The bacteriophage T4 replication system is one of the best-studied of the related mechanisms of DNA replication employed by prokaryotes and eukaryotes. This proposal is focused on the assembly and functioning of the primosome in the context of DNA replication. A complete understanding will require studying the individual proteins of the primosome as a large enzyme machine. The assembly process of the primosome will be interrogated by ensemble and single-molecule FRET equilibrium and pre-steady state studies. Additional structural information on the primosome will be gained by electron microscopy. The functioning of the primosome will by elucidated by cross-linking/footprinting studies and by monitoring protein-protein interactions using FRET during active translocation and priming by the primosome. Completion of this project will result in a description of the assembly and functioning of the primosome in detail similar to that already determined for the polymerase holoenzyme. Since the process of DNA replication has profound implications for human disease, including cancer, understanding the fidelity of DNA replication is of significant humanitarian and economic interest.
