Abstract
Life stores genetic information in the form of DNA. Because of this, investigating the systems involved in DNA replication and damage repair is imperative to understanding cellular function and survival. DNA Polymerase III serves as the premier prokaryotic enzyme in DNA replication and thus its characterization is of great interest. This study looks at the binding of DnaQ (epsilon) with HolE (theta) and separately, DnaQ with YoaA. Both DnaQ and HolE are subunits that, along with DnaE (alpha), make up the two catalytic cores of DNA Polymerase III. The catalytic cores contribute to the polymerase’s function in DNA replication and repair. DnaQ is responsible for the 3’ → 5’ proofreading exonuclease activity, HolE helps stabilize and stimulate DnaQ, and DnaE holds the polymerase active site and binds DnaQ to the rest of the complex. Additionally, YoaA is a helicase that has been implicated in the process of DNA damage repair and found to interact with an additional DNA Polymerase III subunit HolC. DnaQ mutants were made, including site directed mutants and truncations, to interrupt specific interactions with HolE and investigate which domains of DnaQ are required for DnaQ-HolE interaction. DnaQ and DnaQ mutants were also tested against YoaA to probe for and help characterize any interactions. Using Yeast Two Hybrid assays, results showed that DnaQ-HolE interaction requires DnaQ residues F48, D70, and E85 but not E71 or the C-terminal region of DnaQ. A novel DnaQ-YoaA interaction was identified and further tests implicated DnaQ residues F48, F63, D70, and E85 as required for interaction in the Yeast Two Hybrid system but not the E71 residue or C-terminal region of DnaQ.