INTRON-ENCODED ENDONUCLEASE I-TEVI BINDS AS A MONOMER TO EFFECT SEQUENTIAL CLEAVAGE VIA CONFORMATIONAL-CHANGES IN THE TD HOMING SITE

I-TevI, the intron-encoded endonuclease from the thymidylate synthase (td) gene of bacteriophage T4, binds its DNA substrate across the minor groove in a sequence-tolerant fashion, We demonstrate here that the 28 kDa I-TevI binds the extensive 37 bp tn homing site as a monomer and significantly distorts its substrate, In situ cleavage assays and phasing analyses indicate that upon nicking the bottom strand of the rd homing site, I-TevI induces a directed bend of 38 degrees towards the major groove near the cleavage site, Formation of the bent I-TeVI-DNA complex is proposed to promote top-strand cleavage of the homing site. Furthermore, reductions in the degree of distortion and in the efficiency of binding base-substitution variants of the fd homing site indicate that sequences flanking the cleavage site contribute to the I-TevI-induced conformational change, These results, combined with genetic, physical and computer-modeling studies, form the basis of a model, wherein I-TevI acts as a hinged monomer to induce a distortion that widens the minor groove, facilitating access to the top-strand cleavage site, The model is compatible with both unmodified DNA and glucosylated hydroxymethylcytosine-containing DNA, as exists in the T-even phages.