Principal Investigator Stephen Bell
We are using a combination of genetics, protein biochemistry and in vitro assays for pre-RC formation to determine how this essential complex is assembled and how it prepares the origin DNA for replication initiation. Recent studies focusing on the ATP control of pre-RC assembly have revealed how multiple ATP-dependent steps required for pre-RC formation are coordinated and ensure that this event occurs at the correct cell cycle time and chromosomal position. ATP hydrolysis by Cdc6 is activated by origin-bound ORC and is required to load the Mcm2-7 onto origin DNA. In contrast, ORC ATP hydrolysis is not required for the initial Mcm2-7 loading event but is required for this event to be repeated. These and other observations have led us to propose a model for pre-RC formation. Current efforts are aimed at fully reconstituting pre-RC assembly with purified proteins, testing the predictions of our pre-RC assembly model (e.g., What events trigger ATP hydrolysis by ORC and Cdc6? Does the Mcm2-7 ring encircle dsDNA?), and developing new assays for downstream steps in replication initiation.