I-PPOI AND I-CREI HOMING SITE SEQUENCE DEGENERACY DETERMINED BY RANDOM MUTAGENESIS AND SEQUENTIAL IN-VITRO ENRICHMENT

Plasmid libraries containing partially randomized cleavage sites for t he eukaryotic homing endonucleases I-PpoI and I-CreI were constructed, and sites that could be cleaved by I-PpoI or I-CreI were selectively recovered by successive cycles of cleavage and gel separation followed by religation and growth in Escherichia coli. Twenty-one different I- PpoI-sensitive homing sites, including the native homing site, were is olated. These sites were identical at four nucleotide positions within the 15 bp homing site, had a restricted pattern of base substitutions at the remaining 11 positions and displayed a preference for purines flanking the top strand of the homing site sequence. Twenty-one differ ent I-CreI-sensitive homing sites, including the native site, were iso lated. Ten nucleotide positions were identical in homing site variants that were I-CreI-sensitive and required the addition of SDS for effic ient cleavage product release. Four of these ten positions were identi cal in homing sites that did not require SDS for product release. Ther e was a preference for pyrimidines flanking the top strand of the homi ng site sequence. Three of the 24 I-CreI homing site nucleotide positi ons apparently lacked informational content, i.e. were permissive of c leavage when occupied by any nucleotide. These results suggest that I- PpoI and I-CreI make a large number of DNA-protein contacts across the ir homing site sequences, and that different subsets of these contacts may be sufficient to maintain a high degree of sequence-specific homi ng site recognition and cleavage. The sequential enrichment protocol w e used should be useful for defining the sequence degeneracy and infor mational content of other homing endonuclease target sites. (C) 1998 A cademic Press.