Studies On The Mechanism Of Genetic Recombination

基因重组机制的研究

• 批准号: 6673458
• 负责人: KIYOSHI MIZUUCHI
• 金额: --
• 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/6673458
• 关键词: DNA replication; adenosine triphosphate; adenosinetriphosphatase; bacterial genetics; bacterial virus; conformation; fluorescence microscopy; genetic recombination; microorganism culture; molecular chaperones; nucleic acid sequence; protein structure function; thiophosphate; transposon /insertion element; virus genetics

The objective of this project is to uncover the molecular mechanisms of genetic rearrangements. The transposition reaction of bacteriophage Mu is studied as a model system. Critical steps in Mu transposition are a pair of DNA cleavages and strand transfers involving the ends of Mu DNA sequence and a target DNA; these reactions generate a branched DNA intermediate. The two chemical reaction steps take place within higher order protein-DNA complexes called transpososomes, the core of which is composed of two Mu-end DNA segments synapsed by a tetramer of MuA transposase protein. Transpososome assembly is controlled by a number of cofactors: an enhancer type DNA sequence element called IAS that overlaps the Mu operator sequence and the Mu repressor that binds to it, the MuB protein, the E. coli-encoded HU and IHF proteins, ATP, and Mg++. By making use of a simplified transpososome assembly reaction system, we have shown that both the Mu end DNA cleavage and the subsequent strand transfer at one Mu DNA end are catalyzed by the MuA monomer that is bound to the partner Mu DNA end within a transpososome. For Tn10 and Mu transposition, two transposase monomers within the transpososome have been shown to catalyze all the chemical steps at the two transposon ends. By comparing the activity of chiral phosphorothioate containing DNA substrates, we could deduce the change of orientation of the substrate DNA within the transposase active site throughout the successive reaction steps. MuB ATPase controls each of the early steps of 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. Structural and functional aspects of MuB-DNA complex are currently under investigation by using a variety of physical and biochemical techniques. The molecular interactions involved in the transposition complex of phage Mu were studied by using fluorescence labeled proteins and DNA. Fluorescence-based tools have been developed for the assay of transposase-DNA binding, Mu-end pairing, stable synaptic complex formation, and Mu-end DNA deformation. Techniques and instruments have been developed to study these reactions at the single molecule level by using a sensitive fluorescence microscope/CCD camera system. The assembly and disassembly of protein-DNA complexes, and also the conformational changes within the complex during the reaction are currently studied in real time both by ensemble-level and single-molecule experiments. Efforts are continued toward solving the high-resolution structure of the higher order protein-DNA complexes involved in transposition reactions.

该项目的目标是揭示基因重排的分子机制。以噬菌体Mu的转座反应为模型系统进行了研究。Mu转座的关键步骤是一对DNA切割和链转移，涉及Mu DNA序列的末端和靶DNA;这些反应产生分支的DNA中间体。这两个化学反应步骤发生在称为转座体的高级蛋白质-DNA复合物中，其核心由两个MuA转座酶蛋白四聚体突触的Mu末端DNA片段组成。转座体的装配受许多辅因子的控制：一种称为IAS的增强子型DNA序列元件，它与Mu操纵子序列和与之结合的Mu阻遏子重叠;大肠杆菌编码的HU和IHF蛋白、ATP和Mg++。 通过利用一个简化的转座体组装反应系统，我们已经表明，无论是Mu末端DNA切割和随后的链转移在一个Mu DNA末端的催化的MuA单体结合到合作伙伴Mu DNA末端内的转座体内。对于TnlO和Mu转座，转座体内的两个转座酶单体已显示催化两个转座子末端的所有化学步骤。通过比较含有手性硫代磷酸酯的DNA底物的活性，我们可以推断在整个连续的反应步骤中底物DNA在转座酶活性位点内的取向的变化。 MuB ATP酶控制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。MuB-DNA复合物的结构和功能方面目前正在使用各种物理和生物化学技术进行研究。 用荧光标记的蛋白质和DNA研究了噬菌体Mu转座复合物中的分子相互作用。已经开发了基于双链的工具用于测定转座酶-DNA结合、Mu-末端配对、稳定的突触复合物形成和Mu-末端DNA变形。技术和仪器已经开发，通过使用灵敏的荧光显微镜/CCD相机系统在单分子水平上研究这些反应。蛋白质-DNA复合物的组装和分解以及反应过程中复合物内部的构象变化目前都是通过整体水平和单分子水平的实验来进行真实的实时研究的。 继续努力解决涉及转座反应的高阶蛋白质-DNA复合物的高分辨率结构。