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

大肠杆菌复制起始中dna介导的dna - dnac复合体负载以多种方式影响我们的日常生活，众所周知，一些细菌物种会危害健康，但其他细菌则发挥关键的有益作用。众所周知的例子是在乳制品行业，通过帮助固氮来促进粮食作物的生长，以及城市污水的处理。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群，而微生物群随年龄和健康状况而变化。然而，我们对细菌如何复制与其细胞生长直接相关的染色体的理解存在根本性的差距，这限制了我们操纵它们改善人类健康、农业和环境水质的能力。为了深入了解细菌DNA复制及其调控的基本细胞过程，该项目将三个实验室独特但互补的技能协同起来。这项工作的重点是在模式生物大肠杆菌中DNA复制起始阶段所需的关键过程。研究生和本科生以及博士后，包括那些代表性不足的群体，将通过参与工作获得跨学科研究培训。项目参与者将通过演讲和研讨会与非科学家分享这些发现，并将告知他们这项研究如何有助于我们了解自由生物体内的DNA复制，从而造福人类。本项目重点研究了在复制起始阶段解旋酶装载的具体步骤。对大肠杆菌解旋酶装载过程的研究表明，在大肠杆菌的复制起点，需要DnaA结构域1将DnaB复合物装载到DnaC上。在dna盒中形成的核蛋白复合物的分子分析，可能类似于在细菌复制起点形成的亚复合物，表明解旋酶装载的保守机制。本研究对这一模型进行了验证。利用低温电镜和氢/氘交换分析相结合的实验方法，结合生物化学和遗传学方法，将获得在复制起始阶段包含DnaA， DnaB和DnaC的高分辨率结构。该研究将解决有关复制起始机制的关键问题。具体来说，DNA是如何将DNA复合体的负载引导到DNA中特定位置的DnaC上的？DnaC在解旋酶装载中起直接作用吗？每个dna原体的构象是否适合与引物酶相互作用？这一发现可能解释了这两种解旋酶中的一种如何在其他细菌的复制起点上装载。目前，人们对这一过程知之甚少。该奖项反映了美国国家科学基金会的法定使命，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.