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

研究参与各种 DNA 交易的高阶蛋白质 DNA 复合物的动力学

• 批准号: 10250238
• 负责人: KIYOSHI MIZUUCHI
• 金额: $ 14.85万
• 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/10250238
• 关键词: Affect; Attention; Bacterial Chromosomes; Bacteriophage mu; Behavior; Binding Proteins; Cell Nucleus; Cells; Chromosome Segregation; Chromosomes; Complex; DNA; DNA Binding; DNA Integration; DNA Sequence; DNA-Binding Proteins; Development; Distant; Eukaryota; Fluorescence; Gene Rearrangement; HIV; Label; Mitosis; Modeling; Nature; Phase; Physical condensation; Plasmids; Play; Process; Prokaryotic Cells; Property; Proteins; Reaction; Role; Site; Structure; System; Transact; Transposase; Virus; dimer; fluorescence microscope; molecular assembly/self assembly; mu opioid receptors; protein complex; viral DNA

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.

该项目研究了噬菌体 Mu 和 HIV 等转座病毒的目标染色体的动态方面。涉及 Mu 或 HIV DNA 序列末端的一对 DNA 切割和链转移以及它们与染色体靶 DNA 位点的相互作用发生在所涉及的每个 DNA 片段的高阶蛋白质-DNA 组装的背景下。转座病毒DNA末端组装成更高级的蛋白质-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，并由MuA转座酶或HIV IN蛋白的四聚体突触。这些高级蛋白质-DNA 复合物在自然界中的组装是在影响组装过程的其他 DNA 结合蛋白存在的情况下进行的。用于转座的靶DNA也以真核染色体或细菌核的形式作为高级蛋白质-DNA复合物存在。转座病毒DNA复合物的组装过程以及作为转座靶标的染色体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 分子都保持动态浓缩状态，涉及多种 DNA 浓缩蛋白。这些蛋白质要么弯曲/弯曲 DNA，要么通过直接或间接 DNA 结合跨桥遥远的 DNA 片段。在我们的单独项目 (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 蛋白也会以高度受控的方式引起 DNA 缩合，并且它们的功能动态很可能受到与细菌核相关的其他 DNA 缩合蛋白 NAP（类核相关蛋白）的影响。 该项目旨在加深我们对染色体相关蛋白如何通过其对浓缩 DNA 动力学的影响来影响 DNA 事务的理解，例如转座病毒 DNA 整合、转座目标位点搜索以及细菌染色体分离过程。预整合复合体中的 HIV DNA 受到 BAF 蛋白的保护，免遭自我破坏性的自动整合。据信，BAF 会以某种方式浓缩 DNA，使其无法进行自我破坏性的自动整合。通过使用荧光标记的 BAF 和高灵敏度荧光显微镜系统，在单个 DNA 分子水平上研究了 BAF 浓缩 DNA 的机制。 BAF 是一种小型二聚体蛋白，它以接近正交的方向结合两个 DNA 片段，形成交叉桥以浓缩 DNA。 BAF被认为在有丝分裂后细胞核重新组装过程中发挥作用。虽然它不被认为是一般的染色体相关缩合蛋白，但它可作为桥接型 DNA 缩合体的模型，我们研究其与细菌 NAP 和其他 DNA 缩合蛋白的 DNA 相互作用动力学。 目前正在开发实验方法来定量评估原核和真核来源的 DNA 浓缩蛋白对 DNA 结构动力学、高阶蛋白质-DNA 复合物组装过程以及这些大分子组装体的动态特性的影响。