Isolation and characterization of type IIS restriction endonuclease from Neisseria cuniculi ATCC 14688

Neisseria cuniculi produces the restriction enzyme NcuI which is an isoschi zomer of MboII. We have demonstrated that NcuI recognizes a pentanucleotide sequence (5'-GAAGA-3'/3'-CTTCT-5'), and cleaves the DNA 8 and 7 nucleotide s downstream from the recognition site leaving a single 3'-protruding nucle otide. We have purified this enzyme to electrophoretic homogenity using a f our-step chromatographic procedure. NcuI endonuclease is a monomeric protei n with a M-r = 48 000 +/- 1000 under denaturing conditions. The properties of NcuI are consistent with those for MboII, the position of the cleavage s ite being identical and the pH profile and divalent cation requirements bei ng similar. Moreover, NcuI cross-reacts strongly with anti-MboII serum sugg esting the presence of similar antigenic determinants. We have determined t he sequence of 20 N-terminal amino acids for NcuI and concluded that this s equence is identical to the N-terminal portion of the MboII enzyme. (C) 200 1 Federation of European Microbiological Societies. Published by Elsevier S cience B.V. All rights reserved.