Study of the higher order protein DNA complexes involved in DNA rearrangements

本课题研究了噬菌体Mu的转座反应和HIV DNA整合反应。 这些反应中的关键步骤是一对DNA切割和链转移，涉及Mu或HIV DNA序列的末端和靶DNA;这些反应产生分支DNA中间体。 这两个化学反应步骤发生在称为转座体或前整合复合物的高级蛋白质-DNA复合物内，其核心由通过MuA转座酶或HIV IN蛋白的四聚体突触的转座供体DNA的两个末端片段组成。 这些高阶蛋白质-DNA复合物的组装和组装复合物内蛋白质的催化活性受多种因素控制，并非所有因素都很好地理解。 该项目旨在推进我们对病毒DNA整合过程如何由结构组分及其在复合物内的动态相互作用控制的理解。 我们已经表明，Mu末端DNA切割和随后在一个Mu DNA末端的链转移都是由与转座体内的伴侣Mu DNA末端结合的MuA单体催化的。 通过比较含有手性硫代磷酸酯的DNA底物的活性，我们可以在整个连续的反应步骤中监测底物DNA和转座酶活性位点之间的相互作用模式。 这项研究的结果导致了一个机械模型，解释了DNA插入中涉及的连续反应步骤如何在高阶复合物中发生。 用荧光标记的蛋白质和DNA底物研究了Mu转座复合物和HIV前整合复合物中的分子相互作用。已经开发了基于双核苷酸的工具用于测定Mu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。 用于研究艾滋病毒整合前复合物的类似荧光工具正在开发中。 我们已经开发了基于FRET的工具，用于分析这些反应中涉及的高阶复合物，并在FRET数据分析方法上取得了进展，以提高所获得的信息质量。 整合前复合物中的HIV DNA受到BAF蛋白的保护，据信BAF蛋白以这样一种方式浓缩DNA，使其无法进行自我破坏的自动整合。 利用荧光标记的BAF和高灵敏度的荧光显微镜系统，在单DNA分子水平上研究了BAF对DNA的凝聚机理。 研究了蛋白质-DNA相互作用和DNA缩合的动力学性质。

The transposition reaction of bacteriophage Mu and HIV DNA integration reaction are studied in this project. Critical steps in these reactions are a pair of DNA cleavages and strand transfers involving the ends of Mu or HIV DNA sequence and a target DNA; these reactions generate branched DNA intermediates. The two chemical reaction steps take place within higher order protein-DNA complexes called transpososome or preintegration complex, the core of which is composed of two end segments of the transposing donor DNA synapsed by a tetramer of MuA transposase or HIV IN protein. The assembly of these higher order protein-DNA complexes and the catalytic activities of the protein within the assembled complex are controlled by a variety of factors, not all of which are well understood. This project aims to advance our understanding of how the viral DNA integration processes are controlled by the structural components and their dynamic interactions within the complex. 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. By comparing the activity of chiral phosphorothioate containing DNA substrates, we could monitor the mode of interactions between the substrate DNA and the transposase active site throughout the successive reaction steps. The results of this study led to a mechanistic model that explained how the successive reaction steps involved in the DNA insertion take place within the higher order complex. The molecular interactions involved in Mu transposition complex and HIV preintegration complex have been studied by using fluorescence labeled proteins and DNA substrates. Fluorescence-based tools have been developed for the assay of the Mu transposase-DNA binding, Mu-end pairing, stable synaptic complex formation, and Mu-end DNA deformation. Similar fluorescence-based tools for the study of HIV preintegration complex are under development. We have developed FRET based tools for the analysis of the higher order complexes involved in these reactions, and advances have been made on the methods for the FRET data analysis in order to improve the information quality obtained. HIV DNA within a preintegration complex is protected by BAF protein, which is believed to condense the DNA in such a way to make it inaccessible for self-destructive auto-integration. The mechanism of DNA condensation by BAF has been studied at single DNA-molecule level by using fluorescence labeled BAF and a high-sensitivity fluorescence microscope system. Kinetic properties of the protein-DNA interaction and DNA condensation have been investigated.