PURIFICATION OF NMU I AND STUDIES ON THE ROLE OF ARGININE RESIDUE AND METAL ION REQUIREMENT BY RESTRICTION ENZYME

A new restriction enzyme, Nmu I, has been purified from Neisseria mucosa to near homogeneity by the simple procedures of phosphocellulose and heparin agarose column chromatography. The enzyme was pure enough to be used in cloning experiments and DNA sequencing work. The cutting site of Nmu I, 5' -GC(DARR)CGGC- 3', has been determined by computer analysis of the cleavage pattern on substrates. Nmu I has a single cleavage site on SV40 DNA, and it will therefore be useful for eukaryotic gene cloning. In order to study the interaction between the restriction enzymes and nucleic acids, arginyl residues of the specific endonucleases have been modified by butanedione. As an arginine specific reagent, butanedione has been known to possess inhibitory effects in enzymes that contain either a nucleotide or a phosphate binding site. Eighteen enzymes out of twenty one restriction enzymes tested have exhibited a loss of activity upon modification. This might indicate that an arginine residue in proteins has been involved in forming an enzyme/nucleic acid complex as a key element of the catalytic or binding site of restriction endonuclease. It is well accepted that the magnesium ion is required for the action of restriction enzymes, but the exact role of the metal ion is not understood fully. To investigate the effects of metal ions in the process of specific endonuclease action, Mg('++) was replaced with Ca('++) and the transition metal ions, Co('++), Cu('++), Mn('++) and Zn('++). In the presence of Co('++), Mn('++) and Zn('++) most of the restriction enzymes tested demonstrated the same cleavage pattern on the appropriate substrate except Eco RI which has been known to exhibit relaxation of the recognition sequences with Mn('++). It is reasonable to assume that the metal ions form a coordinated complex with the nucleotides in a recognition sequence, which would become a proper target for action of specific endonucleases.