Regions of endonuclease EcoRII involved in DNA target recognition identified by membrane-bound peptide repertoires

Target sequence-specific DNA binding regions of the restriction endonucleas e EcoRII were identified by screening a membrane-bound EcoRII-derived pepti de scan with an EcoRII recognition site (CCWGG) oligonucleotide duplex, Dod ecapeptides overlapping by nine amino acids and representing the complete p rotein were prepared by spot synthesis. Two separate DNA binding regions, a mino acids 88-102 and amino acids 256-273, which share the consensus motif KXRXXK, emerged. Screening 570 single substitution analogues obtained by ex changing every residue of both binding sites for all other amino acids demo nstrated that replacing basic residues in the consensus motifs significantl y reduced DNA binding. EcoRII mutant enzymes generated by substituting alan ine or glutamic acid for the consensus lysine residues in DNA binding site I expressed attenuated DNA binding, whereas corresponding substitutions in DNA binding site II caused impaired cleavage, but enzyme secondary structur e was unaffected. Furthermore, Glu(96), which is part of a potential cataly tic motif and also locates to DNA binding site I, was demonstrated to be cr itical for DNA cleavage and binding. Homology studies of DNA binding site I I revealed strong local homology to SsoII (recognition sequence, CCNGG) and patterns of sequence conservation, suggesting the existence of functionall y related DNA binding sites in diverse restriction endonucleases with recog nition sequences containing terminal C:G or G:C pairs.