The goal of this project is to understand how bacteria make a copy of their DNA. Accurate and efficient DNA replication is critical for the survival of all organisms. However, DNA replicates with occasional spontaneous errors, and in addition, it is constantly damaged by environmental factors. DNA replication enzymes (replicative DNA polymerases) are generally unable to copy damaged DNA; there are special DNA polymerases that are used by cells to copy across the DNA damage. This project studies the accessory proteins, single-stranded DNA binding protein and the beta processivity clamp, and asks how they regulate the activity of replicative and specialized DNA polymerases. Students trained as part of this project will be highly skilled in molecular biology, biotechnology, and biophysics and will be prepared to pursue further education and training, to pursue careers in basic research, or to contribute to the biotechnology and pharmaceutical industries in the local area and beyond, thereby contributing to the life sciences industries. This project will also give high school students the opportunity to gain research experience. Furthermore, by integrating a research project in the Principles of Chemical Biology teaching laboratory, this project will expose more students to research.
Many key aspects of bacterial DNA replication remain poorly understood. The work proposed here will elucidate the factors that control DNA replication in the model bacterium E. coli and will determine how they exert their control. One focus of the project is on single-stranded DNA binding protein (SSB) as a regulator protein. SSB protects single-stranded DNA and is thought to eliminate DNA structures that can inhibit replication. SSB has been shown to inhibit polymerase activity of the pol III alpha subunit and core, of which alpha is a part, but enhance the activity of other DNA polymerases; the work in this proposal will determine the molecular basis for these differential effects. By analyzing the activity of DNA polymerases on undamaged and damaged DNA and with accessory proteins SSB and the beta processivity clamp, kinetic parameters of each polymerase will be determined to provide insights into the dynamic processes of proofreading and polymerase switching. This project combines biochemical, cellular, and biophysical single molecule methods to develop a comprehensive understanding of the mechanisms of DNA replication.
