Direct selection of binding proficient/catalytic deficient variants of BamHI endonuclease

• Published in: Nucleic acids research 1994-03, Vol.22 (6), p.1068-1074
• Main Authors: Dorner, Lydia F., Schildkraut, Ira
• Format: Article
• Language: English
• Subjects: Analytical, structural and metabolic biochemistry; Aspartic Acid; Base Sequence; Binding Sites; Biological and medical sciences; Catalysis; Deoxyribonuclease BamHI - chemistry; Deoxyribonuclease BamHI - genetics; Deoxyribonuclease BamHI - metabolism; DNA - drug effects; Drug Resistance; Enzymes and enzyme inhibitors; Escherichia coli; Fundamental and applied biological sciences. Psychology; Glutamates; Glutamic Acid; Hydrolases; Hydroxylamine; Hydroxylamines - pharmacology; Molecular Sequence Data; Mutagenesis; Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Repressor Proteins; Sequence Analysis; Spectinomycin - pharmacology
• ISSN: 0305-1048; 1362-4962
• DOI: 10.1093/nar/22.6.1068

Variants of BamHI endonuclease in which the glutamate 113 residue has been changed to lysine or the aspartate 94 to asparagine were shown to behave as repressor molecules in vivo. This was demonstrated by placing a BamHI recognition sequence, GGATCC, positioned as an operator sequence in an antlsense promoter for the aadA gene (spectinomycin resistance). Repression of this promoter relieved the inhibition of expression of spectinomycin resistance. This system was then used to select new binding proficient/cleavage deficient BamHI variants. The BamHI endonuclease gene was mutagenized either by exposure to hydroxylamine or by PCR. The mutagenized DNA was reintroduced into E.coil carrying the aadA gene construct, and trartsformants that conferred spectinomycin resistance were selected. Twenty Spr transformants were sequenced. Thirteen of these were newly isolated variants of the previously identified D94 and E113 residues which are known to be involved in catalysis. The remaining seven variants were all located at residue 111 and the glutamate 111 residue was shown to be involved with catalysis.