Genetic analysis of the base-specific contacts of BamHI restriction endonuclease

• Published in: Journal of molecular biology 1999-01, Vol.285 (4), p.1515
• Main Authors: Dorner, L F, Bitinaite, J, Whitaker, R D, Schildkraut, I
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acid Substitution; Base Sequence; Binding Sites - genetics; Deoxyribonuclease BamHI - chemistry; Deoxyribonuclease BamHI - genetics; Deoxyribonuclease BamHI - metabolism; DNA, Bacterial - genetics; DNA, Bacterial - metabolism; Escherichia coli - enzymology; Escherichia coli - genetics; Models, Molecular; Molecular Sequence Data; Mutagenesis, Insertional; Mutation; Protein Conformation
• ISSN: 0022-2836
• DOI: 10.1006/jmbi.1998.2408

Here, we investigate the highly specific interaction of the BamHI endonuclease with its cognate recognition sequence GGATCC by determining which amino acid residues can be substituted at the DNA interface while maintaining specificity. Mutational studies, together with the structural determination of the restriction endonuclease BamHI have revealed the amino acid residues which are involved in DNA catalysis and those which play a role in the specific binding of the enzyme to its cognate DNA recognition sequence. Amino acid residues N116, S118, R122, D154 and R155 are involved in DNA sequence recognition and are located in the major groove in close proximity to the nucleotide bases comprising the recognition sequence. Cassette mutagenesis of these amino acids, together with in vivo transcriptional interference selection, was used to identify an array of substitutions which maintain site-specific binding to the cognate GGATCC sequence. This approach has demonstrated the extent of acceptable variation among amino acid residues which are directly involved in site-specific binding. One variant, double mutant N116H, S118G was found to cleave DNA only when the adenine base in the recognition site is methylated.