DnaA-mediated loading of the DnaB-DnaC complex in replication initiation in Escherichia coli

大肠杆菌中DnaA介导的DnaB-DnaC复合物在复制起始中的负载以多种方式影响我们的日常生活，众所周知，一些细菌物种危害健康，但其他细菌物种发挥关键的有益作用。众所周知的例子是在乳制品工业，通过帮助固氮来种植粮食作物，以及处理城市废水。在个人层面上，细菌的种类及其相对丰度决定了一个人的微生物组，这随着年龄和健康而变化。然而，我们对细菌如何复制染色体的理解存在根本性的差距，这与细胞生长直接相关，限制了我们操纵它们以改善人类健康，农业以及环境水质的能力。为了深入了解细菌DNA复制及其调控的基本细胞过程，该项目协同了三个实验室独特但互补的技能。这项工作的重点是在启动DNA复制的模式生物，大肠杆菌的阶段所需的关键过程。研究生和本科生以及博士后（包括代表性不足的群体）的学生将通过参与这项工作接受跨学科的研究培训。项目参与者将通过演讲和工作坊与非科学家分享研究成果，并告诉他们这项研究如何有助于我们理解自由生活的生物体中的DNA复制，从而造福人类。项目重点是在复制开始阶段加载解旋酶的具体步骤。对E. coli中发现，DnaA的结构域1需要在E. coli复制起点。在DnaA盒，这可能是类似于在细菌复制起点形成的子复合物形成的核蛋白复合物的分子分析表明，一个保守的解旋酶加载机制。研究人员正在测试这个模型。利用冷冻电子显微镜和氢/氘交换分析的实验方法，结合生物化学和遗传学的方法，在复制起始阶段，包含DnaA，DnaB和DnaC组装在DnaA盒序列的核蛋白复合物的高分辨率结构将被获得。这项研究将解决复制启动机制的关键问题。具体来说，DnaA如何引导DnaB与DnaC复合在DNA的特定位点上？DnaC是否在解旋酶加载中起直接作用？每个DnaB原聚体的构象是否适合与引发酶相互作用？这些发现可以解释两种解旋酶中的一种如何加载到其他细菌的复制起点。目前，人们对这一过程知之甚少。该奖项反映了NSF的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

DnaA-mediated loading of the DnaB-DnaC complex in replication initiation in Escherichia coliImpacting our daily lives in a multitude of ways, some bacterial species are well known to imperil health, but others play critical beneficial roles. Well-known examples are found in the dairy products industry, the growth of food crops by aiding in nitrogen fixation, and the treatment of municipal wastewater. On an individual level, the species of bacteria and their relative abundance determine a person's microbiome, which varies with age and health. However, fundamental gaps in our understanding of how bacteria duplicate their chromosomes, which is directly correlated with cell growth, limit our ability to manipulate them to improve human health, agriculture, and also the water quality of our environment. To gain insight into the fundamental cellular processes of bacterial DNA replication and its regulation, this project synergizes the unique but complementary skills of three laboratories. The work focuses on the crucial processes required at the stage of initiation of DNA replication in the model organism, Escherichia coli. Students at the graduate and undergraduate level, and a postdoc, including those in underrepresented groups, will receive interdisciplinary research training by participating in the work. Project participants will share the findings with non-scientists through presentations and workshops, and will inform them about how the research contributes to our understanding of DNA replication in free-living organisms to benefit mankind.The project focuses on the specific step of helicase loading at the stage of replication initiation. Studies of the helicase loading process in E. coli show that domain 1 of DnaA is required to load DnaB complexed to DnaC at the E. coli replication origin. Molecular analysis of a nucleoprotein complex formed at a DnaA box, which may be analogous with a sub-complex formed at bacterial replication origins, suggests a conserved mechanism of helicase loading. The research tests this model. Using the experimental approaches of cryo-electron microscopy and hydrogen/deuterium exchange analysis combined with biochemical and genetic methods, a high-resolution structure of a nucleoprotein complex at the replication initiation stage that contains DnaA, DnaB and DnaC assembled at a DnaA box sequence will be obtained. The study will address critical questions about the mechanism of replication initiation. Specifically, how does DnaA direct the loading of DnaB complexed to DnaC at a specific site in DNA? Does DnaC play a direct role in helicase loading? Is the conformation of each DnaB protomer suitable for interaction with primase? The findings may explain how one of the two helicases loads at a replication origin in other bacteria. At present, this process is poorly understood.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.