Principal Investigator Stephen Bell
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.