Isolation and characterization of type IIS restriction endonuclease from Neisseria cuniculi ATCC 14688.
Neisseria cuniculi produces the restriction enzyme NcuI which is an isoschizomer of MboII. We have demonstrated that NcuI recognizes a pentanucleotide sequence (5'-GAAGA-3'/3'-CTTCT-5'), and cleaves the DNA 8 and 7 nucleotides downstream from the recognition site leaving a single 3'-protruding nucleotide. We have purified this enzyme to electrophoretic homogeneity using a four-step chromatographic procedure. NcuI endonuclease is a monomeric protein 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 site being identical and the pH profile and divalent cation requirements being similar. Moreover, NcuI cross-reacts strongly with anti-MboII serum suggesting the presence of similar antigenic determinants. We have determined the sequence of 20 N-terminal amino acids for NcuI and concluded that this sequence is identical to the N-terminal portion of the MboII enzyme.
Department of Microbiology, University of Gdansk, Kladki 24, 80-822 Gdansk, Poland.
B
B
Furmanek
K
K
Gromek
M
M
Sektas
T
T
Kaczorowski
D000595:Amino Acid Sequence; D002845:Chromatography; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D015280:DNA Restriction-Modification Enzymes; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D009343:Neisseria / Q000201:enzymology*; D020539:Sequence Analysis, Protein; D013379:Substrate Specificity
D015280:DNA Restriction-Modification Enzymes; D007527:Isoenzymes; D004247:DNA; C024413:endodeoxyribonuclease MboII; D015252:Deoxyribonucleases, Type II Site-Specific