GENETIC-ENGINEERING, PRODUCTION AND CHARACTERIZATION OF MONOMERIC VARIANTS OF THE DIMERIC SERRATIA-MARCESCENS ENDONUCLEASE

The Serratia nuclease is a non-specific endonuclease which cleaves sin gle-and double-stranded RNA and DNA, It is a member of a large family of related endonucleases, most of which are dimers of identical subuni ts, with the notable exception of the Anabaena nuclease which is a mon omer, In order to find out whether the dimer state of the Serratia nuc lease is essential for its function we have produced variants of this nuclease which based on the crystal structure (Miller, M.D. and Krause , K.L. (1996), Protein Science 5, 24-33) were expected to he unable to dimerise, We demonstrate here that these variants, H183A, H184N, H181 T and H181R, are monomers and have the same secondary structure, stabi lity towards chemical denaturation and activity as the wild-type enzym e. This allows to conclude that the dimeric state is not essential for the catalytic function of the Serratia nuclease, In contrast, the S17 9C variant which is also a monomer shows little activity, presumably b ecause this amino acid substitution changes the structure of the enzym e. (C) 1998 Federation of European Biochemical Societies.