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

单分子水平DNA转座靶免疫研究

• 批准号: 8553464
• 负责人: KIYOSHI MIZUUCHI
• 金额: $ 30.68万
• 依托单位: NATIONAL INSTITUTE OF DIABETES AND DIGESTIVE AND KIDNEY DISEASES
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8553464
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Active Sites; B-DNA; Bacteriophage mu; Binding; Biochemical; Chromatin Loop; Communication; Complex; Coupled; DNA; DNA Binding; DNA Sequence; DNA Transposons; DNA biosynthesis; DNA-Binding Proteins; Dissociation; Electron Microscopy; Elements; Event; Exhibits; Family; Filament; Genome; Glass; Immunity; Lead; Measures; Mediating; Modeling; Molecular Chaperones; Monitor; Motion; Pattern; Pattern Formation; Polymers; Process; Proteins; Reaction; Risk; Site; Slide; Structure; Surface; System; Techniques; Time; Transposase; base; charge coupled device camera; fluorescence microscope; image reconstruction; instrument; member; mutant; novel; premature; protein distribution; single molecule

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.

一些DNA转座子配备了新的方法来控制插入的靶点选择。这些转座子表现出的转座靶点选择现象称为转座靶免疫；转座子末端序列附近的DNA位点被避免作为同一类型转座子额外插入的靶。噬菌体Mu转座子就是这样一个元件，它使用依赖于ATP的DNA结合蛋白Mub作为靶点选择的中心角色，以减少自毁插入到自己基因组中的风险。 MUB ATPase控制噬菌体Mu DNA转座的每个早期步骤：它帮助转座体组装，参与靶DNA位点的选择，激活MUA转座酶进行链转移反应，并保护转座体不被ClpX伴侣蛋白过早分解，直到链转移完成，转座中间产物为宿主复制蛋白的DNA复制做好准备。反过来，MUb的功能状态受其ATPase循环和与MUA相互作用的控制。我们已经证明，Mu转座靶标的选择涉及到在远离Mu DNA转座酶结合的Mu DNA序列末端的DNA分子上建立Mu转座ATPase的优先分布。 利用灵敏的荧光显微镜/CCD摄像系统和电子显微镜，已经开发出在单分子水平上研究Mub-DNA复合体的结构和功能方面的技术和仪器。使用GFP标记的MUB，在不同的反应条件下，监测了固定在玻片表面的DNA单分子上MUB聚合物的组装和拆解。我们发现，MUB沿着DNA形成不同大小的聚合物，并且依赖于ATP的MUB聚合物的组装涉及随机的DNA结合和成核事件。此外，MUB解离优先发生，但不是唯一的，从聚合物的末端，并紧密耦合到三磷酸腺苷的水解。最后，MUA四聚体在依赖于DNA环介导的MUB聚合物和MUA四聚体的相互作用的过程中加速了MUB从DNA中的解离。为了解释靶免疫影响DNA的距离，超过了平均被动DNA环距离，我们测量了环大小分布作为自由DNA布朗运动时间的函数，在此期间环相互作用可能发生。我们证明了平均DNA环尺寸随着DNA布朗运动时间的增加而增加，从而得到了一个详细的模型，解释了ATP驱动的蛋白质沿DNA分子不均匀分布模式的形成机制。用EM图像重建方法研究了螺旋MUB聚合物的结构。在中心结合的近B型DNA螺旋和包裹DNA的蛋白质螺旋细丝之间发现了独特的螺旋参数不匹配，这一发现具有机制意义。 根据序列比较和对一些定点突变蛋白的详细生化研究，MUb被鉴定为AAA+ATPase家族的成员。将关键残基分配给生化功能有助于更好地理解MUB蛋白齐聚、ATPase活性部位功能、MUA相互作用和DNA结合之间的功能联系。 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用于机械相关反应系统的平行研究。