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

Dna A介导的Dna B-Dna C复合体在大肠杆菌复制起始中的负载在我们的日常生活中以多种方式影响，一些细菌物种众所周知地危害健康，但另一些细菌起着关键的有益作用。众所周知的例子有乳制品工业，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，细菌的种类和它们的相对丰度决定了一个人的微生物群，这一微生物群随年龄和健康而变化。然而，我们对细菌如何复制其染色体(与细胞生长直接相关)的理解存在根本差距，限制了我们操纵它们以改善人类健康、农业以及我们环境的水质的能力。为了深入了解细菌DNA复制的基本细胞过程及其调控，该项目结合了三个实验室独特但互补的技能。这项工作的重点是在模式生物大肠杆菌中启动DNA复制阶段所需的关键过程。研究生和本科生以及博士后，包括代表人数不足的群体，将通过参与这项工作接受跨学科研究培训。项目参与者将通过演讲和研讨会与非科学家分享研究结果，并向他们介绍这项研究如何有助于我们理解自由生命有机体中的DNA复制以造福人类。该项目侧重于复制启动阶段解旋酶负载的具体步骤。对大肠杆菌中解旋酶装载过程的研究表明，DNAA的结构域1需要在大肠杆菌复制起始处装载与DNAC络合的DNAB。对Dna A盒形成的核蛋白复合体的分子分析表明，解旋酶的负载是一种保守的机制，该复合体可能类似于细菌复制起始处形成的亚复合体。研究对这一模型进行了检验。利用低温电子显微镜和氢/氢交换分析的实验方法，结合生化和遗传学方法，将获得复制起始阶段的核蛋白复合体的高分辨结构，该复合体包含Dna A盒序列，Dna A，Dna B和Dna C。这项研究将解决有关复制启动机制的关键问题。具体地说，DNAA是如何在DNA中的特定位置将复杂的DNAB引导到DNAC的？DNAC在解旋酶的负载中起直接作用吗？每个DNAB原基的构象是否适合与底物酶相互作用？这些发现可能解释了两种解旋酶中的一种是如何在其他细菌的复制起始处加载的。目前，这一过程还没有得到很好的理解。这一奖项反映了NSF的法定使命，并通过使用基金会的智力优势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.