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

Here, we investigate the highly specific interaction of the BamHI endonucle ase with its cognate recognition sequence GGATCC by determining which amino acid residues can be substituted at the DNA interface while maintaining sp ecificity. Mutational studies, together with the structural determination o f the restriction endonuclease BamHI have revealed the amino acid residues which are involved in DNA catalysis and those which play a role in the spec ific binding of the enzyme to its cognate DNA recognition sequence. Amino a cid residues N116, S118, R122, D154 and R155 are involved in DNA sequence r ecognition and are located in the major groove in close proximity to the nu cleotide bases comprising the recognition sequence. Cassette mutagenesis of these amino acids, together with in vivo transcriptional interference sele ction, was used to identify an array of substitutions which maintain site-s pecific binding to the cognate GGATCC sequence. This approach has demonstra ted the extent of acceptable variation among amino acid residues which are directly involved in site-specific binding. One variant, double mutant N116 H, S118G was found to cleave DNA only when the adenine base in the recognit ion site is methylated. (C) 1999 Academic Press.