The successful duplication of genomic information is essential for cell division and viral propagation. Research that deepens our understanding of genomic replication is crucial for advance in the treatment of cancer as well as infection by bacteria, parasites, and viruses. The bacterium Escherichia coli (E. coli) is a model system for studying DNA replication due to its ease of handling and genetic manipulation, the large amount of information available about this organism, and its many mechanistic similarities with eukaryotic replication systems. The E. coli replicase, DNA Pol III holoenzyme, is composed of 10 different protein subunits. While the stoichiometry, placement, and general function have been determined for many subunits, the significance of dynamic interactions between subunits has yet to be fully understood. The goal of the research proposed here is to further elucidate the communication mechanisms within the DNA Pol III replicase that affect coordination of the leading and lagging strand polymerases at the replication fork. A reconstituted DNA replication system will be used in conjunction with BIAcore biosensor and fluorescence anisotropy techniques to identify the effectors that signal polymerase cycling on the lagging strand. The rates at which different signaling mechanisms cause polymerase dissociation from DNA will be measured in order to determine their physiological relevance. Additionally, communication between the two polymerases at the replication fork will be studied using a novel rolling circle replication system in which the affects on leading strand synthesis can be measured while the behavior of the lagging strand polymerase is varied.
