MnlI--The member of H-N-H subtype of Type IIS restriction endonucleases.
The Type IIS restriction endonuclease MnlI recognizes the non-palindromic nucleotide sequence 5'-CCTC(N)7/6 downward arrow and cleaves DNA strands as indicated by the arrow. The genes encoding MnlI restriction-modification system were cloned and sequenced. It comprises N6-methyladenine and C5-methylcytosine methyltransferases and the restriction endonuclease. Biochemical studies revealed that MnlI 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 MnlI 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 306Rx3ExHHx14Nx8H greatly reduced specific activity of MnlI, and some mutations even completely inactivated the enzyme. However, none of these mutations had effect on MnlI binding to the specific DNA, and on its oligomerisation state as well. We interpret the presented experimental evidence as a suggestion that the motif 306Rx3ExHHx14Nx8H represents the active site of MnlI. Consequentially, MnlI seems to be the member of Type IIS with the active site of the H-N-H type.
Kriukiene
Edita
E
Institute of Biotechnology, Graiciuno 8, Vilnius LT-02241, Lithuania.
Lubiene
Judita
J
Lagunavicius
Arunas
A
Lubys
Arvydas
A
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution / Q000235:genetics; D010582:Bacteriophage lambda / Q000235:genetics; D002384:Catalysis; D002413:Cations, Divalent / Q000737:chemistry; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D015280:DNA Restriction-Modification Enzymes / Q000235:genetics / Q000378:metabolism; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000737:chemistry / Q000378:metabolism; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009016:Moraxella / Q000201:enzymology* / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D016366:Open Reading Frames / Q000235:genetics; D011485:Protein Binding; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity / Q000235:genetics
D002413:Cations, Divalent; D015280:DNA Restriction-Modification Enzymes; D004269:DNA, Bacterial; D004277:DNA, Single-Stranded; D011994:Recombinant Proteins; C052155:endodeoxyribonuclease MnlI; D015252:Deoxyribonucleases, Type II Site-Specific