Selection of a remote cleavage site by I-tevI, the td intron-encoded endonuclease

Abstract

I-TevI, a double-strand DNA endonuclease involved in the mobility of the td intron of phage T4, is highly unusual in that it binds and cleaves intronless td alleles (td homing sites) in a site-specific but sequence-tolerant manner. The endonuclease binds to sequences flanking the intron insertion site and near the remote cleavage site, located 23 and 25 nucleotides away on the top and bottom strands, respectively. Mapping studies indicate that I-TevI has both sequence and distance sensors that function during cut-site selection. Although I-TevI cleavage of many insertion and deletion variants of the homing site is impaired, double-strand breaks are generated at positions that collectively span two turns of the helix, indicating that the interaction is extraordinarily flexible. However, the endonuclease does exhibit spacing preferences between its binding domains, and sequence preferences near the cleavage site, with the G:C pair at -23 implicated as a cleavage determinant. Furthermore, I-TevI appears to function through interactions across the minor groove at the cleavage site, as it does at the intron insertion site, and to be capable of cleaving sequentially, first on the bottom and then on the top strand. These properties of I-TevI are incorporated in a model wherein the endonuclease effects distant cleavage via a flexible hinge.