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

作为转座病毒（如噬菌体Mu和HIV）目标的染色体的动态方面在本项目中进行了研究。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相关DNA片段的高阶蛋白质-DNA组装的背景下。转座病毒DNA末端组装成更高阶的蛋白质-DNA复合物，称为转座体（对于Mu）或预整合复合物（对于HIV），其核心由转座病毒DNA的两个末端片段组成，由MuA转座酶或HIV IN蛋白的四聚体连接。在自然界中，这些高阶蛋白质-DNA复合物的组装发生在影响组装过程的附加DNA结合蛋白的存在下。转位的靶DNA也以真核生物染色体或细菌类核的高阶蛋白质-DNA复合物的形式存在。转座病毒DNA复合体的组装过程和作为转座靶的染色体DNA的活性如何受到一般染色体/类核相关蛋白的影响，从而影响DNA的动态行为，目前尚不清楚。在原核生物和真核生物中，染色体DNA分子保持在动态缩合状态，涉及大量的DNA缩合蛋白。这些蛋白质要么弯曲/弯曲DNA，要么通过直接或间接的DNA结合来桥接遥远的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/质粒分割系统的机制。ParA和ParB蛋白也以高度可控的方式引起DNA凝聚，它们的功能动力学很可能受到与细菌类核相关蛋白（nap）相关的其他DNA凝聚蛋白的影响。

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.