MnlI - The member of H-N-H subtype of Type IIS restriction endonucleases

The Type IIS restriction endonuclease Mull recognizes the non-palindromic nucleotide sequence 5'-CCTC(N)7/6 down arrow and cleaves DNA strands as indicated by the arrow. The genes encoding Mull restriction-modification system were cloned and sequenced. It comprises N6-methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease. Biochemical studies revealed that Mull restriction endonuclease cleaves double- and single-stranded DNA, and that it prefers different metal ions for hydrolysis of these substrates. Mg2+ ions were shown to be required for the specific cleavage of double-stranded DNA, whereas Ni2+ and some other transition metal ions were preferred for nonspecific cleavage of single-stranded DNA. The C-terminal part of Mull restriction endonuclease revealed an intriguing similarity with the H-N-H type nucleolytic domain of bacterial toxins, Colicin E7 and Colicin E9. Alanine replacements in the conserved sequence motif 306Rx(3)ExHHx(14)Nx(8)H greatly reduced specific activity of Mull, and some mutations even completely inactivated the enzyme. However, none of these mutations had effect on MnII binding to the specific DNA, and on its oligomerisation state as well. We interpret the presented experimental evidence as a suggestion that the motif 306Rx(3)ExHHx(14)Nx(8)H represents the active site of Mull. Consequentially, MnII seems to be the member of Type IIS with the active site of the H-N-H type. (c) 2005 Elsevier B.V. All rights reserved.