A mutant of BamHI restriction endonuclease which requires N6-methyladeninefor 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, th e variants N116H, N116H/S118G and S118G were purified and characterized. Th e variants N116H and N116H/S118G were found to have lost their ability to c leave unmethylated GGATCC sequences by more than two orders of magnitude, w hile maintaining nearly wild-type levels of activity on the N6-methyladenin e-containing sequence, GGmATCC. in contrast, wild-type BamHI and variant S1 18G have only a three- to fourfold lower activity on unmethylated GGATCC se quences compared with GGmATCC sequences. The N116 to H116 mutation has effe ctively altered the specificity of BamHI from an endonuclease which recogni zes and cleaves GGATCC and GGmATC, to an endonuclease which only cleaves GG mATCC. The N116H change of specificity is due to the lowered binding affini ty for the unmethylated sequence because of the loss of two asparagine-DNA hydrogen bonds and the introduction of a favorable van der Waals contact be tween the imidazole group of histidine and the N6-methyl group of adenine. (C) 1999 Academic Press.