This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
To date, E. coli serves as the only cellular system for which a complete replication fork can be reconstituted from purified proteins. In this project, a highly divergent bacterium, Baccilus subtilis, will be explored to learn what principles established from the E. coli model are general and what variations can be employed for the rapid, processive, coordinated replication of a chromosome. B. subtilis is an attractive system in that regard. Through genetic, molecular and cell biology work it is known that two distinct replicases are required, unlike the E. coli system where the DNA polymerase III holoenzyme alone suffices. The two-replicase system is reminiscent of eukaryotic systems. It is also known that two proteins are required to load the helicase, unlike E. coli, but perhaps similar to the suspected Orc6/Cdt1 requirement in eukaryotes. B. subtilis also employs two novel proteins, DnaD and DnaB, that have no homologs in E. coli. Thirteen proteins, predicted from genetics, have been expressed and purified and have been used to reconstitute a robust, rolling circle replicative reaction that recapitulates the general features of B. subtilis chromosomal replication in vivo. A rigorous study will be conducted to elucidate the cooperative interactions of replication proteins that participate in the helicase assembly process. The function of the two replicases, DnaE and PolC, will be perturbed to gain fundamental information regarding their function.
Completion of the program will lead to an important mechanistic understanding of DNA replication in a diverse model organism. Fundamental knowledge will also serve as a prototype for comparative studies in less tractable eukaryotic systems. The program will increase training opportunities for undergraduate and graduate researchers, including groups underrepresented in science.
