Cell cycle regulation of complex formation at origins of DNA replications

Abstract

This suggests that the S phase cyclin, which first activates DNA replication, then acts locally at the origin to prevent pre-RC formation.

Eukaryotic DNA replication is regulated by the formation of protein complexes over the initiator protein, the Origin Recognition Complex (ORC). In the G1 phase of the cell cycle, the pre-Replicative Complex (pre-RC) associates with ORC at the origin, priming the origin for initiation of DNA replication. Upon entry into S phase, the pre-RC is activated by the combined activities of two protein kinases, the S phase specific Dbf4 Dependent Kinase (DDK) Cdc7 and the Cyclin Dependent Kinase (CDK) Cdc28. After the origin initiates or is replicated over in S phase, a complex called the post-replicative Complex (post-RC) is left at the origin until CDK levels drop and pre-RCs reform in the next G1. In this work, both the cis acting sequences necessary at the origin to form pre-RCs and the trans acting proteins that regulate pre-RC formation were studied. Along with an essential ORC binding site, the well characterized yeast origin ARS1 requires specific sequences at an auxiliary sequence element, the B2 element, to load pre-RCs. These sequences resemble an extra ORC binding site, but do not bind a second ORC in vitro. Mutations in these sequences are rescued by overexpression of the pre-RC component Cdc6. Together, these results suggest that one of the proteins in the pre-RC interacts with this DNA sequence to facilitate complex formation. An interaction between the smallest subunit of ORC, Orc6, and the S phase cyclin Clb5 was documented. The molecular domains responsible for this interaction are a hydrophobic patch on Clb5 and an RXL motif and CDK phosphorylation sites on Orc6. Clb5 associates stably with origins after they replicate, forming part of the post-RC. This interaction aids in prevention of re-replication within a single cell cycle.