Functional Dynamics of Large Molecular Machines. The Primosome.

单链DNA是DNA复制过程中至关重要的活性中间体， 重组和修复，这对遗传信息的传递至关重要。该中间体 由双链DNA的瞬时解旋形成，这是由一类称为解旋酶的酶催化的。 解旋酶在DNA和RNA代谢的各个方面都是不可或缺的，也充当分子泵 以及作为大型多蛋白质机器的生物马达，包括复制体和前引发体。 由于环境和细胞因素，基因组DNA损伤在细胞中不断发生， 导致形成停滞的复制叉。停止的fork的重新启动通过 组装大分子机器，即前原体，这是保护完整性的关键过程。 遗传信息的。在大肠大肠杆菌，两种解旋酶，DnaB和PriA蛋白，以及DnaC，DnaT， PriB和PriC蛋白参与前原体组装和功能。 解旋酶机制的阐明对于理解解旋酶的基本功能是至关重要的。 核酸代谢的过程以及为什么这些过程功能障碍，例如，癌和人类 遗传病研究分子机制将提供必要的知识， 调节和控制这些过程，在设计有效的疾病治疗方法方面非常宝贵。阐发 前原体组装和活动的机制对于理解这些规则至关重要 控制着停滞的分叉的重新启动，以及一般来说，控制着大型分子机器的运作。 急诊coli DnaB蛋白是六聚体复制解旋酶的范例模型。六聚体复制的 解旋酶最初被分类为DnaB样酶家族。原始体是一种原型 不同马达蛋白和大型分子机器协同作用的分子系统。的 PriA蛋白反过来又是该因子的原型模型，它能感知到停滞的分叉的存在， 启动前原体的组装。 该项目的长期目标是建立复制解旋酶的分子机制， 参与的前primosome，和规则，其中管理大分子相互作用的前primosome， 原体机器这将通过热力学、动力学、 和多种蛋白质-蛋白质和蛋白质-DNA复合物的结构，使用分析超离心， 动态光散射，化学快速猝灭流，荧光停止流，荧光各向异性， 荧光能量转移、晶体学、电子显微镜和生物化学方法。

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 pre- primosome 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.