Functional Dynamics of Large Molecular Machines. The Primosome.

描述(申请人提供)：单链DNA是DNA复制、重组和修复过程中的关键和活跃的中间体，而DNA复制、重组和修复是遗传信息传递所必需的。这种中间体是由双链DNA瞬间解离形成的，双链DNA由一种名为解旋酶的酶催化。解旋酶在DNA和RNA新陈代谢的各个方面都是不可或缺的，也是大型多蛋白质机器的分子泵和生物马达，包括复制体和前原粒。由于环境和细胞因素，基因组DNA损伤不断地发生在细胞中，导致停滞不前的复制叉的形成。停滞不前的分叉的重新启动是通过组装大分子机器--前原粒，这是保护遗传信息完整性的关键过程。在大肠杆菌中，两种解旋酶DNAB和PriA蛋白与DNAC、DNAT、Prib和PrIC蛋白一起参与了前原粒的组装和功能。解旋酶机制的阐明对于理解核酸新陈代谢的基本过程以及为什么这些过程在癌症和人类遗传性疾病中功能障碍至关重要。研究分子机制将为如何调节和控制这些过程提供必要的知识，对于设计有效的疾病治疗方法是非常宝贵的。阐明前原粒组装和活动的机制对于理解控制失速的叉子重新启动的规则以及一般大分子机器的功能是至关重要的。大肠杆菌DNAB蛋白是一个六聚体复制型解旋酶的范例模型。六聚体复制解旋酶最初被归类为DNAB样酶家族。原始体是不同马达蛋白和大分子机器协同作用的原型分子系统。反过来，PriA蛋白是因子的原型模型，它感知停滞不前的叉子的存在，并启动前原粒的组装。该项目的长期目标是建立复制解旋酶的分子机制及其在前原粒体内的参与，以及控制前原粒体内大分子相互作用的规则。这将通过定量研究多种蛋白质-蛋白质和蛋白质-DNA复合体的热力学、动力学和结构来实现，使用分析超速离心法、动态光散射、化学快速猝灭流、荧光停流、荧光各向异性、荧光能量转移、结晶学、电子显微镜和生化方法。与公众健康相关解旋酶在DNA和RNA新陈代谢的各个方面都是至关重要的，它催化活性单链中间体的形成，并充当核酸代谢的大分子机器的发动机。停滞的分叉的重新启动是保护遗传信息完整性的关键过程，通过组装前原粒发生。解旋酶机制的阐明对于理解核酸代谢的基本过程以及为什么这些过程在癌症和人类遗传疾病中功能障碍是至关重要的，而阐明前原粒组装和活动的机制对于理解规则至关重要，这些规则支配着停滞不前的分叉的重新启动，通常是大分子机器的功能。

DESCRIPTION (provided by applicant): The single-stranded DNA is a crucial and active intermediate in the processes of DNA replication, recombination, and repair, which are essential for the transmission of genetic information. This intermediate is formed by a transient unwinding of the duplex DNA, which is catalyzed by a class of enzymes called helicases. The helicases are indispensable in all aspects of DNA and RNA metabolism, serving also as molecular pumps and as biological motors for large multiple-protein machines, including the replisome and pre-primosome. Genomic DNA damages, due to environmental and cellular factors, are constantly occurring in the cell, leading to the formation of the stalled replication fork. The restart of the stalled fork occurs through the assembly of the large molecular machine, the pre-primosome, which is a key process in defending the integrity of the genetic information. In E. coli, two helicases, the DnaB and PriA proteins, together with the DnaC, DnaT, PriB, and PriC proteins, are engaged in the pre-primosome assembly and functions. Elucidation of the helicase mechanisms is of paramount importance for understanding the fundamental processes of the nucleic acid metabolism and why such processes dysfunction in, e.g., cancer and human genetic diseases. Studying the molecular mechanisms will provide the necessary knowledge as to how to regulate and control these processes and is invaluable in designing efficient therapies for diseases. Elucidation of the mechanisms of the pre-primosome assembly and activities is crucial for understanding the rules governing the restart of the stalled fork and, in general, the functioning of large molecular machines. The E. coli DnaB protein is a paradigm model of a hexameric replicative helicase. Hexameric replicative helicases were originally classified as the DnaB-like family of enzymes. The primosome is an archetype molecular system for the collaborative action of different motor proteins and of a large molecular machine. The PriA protein is, in turn, a prototype model for the factor, which senses the presence of the stalled fork and initiates the assembly of the pre-primosome. The long-term goal of this project is to establish molecular mechanisms of replicative helicases and their engagement in the pre-primosome, and the rules, which govern the macromolecular interactions in the preprimosome machine. This will be accomplished through quantitative studies of the thermodynamics, kinetics, and structures of multiple protein-protein and protein-DNA complexes, using the analytical ultracentrifugation, dynamic light scattering, chemical rapid quench-flow, fluorescence stopped-flow, fluorescence anisotropy, fluorescence energy transfer, crystallography, electron microscopy, and biochemical methods. PUBLIC HEALTH RELEVANCE The helicases are crucial in all aspects of DNA and RNA metabolism by catalyzing the formation of the active single-stranded intermediate and serving as motors for large molecular machines of the nucleic acid metabolism. The restart of the stalled fork is a key process in the defense of the genetic information integrity and occurs through the assembly of the pre-primosome. Elucidation of the helicase mechanisms is of paramount importance for understanding fundamental processes of the nucleic acid metabolism and why such processes dysfunction in, e.g., cancer and human genetic diseases, while elucidation of the mechanisms of the pre-primosome assembly and activities is crucial for understanding the rules, governing the restart of the stalled fork and, in general, the functioning of large molecular machines.