Principal Investigator Stephen Bell
The short (100-120 bp), well characterized origins of DNA replication derived from S. cerevisiae chromosomes and the ability to combine biochemical and genetic manipulation have led us to study chromosome duplication in this model eukaryote. During each round of cell division, the cell cycle regulated assembly of multi-protein complexes at the origin DNA culminates in the formation of a bi-directional replication machine called a replisome. These events are nucleated by the recognition of a conserved element within the origin DNA by the six-protein origin recognition complex (ORC). Once bound to the origin, ORC and two other replication factors (Cdc6 and Cdt1) assemble the eukaryotic DNA unwinding enzyme (Mcm2-7 complex) onto the origin DNA. The resulting complex marks all potential sites of replication initiation across the genome and is called the pre-replicative complex (pre-RC). Importantly, the correct regulation of pre-RC formation and activation is essential to ensure that eukaryotic chromosomes are replicated exactly once per cell cycle.
Assembly of DNA Replication Complexes: 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. The current efforts are aimed at fully reconstituting pre-RC assembly with purified proteins, testing the predictions of the 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.
Prevention of re-replication: We are investigating the mechanisms that ensure the genome is replicated exactly once per cell cycle. Cyclin-dependent kinase (CDKs) modulation of pre-RC formation and activation is central to this regulation. During S phase, high CDK levels direct pre-RC activation and prevent new pre-RC formation. In contrast, the low CDK levels present during G1 allow pre-RC formation but not activation. ORC is one of three CDK targets whose modification mediates CDK inhibition of pre-RC formation. In collaboration with Fred Cross’s lab (Rockefeller University), we found that the S-phase cyclin Clb5 binds ORC but only after initiation has occurred. Our findings support a model in which Clb5 binding to ORC provides an origin-localized switch that specifically prevents re-initiation at replicated origins. Using mutants in ORC that alter either its Clb5 interaction or its CDK phosphorylation we are determining how these associations/modifications alter ORC’s ability to direct pre-RC formation.
We have also mapped the sites of pre-RC formation and replication initiation in cells that are undergoing re-replication using high-density genomic arrays. We found that only a subset of the origins used in a normal S phase is used during re-replication. Intriguingly, we also found that not all origins that assemble a pre-RC during re-replication initiate from that site suggesting that the mechanisms that control re-replication target events after pre-RC formation.