10I, SacII and BssHII), and the comparison of the n t sequences of its variable regions allowed us to determine the putati ve TRD of (M) over dot Eco29kI. (C) 1998 Elsevier Science B.V.