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是必不可少的。这个中间体是 由双链DNA瞬间解开形成，这种双链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，也是分子泵。 作为大型多蛋白质机器的生物马达，包括复制体和前原始体。 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子。重启停止的分叉是通过 组装大分子机器--前原粒，这是维护完整性的关键步骤 遗传信息。在大肠杆菌中，两种解旋酶，DNAB和PriA蛋白，以及DNAC，DNAT， Prib和PrIC蛋白参与了前原粒的组装和功能。 解旋酶机制的阐明对于理解解旋酶的基本原理至关重要。 例如，在癌症和人类中，核酸代谢的过程以及为什么这些过程功能障碍 遗传病。研究分子机制将提供必要的知识，如如何 控制和控制这些过程，对于设计有效的疾病治疗方法是非常宝贵的。澄清 对前原粒组装和活动的机制的了解对于理解规则至关重要 管理停滞不前的叉子的重新启动，以及通常情况下，大分子机器的运行。 大肠杆菌DNAB蛋白是一个六聚体复制型解旋酶的范例模型。六聚体复制体 解旋酶最初被归类为DNAB样酶家族。原始体是一个原型 不同马达蛋白质协同作用的分子系统和大分子机器。这个 反过来，PriA蛋白是该因子的原型模型，它感知失速的叉子的存在，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 在前原粒中的相互作用，以及控制前原粒中大分子相互作用的规则。 原始体机器。这将通过对热力学、动力学、 以及多个蛋白质-蛋白质和蛋白质-DNA复合体的结构，使用分析超速离心法， 动态光散射，化学急流，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。

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.