Study of the dynamics of higher order protein DNA complexes involved in variety of DNA transactions

在这个项目中，研究了作为病毒转座目标的染色体的动态方面，如噬菌体Mu和HIV。涉及Mu或HIV DNA序列末端的一对DNA切割和链转移及其与染色体靶DNA位点的相互作用发生在每个DNA片段的高阶蛋白质-DNA组装的背景下。转座病毒DNA末端组装成高阶蛋白质-DNA复合体，称为转座体(Mu)或前整合复合体(HIV)，其核心由转座病毒DNA的两个末端片段组成，这些末端片段由MUA转座酶或HIV IN蛋白的四聚体突触。自然界中这些高阶蛋白质-DNA复合体的组装是在额外的DNA结合蛋白存在的情况下进行的，这些蛋白质影响组装过程。转座的靶DNA也以高阶蛋白质-DNA复合体的形式存在，以真核染色体或细菌类核的形式存在。一般的染色体/类核相关蛋白如何影响转座病毒DNA复合体的组装过程和作为转座目标的染色体DNA的活性，从而影响DNA的动态行为，目前还不清楚。在原核生物和真核生物中，染色体DNA分子都处于动态凝聚状态，涉及多种DNA凝聚蛋白。这些蛋白质要么弯曲/弯曲DNA，要么通过直接或间接的DNA结合跨越遥远的DNA片段。在我们单独的项目(DK036165)中，我们还研究了涉及Para/B/S类系统组件的细菌染色体/质粒分配系统的机制。ParA和PARB蛋白也以高度受控的方式引起DNA缩合，它们的功能动力学很可能受到与细菌类核相关的其他DNA缩合蛋白NAP(NAP)的影响。 该项目旨在加深我们对染色体相关蛋白如何通过影响浓缩DNA动态来影响DNA交易的理解，如转座病毒DNA整合、转座靶点搜索以及细菌染色体分离过程。前整合复合体中的HIV DNA受到BAF蛋白的保护，使其免受自毁自整合的影响。据信，BaF以一种使DNA无法自我破坏的方式凝聚DNA。利用荧光标记的生物滤池和高灵敏度的荧光显微镜系统，在单DNA分子水平上研究了生物滤池与DNA的缩合机理。BAF是一种小的二聚体蛋白，它以接近垂直的方向结合两个DNA片段，形成跨桥来浓缩DNA。BAF被认为在有丝分裂后的核重组过程中发挥作用。虽然它不被认为是一个普遍的与染色体相关的凝聚蛋白，但它是一个模型桥接型DNA凝聚器，我们研究了它与细菌NAP和其他DNA凝聚蛋白的DNA相互作用动力学。目前正在开发实验方法，以定量评估来自原核和真核来源的DNA凝聚蛋白对DNA结构动力学的影响，对高阶蛋白质-DNA复合体组装过程的影响，以及这些大分子组装的动态性质。

Dynamic aspects of the chromosomes that are the target of transposing viruses such as bacteriophage Mu and HIV are studied in this project. A pair of DNA cleavages and strand transfers involving the ends of Mu or HIV DNA sequence and their interaction with chromosomal target DNA sites take place within a context of higher order protein-DNA assemblies for each DNA segments involved. The transposing viral DNA ends are assembled into higher order protein-DNA complexes called transpososome (for Mu) or preintegration complex (for HIV), the core of which is composed of two end segments of the transposing viral DNA synapsed by a tetramer of MuA transposase or HIV IN protein. The assembly of these higher order protein-DNA complexes in nature takes place in the presence of additional DNA binding proteins that influence the assembly process. The target DNA for transposition also exists as higher order protein-DNA complexes in the form of eukaryotic chromosomes or bacterial nucleoid. How the transposing viral DNA complex assembly process and the activity of the chromosomal DNA as the target of transposition are influenced by general chromosome/nucleoid associated proteins, which impact the dynamic behavior of DNA is not well understood. In both prokaryotes and eukaryotes, chromosomal DNA molecules are kept in dynamic condensed states involving large variety of DNA condensation proteins. These proteins either bend/flex DNA, or cross-bridge distant DNA segments through direct, or indirect DNA binding. In our separate project (DK036165), we also study the mechanism of bacterial chromosome/plasmid partition systems that involve the ParA/B/S class of system components. ParA and ParB proteins also cause DNA condensation in highly controlled fashion, and their functional dynamics most likely are impacted by other DNA condensation proteins associated with bacterial nucleoid, NAPs (nucleoid associated proteins). This project aims to advance our understanding of how the chromosome-associated proteins, through their influence on condensed DNA dynamics, affect DNA transactions such as transposing viral DNA integration, transposition target site search, as well as bacterial chromosome segregation processes. HIV DNA within a preintegration complex is protected by BAF protein from self-destructive auto-integration. BAF is believed to condense DNA in a way that makes it inaccessible for self-destructive auto-integration. The mechanism of DNA condensation by BAF was studied at a single DNA-molecule level by using fluorescence labeled BAF and a high-sensitivity fluorescence microscope system. BAF is a small dimeric protein that binds two DNA segments in near orthogonal orientations, forming cross-bridges to condense DNA. BAF is thought to play roles after mitosis during re-assembly of nucleus. While it is not considered to be a general chromosome-associated condensation protein, it serves as a model bridging-type DNA condenser, and we study its DNA interaction dynamics along with bacterial NAPs and other DNA condensing proteins. Experimental approaches are currently under development to quantitatively evaluate the impacts of DNA-condensing proteins, from both prokaryotic and eukaryotic origins, on the structural dynamics of DNA, on higher order protein-DNA complex assembly process, and the dynamic properties of these large molecular assemblies.