Sakai J, Chalmers R M, Kleckner N
Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138, USA.
EMBO J. 1995 Sep 1;14(17):4374-83. doi: 10.1002/j.1460-2075.1995.tb00112.x.
The Tn10 transposition reaction has been reconstituted in vitro on short linear substrate fragments encoding transposon ends. This permits the direct detection of protein-DNA complexes formed during transposition by gel retardation analysis. We demonstrate that a stable synaptic complex containing transposase and a pair of transposon ends forms rapidly and efficiently, prior and prerequisite to the double-strand cleavages involved in transposon excision. These observations extend the general analogies between the Tn10 and Mu transposition reactions, and also reveal significant differences between the two cases. The speed and simplicity of synaptic complex formation in the Tn10/IS10 reaction is suitable for a modular insertion sequence. In contrast, the relative slowness and complexity of this process in the Mu is necessary to permit transposition immunity and control of transposition by Mu repressor protein, two features specifically important for a temperate bacteriophage. Further dissection of the reaction leads to a tentative working model for events preceding the first double-strand cleavage.
Tn10转座反应已在体外于编码转座子末端的短线性底物片段上重建。这使得通过凝胶阻滞分析能够直接检测转座过程中形成的蛋白质-DNA复合物。我们证明，在转座子切除所涉及的双链切割之前且作为其先决条件，一种包含转座酶和一对转座子末端的稳定突触复合物能快速且高效地形成。这些观察结果扩展了Tn10和Mu转座反应之间的一般相似性，同时也揭示了两者之间的显著差异。Tn10/IS10反应中突触复合物形成的速度和简易性适合于模块化插入序列。相比之下，Mu反应中这一过程的相对缓慢和复杂对于实现转座免疫以及由Mu阻遏蛋白控制转座是必要的，这两个特征对于温和噬菌体尤为重要。对该反应的进一步剖析得出了第一个双链切割之前事件的初步工作模型。
