Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences
Tn5 transposase loops DNA in the absence of Tn5 transposon end sequences
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DOI：
10.1111/j.1365-2958.2006.05471.x
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发表时间：
2006-12-01
影响因子：
3.6
通讯作者：
Reznikoff, William S.
中科院分区：
文献类型：
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作者：
Adams, Christian D.;Schnurr, Bernhard;Reznikoff, William S.
转座酶首先通过结合定义转座元件的特定DNA末端序列，然后通过将蛋白质和DNA组织成高度结构和稳定的核蛋白‘突触’复合体来介导转座。突触复合体组装是许多转位机制中的中心检查点。Tn5突触复合体包含两个Tn5转座酶亚基和两个Tn5转座子末端序列，具有广泛的蛋白质末端序列DNA接触，是DNA环的节点。利用单分子和整体生化方法，我们发现在没有Tn5转座子末端序列的情况下，Tn5转座酶组装成一个稳定的核蛋白复合体。令人惊讶的是，这个末端序列独立的复合体在结构上与突触复合体有相似之处。这个复合体是DNA环的节点；转座酶二聚体和DNA特异性突变体影响它的组装；它可能具有与突触复合体相同数量的蛋白质和DNA分子。此外，我们的结果表明，Tn5转座酶优先结合和循环非Tn5末端序列的子集。末端非依赖于序列的核蛋白复合体的组装可能在体内下调转座和许多细菌转座酶的顺式转座偏向中发挥作用。
Transposases mediate transposition first by binding specific DNA end sequences that define a transposable element and then by organizing protein and DNA into a highly structured and stable nucleoprotein 'synaptic' complex. Synaptic complex assembly is a central checkpoint in many transposition mechanisms. The Tn5 synaptic complex contains two Tn5 transposase subunits and two Tn5 transposon end sequences, exhibits extensive protein-end sequence DNA contacts and is the node of a DNA loop. Using single-molecule and bulk biochemical approaches, we found that Tn5 transposase assembles a stable nucleoprotein complex in the absence of Tn5 transposon end sequences. Surprisingly, this end sequence-independent complex has structural similarities to the synaptic complex. This complex is the node of a DNA loop; transposase dimerization and DNA specificity mutants affect its assembly; and it likely has the same number of proteins and DNA molecules as the synaptic complex. Furthermore, our results indicate that Tn5 transposase preferentially binds and loops a subset of non-Tn5 end sequences. Assembly of end sequence-independent nucleoprotein complexes likely plays a role in the in vivo downregulation of transposition and the cis-transposition bias of many bacterial transposases.