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 mino r 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 an d significantly distorts its substrate, In situ cleavage assays and ph asing 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 effici ency of binding base-substitution variants of the fd homing site indic ate 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, whe rein I-TevI acts as a hinged monomer to induce a distortion that widen s the minor groove, facilitating access to the top-strand cleavage sit e, The model is compatible with both unmodified DNA and glucosylated h ydroxymethylcytosine-containing DNA, as exists in the T-even phages.