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

DnaA 介导的 DnaB-DnaC 复合物在大肠杆菌复制起始过程中的负载

• 批准号: 1935089
• 负责人: Jon Kaguni
• 金额: $ 82万
• 依托单位: Michigan State University
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-01 至 2025-07-31
• 项目状态: 未结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1935089&HistoricalAwards=false
• 关键词: DnaA; mediated; loading; DnaB; DnaC; DnaA; mediated; loading; DnaB; DnaC

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.

DNAA介导的DNAB-DNAC复合物在大肠杆菌的复制启动中的负载，以多种方式使我们的日常生活施加，某些细菌物种是众所周知的，但有些细菌却发挥了至关重要的作用。在乳制品行业中发现了众所周知的例子，粮食作物的生长是通过固定固定的，以及对市政废水的处理。在个体水平上，细菌的物种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。但是，我们对细菌如何复制其染色体的理解的根本差距，这与细胞的生长直接相关，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复制的基本细胞过程及其调节，该项目使三个实验室的独特但互补的技能协同作用。这项工作着重于模型生物体大肠杆菌中DNA复制阶段所需的关键过程。研究生和本科生的学生以及包括代表性不足的小组在内的博士后，将通过参与工作来接受跨学科的研究培训。项目参与者将通过演讲和讲习班与非科学家分享调查结果，并将告知他们研究如何有助于我们对自由生活生物中DNA复制的理解，以使人类受益。大肠杆菌中解旋酶负荷过程的研究表明，DNAA的结构域1必须在大肠杆菌复制起源上加载与DNAC复合的DNAB。在DNAA盒中形成的核蛋白络合物的分子分析，该核蛋白复合物可能与在细菌复制起源形成的亚复合物类似，这表明有解旋酶负荷的保守机制。研究测试了该模型。使用冷冻电子显微镜和氢/氘交换分析的实验方法与生化和遗传学方法相结合，将获得包含DNAA，DNAB和DNAC在DNAA Box序列组装的DNAA，DNAB和DNAC的高分辨率结构。该研究将解决有关复制启动机制的关键问题。具体而言，DNAA如何将DNAB的载荷引导到DNA中特定位点的DNAC中？ DNAC在解旋酶载荷中起着直接的作用吗？每个DNAB原始物的构象是否适合与原始酶相互作用？这些发现可能解释了两个解旋酶之一如何在其他细菌中复制起源。目前，此过程对此尚未理解。该奖项反映了NSF的法定任务，并被认为是使用基金会的知识分子优点和更广泛影响审查标准的评估值得支持的。