Restriction endonucleases are of great importance to molecular DNA cloning, DNA diagnostics and genetic engineering. There is an ever increasing demand for a larger repertoire of recognition specificities. The restriction endonuclease field is at point where the engineering of cleavage specificities is approachable. BamHI is a good candidate for engineering new specificities. It has been cloned, overexpressed and crystallized. BamHI binding proficient/cleavage deficient (cat-) variants have been isolated by using an in vivo transcriptional interference selection. The selection demands that only cats variants can lead to spectinomycin resistance (spR). By inserting the BamHI recognition sequence GGATCC into an antisense promoter, transcription can be regulated. Binding of the cats BamHI to this """"""""operator"""""""" prevents transcription, thereby relieving the transcriptional interference resulting in spR. The 1.95 Angstroms resolution three dimensional structure of BamHI has led to a determination of four amino acid residues within a seven amino acid loop involved in DNA recognition. Cassette mutagenesis which varies all four DNA recognizing residues of a cats BamHI variant should yield relatively few mutants (-160,000). Among these mutants, those which bind to anti-sense promoter operator sequences other than GGATCC can easily be selected. After selection of altered binding variants, cleavage will be restored by reversing the car mutation.
Potentially can lead to custom designed restriction endonucleases for cleavage at any specific DNA sequence.
