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

该项目研究了作为转座病毒（如噬菌体Mu和HIV）目标的染色体的动态方面。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所涉及的每个DNA片段的更高级蛋白质-DNA组装的背景下。转座病毒DNA末端组装成称为转座体（对于Mu）或前整合复合物（对于HIV）的高级蛋白质-DNA复合物，其核心由通过MuA转座酶或HIV IN蛋白的四聚体突触的转座病毒DNA的两个末端片段组成。这些高级蛋白质-DNA复合物在自然界中的组装在影响组装过程的额外DNA结合蛋白的存在下发生。用于转座的靶DNA也以真核生物染色体或细菌类核的形式作为高级蛋白质-DNA复合物存在。转座病毒DNA复合物组装过程和作为转座靶的染色体DNA的活性如何受到影响DNA动力学行为的一般染色体/类核相关蛋白的影响还没有很好地理解。在原核生物和真核生物中，染色体DNA分子保持在动态凝聚状态，涉及大量的DNA凝聚蛋白。这些蛋白质通过直接或间接的DNA结合使DNA弯曲/弯曲，或跨桥连接远处的DNA片段。在我们单独的项目（DK 036165）中，我们还研究了涉及帕拉/B/S类系统组件的细菌染色体/质粒分配系统的机制。帕拉和ParB蛋白也以高度受控的方式引起DNA缩合，并且它们的功能动力学最有可能受到与细菌类核相关的其他DNA缩合蛋白NAP（类核相关蛋白）的影响。 该项目旨在促进我们对染色体相关蛋白如何通过其对凝聚DNA动力学的影响来影响DNA交易的理解，例如转座病毒DNA整合，转座靶位点搜索以及细菌染色体分离过程。整合前复合物中的HIV DNA受到BAF蛋白的保护，不会自我破坏性的自动整合。BAF被认为以一种使其无法进行自我破坏性自动整合的方式浓缩DNA。利用荧光标记的BAF和高灵敏度的荧光显微镜系统，从单DNA分子水平上研究了BAF对DNA的凝聚机理。BAF是一种小的二聚体蛋白，其以接近正交的方向结合两个DNA片段，形成交叉桥以使DNA缩合。BAF被认为在有丝分裂后的细胞核重组过程中发挥作用。虽然它不被认为是一个一般的染色体相关的凝聚蛋白，它作为一个模型的桥接型DNA冷凝器，我们研究了它的DNA相互作用动力学沿着与细菌NAP和其他DNA凝聚蛋白。 实验方法目前正在开发中，以定量评估的影响，DNA凝聚蛋白质，从原核和真核生物的起源，对DNA的结构动力学，对高阶蛋白质-DNA复合物的组装过程，以及这些大分子组装的动力学特性。 最近，我们致力于了解CTP依赖的F-质粒分配复合物通过F-质粒的着丝粒结合蛋白ParB缩合的机制。二十多年来，人们已经知道F-质粒的ParB蛋白及其同源物不仅与其同源的parS DNA序列特异性结合，而且ParB结合扩散到附近的非特异性DNA区域，形成含有大量ParB分子的浓缩分区蛋白-DNA复合物。然而，这些复合物是如何组装的仍然是一个谜。最近，已发现该家族的ParB蛋白结合CTP并具有CTP活性。利用磁镊监测DNA的凝聚，我们发现含有parS的DNA被ParB以CTP依赖的方式凝聚。对于观察到的DNA缩合，parS DNA序列必须以顺式存在。DNA缩合过程的进一步细节和复杂的动态特性的表征正在调查中。

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. Most recently, we focused our effort to understand the mechanism of CTP-dependent F-plasmid partition complex condensation by the centromere-binding protein, ParB of F-plasmid. For more than two decades, it has been known that ParB protein of the F-plasmid and its homologues not only bind sequence-specifically to their cognate parS DNA sequence, ParB-binding spreads to nearby non-specific DNA areas, forming condensed partition protein-DNA complexes containing large numbers of ParB molecules. However, how such complexes are assembled remained a mystery. Recently, ParB proteins of this family have been found to bind CTP and have CTPase activity. By making use of magnetic tweezers to monitor DNA condensation, we found parS-containing DNA is condensed by ParB in CTP-dependent manner. The parS DNA sequence must be present in cis for the observed DNA condensation. Further details of the DNA condensation process and characterization of the dynamic properties of the complex are under investigation.