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: 7593577
• 负责人: KIYOSHI MIZUUCHI
• 金额: $ 37.22万
• 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/7593577
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Bacteriophage mu; Binding; Biological Models; Chromatin Loop; Class; Complex; Condition; Coupled; DNA; DNA Sequence; DNA Transposons; DNA biosynthesis; DNA-Binding Proteins; Dissociation; Elements; Event; Exhibits; Genome; Glass; Immunity; Learning; Mediating; Molecular Chaperones; Monitor; Pattern; Polymers; Process; Proteins; Reaction; Risk; Site; Slide; Specificity; Structure; Surface; System; Techniques; Transposase; charge coupled device camera; fluorescence microscope; instrument; molecular recognition; novel; single molecule; size

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.

几个DNA转座子配备了新的方式，通过它们控制插入的目标位点。这些转座子所表现出的转座标位选择现象被称为转座标免疫；转座子末端序列附近的DNA位点被避免作为转座子插入的目标。噬菌体Mu转座子就是这样一种元件，它使用MuB atp依赖的DNA结合蛋白作为目标位点选择的中心角色，以降低插入自身基因组的自毁性风险。Tn7是另一个表现出目标免疫的转座子的例子，它具有选择性插入特定DNA序列或结构的独特能力。