STRUCTURAL ASPECTS OF A HIGHER-ORDER NUCLEOPROTEIN COMPLEX - INDUCTION OF AN ALTERED DNA-STRUCTURE AT THE MU-HOST JUNCTION OF THE MU-TYPE-1 TRANSPOSOSOME
STRUCTURAL ASPECTS OF A HIGHER-ORDER NUCLEOPROTEIN COMPLEX - INDUCTION OF AN ALTERED DNA-STRUCTURE AT THE MU-HOST JUNCTION OF THE MU-TYPE-1 TRANSPOSOSOME
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DOI：
10.1002/j.1460-2075.1991.tb07856.x
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发表时间：
1991-10-01
期刊：
影响因子：
11.4
通讯作者：
CHACONAS, G
中科院分区：
文献类型：
--
作者：
LAVOIE, BD;CHAN, BS;CHACONAS, G
Mu 体外链转移反应通过两个稳定的高阶核蛋白复合物（1 型和 2 型转座体）进行。 Mu A 蛋白负责 1 型转座体的结构和功能完整性。我们通过化学交联实验研究了该复合物中Mu A蛋白的四级结构，发现其基本结构单元是A四聚体。转座体中的三个 Mu A 结合位点受到 DNase I 足迹的保护：最外面的 A 结合位点 L1 和 R1，以及 R2。还提出了遗传证据证实了这一结果。 1 型复合物的有效形成发生在删除 L3 或 R3 位点或 L2 位点被取代的 mini-Mus 中；但在没有R2的情况下不会发生反应。 L1 和 R1 位点的保护延伸超出 Mu 宿主连接处 12-13 bp，如 DNase I 和甲锭丙基-EDTA.Fe(II) [MPE.Fe(II)] 足迹所示，表明 Mu A 与转座体中的侧翼宿主序列接触，但不在线性 DNA 上接触；此外，羟基自由基足迹显示连续链上前所未有的大幅增强，位于 Mu 右端外侧切口位点之外 2 bp，这表明在 1 型复合物形成时诱导了 DNA 结构的改变。
The Mu in vitro strand transfer reaction proceeds via two stable higher order nucleoprotein complexes, the Type 1 and Type 2 transpososomes. The Mu A protein is responsible for the structural and functional integrity of the Type 1 transpososome. We have investigated the quaternary structure of the Mu A protein within this complex by chemical cross-linking experiments and found that the basic structural unit is an A tetramer. Three Mu A binding sites in the transpososome are protected by DNase I footprinting: the outermost A binding sites L1 and R1, as well as R2. Genetic evidence is also presented which corroborates this result. Efficient formation of Type 1 complexes occurs in mini-Mus with the L3 or R3 sites deleted or when the L2 site has been substituted; but no reaction occurs in the absence of R2. The protection at the L1 and R1 sites extends 12-13 bp beyond the Mu-host junctions as seen by DNase I and methidiumpropyl-EDTA.Fe(II) [MPE.Fe(II)] footprinting, indicating Mu A contacts with the flanking host sequences in the transpososome but not on linear DNA; furthermore, hydroxyl radical footprinting shows an unprecedentedly large enhancement on the continuous strand, 2 bp beyond the nick site outside the Mu right end, which suggests that an altered DNA structure is induced upon Type 1 complex formation.