Even after decades of study, remarkably little is known about the structural basis for the functional coupling of leading- and lagging-strand synthesis in DNA replication. A fundamental question regards how the polymerase active site couples to other subunits to provide proper proofreading and coordination between leading- and lagging-strand syntheses. My objective is to understand the molecular underpinnings of this complex and dynamic process in the bacterial polymerase III system using a combination of structural and biochemical studies and real-time visualization.
The specific aims of this proposal are to: 1) Determine the structural basis for the processivity switch in polymerase recycling. The structure of the complex of tauC bound to the polymerase subunit, a, will be determined. 2) Elucidate the mechanism by which the proofreading subunit modulates pol III activity. Preliminary data shows that there is likely a functional connectivity between the proofreading subunit and polymerase active site. Intrinsic polymerization activity will be measured in the presence and absence of the proofreading subunit, e, to test the hypothesis that the proofreading unit activates polymerization. Polymerase hybrids will be made and characterized to understand active site regulation principles. Finally, a single molecule assay will be developed in order to understand the interplay between the polymerase and proofreading. 3) Determine the structural basis for activation of replication and proofreading by the e subunit. The structure of the a-e complex will be solved in order to understand the mechanism by which the proofreading subunit, e, influences polymerase activity, and the structural basis for the switch from polymerization to exonucleolysis in pol III.
Understanding the structural basis for regulation of DNA replication will have great therapeutic utility. Small molecules that bind and inhibit the polymerase could prove useful as antibiotics. Moreover, defects in DNA replication have been associated with cancer and neurodegenerative disorders, further highlighting the importance of this process to health science.
| Kelch, Brian A; Makino, Debora L; O'Donnell, Mike et al. (2011) How a DNA polymerase clamp loader opens a sliding clamp. Science 334:1675-80 |
