Intron-encoded endonuclease I-TevI binds as a monomer to effect sequential cleavage via conformational changes in the td homing site.

AUTOR(ES)

Mueller, J E

RESUMO

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 td 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 td 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 td 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.

Documentos Relacionados

• Interaction of the intron-encoded mobility endonuclease I-PpoI with its target site.
• I-TevI, the endonuclease encoded by the mobile td intron, recognizes binding and cleavage domains on its DNA target.
• Zinc finger as distance determinant in the flexible linker of intron endonuclease I-TevI
• The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target.
• The td intron endonuclease I-TevI makes extensive sequence-tolerant contacts across the minor groove of its DNA target.