Impacting 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.
