A mutant of BamHI restriction endonuclease which requires N6-methyladenine for cleavage

Amino acid residues Asn116 and Ser118 of the restriction endonuclease BamHI make several sequence-specific and water-bridged contacts to the DNA bases. An in vivo selection was used to isolate BamHI variants at position 116, 118 and 122 which maintained sequence specificity to GGATCC sites. Here, the variants N116H, N116H/S118G and S118G were purified and characterized. The variants N116H and N116H/S118G were found to have lost their ability to cleave unmethylated GGATCC sequences by more than two orders of magnitude, while maintaining nearly wild-type levels of activity on the N6-methyladenine-containing sequence, GGmATCC. In contrast, wild-type BamHI and variant S118G have only a three- to fourfold lower activity on unmethylated GGATCC sequences compared with GGmATCC sequences. The N116 to H116 mutation has effectively altered the specificity of BamHI from an endonuclease which recognizes and cleaves GGATCC and GGmATC, to an endonuclease which only cleaves GGmATCC. The N116H change of specificity is due to the lowered binding affinity for the unmethylated sequence because of the loss of two asparagine-DNA hydrogen bonds and the introduction of a favorable van der Waals contact between the imidazole group of histidine and the N6-methyl group of adenine.