title: Rational design of a chimeric endonuclease targeted to NotI recognition site.
abstract: A cleavage-deficient variant of NotI restriction endonuclease (GCGGCCGC) was isolated by random mutagenesis of the notIR gene. The NotI variant D160N was shown to bind DNA and protect plasmid DNA from EagI (CGGCCG) and NotI digestions. The EDTA-resistant BmrI restriction endonuclease cleaves DNA sequence ACTGGG N5/N4. The N-terminal cleavage domain of BmrI (residues 1-198) with non-specific nuclease activity was fused to the NotI variant D160N with a short linker. The engineered chimeric endonuclease (CH-endonuclease) recognizes NotI sites specifically in the presence of high salt (100-150 mM NaCl) and divalent cations Mg++ or Ca++. In contrast to wild-type NotI, which cuts within its recognition sequence, BmrI198-NotI (D160N) cleaves DNA outside of NotI sites, resulting in deletion of the NotI site and the adjacent sequences. The fusion of the BmrI cleavage domain to cleavage-deficient variants of Type II restriction enzymes to generate novel cleavage sites will provide useful tools for DNA manipulation.
authors.affiliation: New England Biolabs, Inc., 240 County Road, Ipswich, MA 01938, USA.
authors.forename: Penghua
authors.initials: P
authors.lastname: Zhang
authors.forename: Yongming
authors.initials: Y
authors.lastname: Bao
authors.forename: Lauren
authors.initials: L
authors.lastname: Higgins
authors.forename: Shuang-yong
authors.initials: SY
authors.lastname: Xu
mesh_terms: D001483:Base Sequence; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism*; D015195:Drug Design; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D050506:Mutant Chimeric Proteins / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D050506:Mutant Chimeric Proteins; D011993:Recombinant Fusion Proteins; C046751:endodeoxyribonuclease XmaIII; D015252:Deoxyribonucleases, Type II Site-Specific; C038565:GCGGCCGC-specific type II deoxyribonucleases