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 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.
Molecular Genetics Program, Wadsworth Center and School of Public Health, New York State Department of Health, Albany 12201-2002, USA.
J E
JE
Mueller
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D
Smith
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M
Bryk
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Belfort
D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004249:DNA Damage; D017931:DNA Primers; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D007438:Introns; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D013940:Thymidylate Synthase / Q000235:genetics*
D017931:DNA Primers; D004247:DNA; D013940:Thymidylate Synthase; D004706:Endodeoxyribonucleases; C065074:I-TEVI endonuclease