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

一些DNA转座子配备有新的方法来控制插入的靶位点选择。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位点作为同种转座子的额外插入的靶。噬菌体Mu转座子就是这样一种元件，它使用ATP依赖性DNA结合蛋白MuB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。 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，它参与靶DNA位点选择，它激活MuA转座酶进行链转移反应，并且它保护转座体免受ClpX伴侣蛋白过早分解，直到链转移完成并且转座中间体准备好用于宿主复制蛋白的DNA复制。反过来，MuB的功能状态由其ATP酶循环和与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 DNA序列末端的优先分布。 通过使用灵敏的荧光显微镜/CCD照相系统和电子显微镜，已经开发出在单分子水平上研究MuB-DNA复合物的结构和功能方面的技术和仪器。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。我们发现，MuB形成聚合物的异质尺寸沿着的DNA和ATP依赖的组装MuB聚合物涉及随机DNA结合和成核事件。此外，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。最后，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。为了解释受靶免疫力影响的DNA的沿着距离，其超过平均被动DNA成环距离，我们测量了环大小分布作为自由DNA布朗运动的时间的函数，在此期间可能发生成环相互作用。我们证明了随着DNA布朗运动的时间的平均DNA环的大小的增加，导致ATP驱动的不均匀的蛋白质分布模式沿着DNA分子形成的机制的详细模型。通过EM图像重建研究了螺旋MuB聚合物的结构。独特的螺旋参数之间的不匹配被发现的近B-形式的DNA螺旋结合在中心和蛋白质螺旋丝，包住的DNA，一个发现与机械的影响。 通过对一些定点突变蛋白的序列比较和详细的生化研究，将MuB鉴定为AAA+ ATPase家族的成员。关键残基的生化功能的分配导致更好地理解MuB蛋白寡聚化，ATP酶活性位点功能，MuA相互作用和DNA结合之间的功能通信。 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 to control target site selection for insertion. A transposition target site selection phenomena exhibited by these transposons are referred to as transposition target immunity; DNA sites near a transposon end sequence are avoided as a target for additional insertion by the same kind of transposon. Phage Mu transposon is one such element and it uses an ATP-dependent DNA binding protein, MuB, as the central player in the target site selection to reduce the risk of self destructive insertion into its own genome. MuB ATPase controls each of the early steps of phage Mu DNA transposition: it assists transpososome assembly, it is involved in the target DNA site selection, it activates the MuA transposase for the strand transfer reaction, and it protects the 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 its ATPase cycle and by interaction with MuA. We have demonstrated that Mu transposition target selection involves establishment of a preferential distribution of the MuB ATPase along DNA molecules away from a Mu DNA sequence end 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 and also by electron microscopy. Using GFP-tagged MuB, assembly and disassembly of MuB polymers on single molecules of DNA immobilized on a slide glass surface were monitored under a variety of reaction conditions. We found that MuB forms polymers of heterogeneous sizes along the DNA and ATP-dependent assembly of MuB polymers involves stochastic DNA binding and nucleation events. Also, MuB dissociation takes place preferentially, but not exclusively, from the ends of a polymer and is tightly coupled to ATP hydrolysis. Finally, the MuA tetramer accelerates dissociation of MuB from DNA in a process dependent on a DNA-looping-mediated interaction of the MuB polymer and MuA tetramer. To explain the distance along the DNA that is influenced by target immunity, which exceeds the average passive DNA-looping distance, we measured the loop size distribution as a function of the time given for free DNA Brownian motion during which the looping interactions could take place. We demonstrated an increase of the average DNA loop size with the time of DNA Brownian motion, leading to a detailed model of the mechanism of ATP-driven uneven protein distribution pattern formation along a DNA molecule. The structure of the helical MuB polymer is studied by EM image reconstruction. Unique helical parameter mismatch was found between the near-B-form DNA helix bound in the center and the protein helical filament that encase the DNA, a finding with mechanistic implications. Based on the sequence comparison and detailed biochemical study of a number of site-directed mutant proteins, MuB was identified as a member of AAA+ ATPase family. Assignment of critical residues to biochemical functions lead to better understanding of the functional communication among MuB protein oligomerization, ATPase active site function, MuA interaction and DNA binding. The reaction system studied here is an example of simple biomolecular patterning reactions, and the experimental techniques developed here will be exploited for parallel studies of mechanistically related reaction systems.