SELECTION OF A REMOTE CLEAVAGE SITE BY I-TEVI, THE TD INTRON-ENCODED ENDONUCLEASE

1-TevI, a double-strand DNA endonuclease involved in the mobility of t he td intron of phage T4, is highly unusual in that it binds and cleav es intronless td alleles (td homing sites) in a site-specific but sequ ence-tolerant manner. The endonuclease binds to sequences flanking the intron insertion site and near the remote cleavage site, located 23 a nd 25 nucleotides away on the top and bottom strands, respectively Map ping studies indicate that I-TevI has both sequence and distance senso rs that function during cut-site selection. Although I-TevI cleavage o f many insertion and deletion variants of the homing site is impaired, double-strand breaks are generated at positions that collectively spa n two turns of the helix, indicating that the interaction is extraordi narily flexible. However, the endonuclease does exhibit spacing prefer ences between its binding domains, and sequence preferences near the c leavage site, with the G:C pair at -23 implicated as a cleavage determ inant. Furthermore, I-TevI appears to function through interactions ac ross the minor groove at the cleavage site, as it does at the intron i nsertion site, and to be capable of cleaving sequentially, first on th e bottom and then on the top strand. These properties of I-TevI are in corporated in a model wherein the endonuclease effects distant cleavag e via a flexible hinge.