Single-molecule enzymology of the replisome

复制体的单分子酶学

• 批准号: 7796850
• 负责人: ANTOINE M VAN OIJEN
• 金额: $ 17.28万
• 依托单位: GRONINGEN UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-04-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7796850
• 关键词: Bacteriophage T7; Binding; Biochemical; Biological Assay; Biological Models; Complex; Coupled; DNA; DNA Primase; DNA biosynthesis; DNA-Directed DNA Polymerase; Dissociation; Enzymatic Biochemistry; Frequencies; Genome Stability; Genomics; Goals; Hand; In Vitro; Individual; Kinetics; Label; Malignant Neoplasms; Methodology; Molecular; Monitor; Movement; Nature; Nucleotides; Okazaki fragments; Polymerase; Process; Property; Proteins; Reaction; Recycling; Relative (related person); Research Personnel; System; Techniques; Testing; Time; design; helicase; insight; movie; novel; numb protein; polymerization; prevent; programs; public health relevance; reconstitution; research study; single molecule; single-molecule FRET

DESCRIPTION (provided by applicant): Replication of genomic DMA involves the coordinated activity of a large number of proteins. The replisome, the molecular machinery of DMA replication, unwinds the double-stranded DMA, provides primers to initiate synthesis, and polymerizes nucleotides onto each of the two growing strands. Remarkable progress has been made in characterizing the structural and functional properties of the individual components; their coordination at the replication fork is less well understood. The dynamic nature of the replisome makes it difficult to probe these processes with ensemble-averaging techniques. We propose to use single molecule techniques to study the mechanisms that coordinate replication in fully assembled and functional replication complexes. We will use in vitro reconstituted bacteriophage T7 replisomes as a model system and study the kinetics of both leading- and lagging-strand synthesis on a single-molecule level. In particular, we will determine how the continuous polymerization at the leading-strand is coupled to the frequently interrupted lagging-strand synthesis and what enzymatic step triggers the recycling of the lagging-strand DMA polymerase. Furthermore, we will use single-molecule fluorescence resonance energy transfer between labeled components of the replisome during replication to determine how the large conformational changes required for lagging-strand DNA polymerase recycling are facilitated. Public Health relevance: The ability of DNA replication proteins to quickly and accurately replicate DNA without stalling or dissociating is crucial to maintain genomic stability and thus to prevent cancer. By observing individual replication complexes and recording "molecular movies" of their enzymatic activities, we will provide new insights into the general mechanisms underlying the coordination of proteins at the replication fork. Furthermore, the novel single-molecule methodologies we propose to develop will be directly applicable to studying the dynamics of other, more complex systems with unprecedented detail.

描述（由申请人提供）：基因组DMA的复制涉及大量蛋白质的协调活性。 DMA复制的分子机制解释了双链DMA，为启动合成提供了引物，并将核苷酸聚合到两个生长链中的每个链上。在表征各个组件的结构和功能特性方面取得了显着的进步。他们在复制叉上的协调不太了解。重置体的动态性质使得很难通过合奏平均技术来探测这些过程。我们建议使用单分子技术来研究在完全组装和功能复制复制复合物中协调复制的机制。我们将使用体外重构的噬菌体T7重新分裂作为模型系统，并在单分子水平上研究铅和滞后链合成的动力学。特别是，我们将确定前链处的连续聚合如何与经常中断的滞后链合成以及哪种酶促步长触发落后链DMA聚合酶的回收利用。此外，我们将在复制过程中使用单分子荧光共振能量转移，以确定如何促进滞后链链DNA聚合酶回收所需的大构象变化。公共卫生相关性：DNA复制蛋白快速准确地复制DNA而不停滞或解离的能力对于维持基因组稳定性至关重要，从而预防癌症。通过观察单个复制复合物并记录其酶活性的“分子电影”，我们将提供有关在复制叉上蛋白质协调的一般机制的新见解。此外，我们建议开发的新型单分子方法将直接适用于研究其他具有前所未有的细节的其他更复杂系统的动力学。