Study of the DNA transposition target immunity at the single-molecule level

几种DNA转座子具有控制插入靶位点的新方法。 这些转座子表现出的转座靶位点选择现象被称为转座靶免疫;避免转座子末端序列附近的DNA位点作为转座子插入的靶。 噬菌体Mu转座子就是这样一种元件，它使用MuB ATP依赖性DNA结合蛋白作为靶位点选择的中心参与者，以降低自我破坏性插入自身基因组的风险。 Tn 7是表现出靶免疫性的转座子的另一个实例，沿着其在特定DNA序列或结构处选择性插入的独特能力。 MuB ATP酶控制噬菌体Mu DNA转座的每个早期步骤：它协助转座体组装，参与靶DNA位点选择，激活MuA转座酶进行链转移反应，并保护转座体免受ClpX伴侣蛋白过早分解，直到链转移完成，转座中间体准备好由宿主复制蛋白进行DNA复制。 反过来，MuB的功能状态由ATP酶循环及其与MuA的相互作用控制。 我们已经证明，Mu转座靶选择涉及建立MuB ATP酶沿着DNA分子远离MuA转座酶结合的Mu末端DNA序列的优先分布。 利用灵敏的荧光显微镜/CCD摄像系统，在单分子水平上研究MuB-DNA复合物的结构和功能的技术和仪器已经发展起来。 使用GFP标记的MuB，在各种反应条件下监测固定在载玻片表面上的单分子DNA上的MuB聚合物的组装和拆卸。我们了解到：MuB并不均匀地包裹DNA，相反，它沿着DNA形成沿着不同大小的簇。 MuB聚合物的ATP依赖性组装涉及优先在A/T富集区域的随机成核事件，其中优选的Mu转座位点位于该区域。 MuB解离优先发生，但不排他地，从聚合物的末端，并紧密耦合到ATP水解。 MuA四聚体在依赖于MuB聚合物和MuA四聚体的DNA环介导的相互作用的过程中加速MuB从DNA的解离。 这里研究的反应系统是一个简单的生物分子图案化反应的例子，这里开发的实验技术将被利用的机械相关的反应系统的平行研究。

Several DNA transposons are equipped with novel ways by which they control the target site for insertion. A transposition target site selection phenomenon exhibited by these transposons is referred to as transposition target immunity; DNA sites near a transposon end sequence are avoided as a target for transposon insertion. Phage Mu transposon is one such element and it uses MuB ATP-dependent DNA binding protein as the central player in the target site selection to reduce the risk of self destructive insertion into its own genome. Tn7 is another example of transposons that exhibit target immunity, along with its unique ability of selective insertion at specific DNA sequence or structure. MuB ATPase controls each of the early steps of phage Mu DNA transposition: it assists transpososome assembly, is involved in the target DNA site selection, activates the MuA transposase for strand transfer reaction, and protects transpososome from premature disassembly by ClpX chaperon protein until strand transfer is completed and the transposition intermediate is ready for DNA replication by the host replication proteins. In turn, the functional state of MuB is controlled by the ATPase cycle and by its interaction with MuA. We have demonstrated that Mu transposition target selection involves establishment of preferential distribution of the MuB ATPase along DNA molecules away from a Mu end DNA sequence to which MuA transposase binds. Techniques and instruments have been developed to study the structural and functional aspects of MuB-DNA complex at the single molecule level by using a sensitive fluorescence microscope/CCD camera system. Using GFP-tagged MuB, assembly and disassembly of MuB polymers on single molecules of DNA immobilized on a slide glass surface was monitored under a variety of reaction conditions. We learned that: MuB does not uniformly coat DNA, instead, it forms clusters of heterogeneous sizes along the DNA. ATP-dependent assembly of MuB polymers involves stochastic nucleation event preferentially at A/T rich regions where preferred Mu transposition sites are located. MuB dissociation takes place preferentially, but not exclusively, from the ends of a polymer and is tightly coupled to ATP hydrolysis. MuA tetramer accelerates dissociation of MuB from DNA in a process dependent on DNA-looping-mediated interaction of the MuB polymer and MuA tetramer. The reaction system studied here is an example of simple biomolecular patterning reactions, and the experimental techniques developed here will be exploited for the parallel studies of mechanistically related reaction systems.