Mueller J E, Smith D, Bryk M, Belfort M
Molecular Genetics Program, Wadsworth Center and School of Public Health, New York State Department of Health, Albany 12201-2002, USA.
EMBO J. 1995 Nov 15;14(22):5724-35. doi: 10.1002/j.1460-2075.1995.tb00259.x.
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.
I-TevI是一种来自噬菌体T4胸苷酸合酶（td）基因的内含子编码内切核酸酶，它以序列耐受的方式跨小沟结合其DNA底物。我们在此证明，28 kDa的I-TevI以单体形式结合37 bp的td归巢位点，并使其底物发生显著扭曲。原位切割分析和定相分析表明，在切割td归巢位点的底部链时，I-TevI会在切割位点附近诱导向大沟方向38度的定向弯曲。弯曲的I-TevI-DNA复合物的形成被认为促进了归巢位点的顶部链切割。此外，td归巢位点碱基取代变体的扭曲程度和结合效率的降低表明，切割位点两侧的序列有助于I-TevI诱导的构象变化。这些结果与遗传学、物理学和计算机建模研究相结合，形成了一个模型的基础，其中I-TevI作为一个铰链单体诱导扭曲，拓宽小沟，便于进入顶部链切割位点。该模型与未修饰的DNA以及T偶数噬菌体中存在的含葡萄糖基化羟甲基胞嘧啶的DNA均兼容。
