ASN(141) IS ESSENTIAL FOR DNA RECOGNITION BY ECORI RESTRICTION-ENDONUCLEASE

The amino acid residue Asn(141) of the restriction endonuclease EcoRI was proposed to make three hydrogen bonds to both adenine residues wit hin the recognition sequence -GAATTC-. We have mutated Asn(141) to ala nine, aspartate, serine, and tyrosine, Only the serine mutant is activ e under normal buffer conditions although 1000-fold less than wild-typ e EcoRI, The alanine and aspartate mutants can be activated by Mn2+, A t acidic pH the latter mutant becomes even mure active than the wild-t ype enzyme in the presence of Mn2+, We conclude that: Asn(141) is esse ntial for DNA recognition and that serine can partly substitute it. (C ) 1998 Federation of European Biochemical Societies.