INTERACTION OF THE INTRON-ENCODED MOBILITY ENDONUCLEASE I-PPOI WITH ITS TARGET SITE
INTERACTION OF THE INTRON-ENCODED MOBILITY ENDONUCLEASE I-PPOI WITH ITS TARGET SITE
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DOI：
10.1128/mcb.13.12.7531
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发表时间：
1993-12-01
影响因子：
5.3
通讯作者：
VOGT, VM
中科院分区：
文献类型：
--
作者：
ELLISON, EL;VOGT, VM
由可移动的I组内含子编码的内切酶是高度特异性的dna酶，它在内含子移动的位置附近诱导双链断裂。来自脱细胞黏菌多头绒泡菌的I-PpoI介导该生物染色体外核糖体DNA中内含子3 （Pp LSU 3）的迁移。我们之前已经证明，i - pol的切割在内含子插入点附近产生了一个四碱基交错切割。我们现在已经描述了核酸内切酶的几个进一步的性质。通过缺失分析，I-Ppol识别的最小目标位点是一个13 ~ 15 bp的序列，横跨切割位点。纯化后的蛋白在沉淀和凝胶过滤中表现为球状二聚体。在EDTA存在下的凝胶迁移迁移实验中，i - pol与DNA形成稳定的特异性复合物，半衰期为45分钟。通过足迹和干扰分析，甲巯丙基edta -铁（II）， i - pol接触了22- 24bp的DNA片段。核酸内切酶保护DNA螺旋主槽和小槽中的大多数嘌呤免受硫酸二甲酯（DMS）修饰。然而，对DMS的反应性在一些嘌呤上增强，表明结合导致DNA的构象改变。在识别序列的两个部分对称的半部分中，DMS保护的模式根本不同。
Endonucleases encoded by mobile group I introns are highly specific DNases that induce a double-strand -break near the site to which the intron moves. I-PpoI from the acellular slime mold Physarum polycephalum mediates the mobility of intron 3 (Pp LSU 3) in the extrachromosomal nuclear ribosomal DNA of this organism. We showed previously that cleavage by I-Ppol creates a four-base staggered cut near the point of intron insertion. We have now characterized several further properties of the endonuclease. As determined by deletion analysis, the minimal target site recognized by I-Ppol was a sequence of 13 to 15 bp spanning the cleavage site. The purified protein behaved as a globular dimer in sedimentation and gel filtration. In gel mobility shift assays in the presence of EDTA, I-Ppol formed a stable and specific complex with DNA, dissociating with a half-life of 45 min. By footprinting and interference assays with methidiumpropyl-EDTA-iron(II), I-Ppol contacted a 22- to 24-bp stretch of DNA. The endonuclease protected most of the purines found in both the major and minor grooves of the DNA helix from modification by dimethyl sulfate (DMS). However, the reactivity to DMS was enhanced at some purines, suggesting that binding leads to a conformational change in the DNA. The pattern of DMS protection differed fundamentally in the two partially symmetrical halves of the recognition sequence.