Cyclin E-dependent mechanisms linking centrosome duplication with DNA replication and cell cycle progression

Centrosome duplication and DNA replication are fundamentally similar processes in that both occur once-and-only-once during each cell cycle. Both events initiate at the GUS transition and require an increase in Cdk2 activity. It has long been evident that the centrosome duplication and DNA replication cycles must not only be highly regulated but also temporally coordinated to prevent inappropriate re-replication. Abnormal centrosome numbers are described in virtually all human malignancies and have been shown to be a probable cause of chromosomal instability. Previous work in this laboratory defined a 20 amino acid modular centrosomal localization signal (CLS) in cyclin E that is both necessary and sufficient for centrosomal localization. Furthermore, it was shown that expression of the CLS displaces both endogenous cyclins E and A from centrosomes and prevents BrdU incorporation into DNA. To identify interactions mediated by the CLS, a HeLa cDNA library was screened using a bacterial two-hybrid assay with the CLS as bait. In this screen MCM5, an essential DNA replication factor, was isolated. Co-immunoprecipitation experiments and GST-pulldown assays verified a direct, in vivo interaction between MCM5 and cyclin E that is dependent on a wild-type CLS but independent of Cdk2. Interaction with cyclin E accounts for MCM5 localization on centrosomes in mammalian cells, and ectopic expression of MCM5 inhibits centrosome over-duplication in S phase-arrested CHO cells. MCM5 and cyclin A also interact in a manner dependent on a functional CLS domain but not on Cdk binding. Expression of the cyclin E CLS in G1/S synchronized cells inhibits S phase due to a lack of chromatin loading of proteins essential for the initiation of DNA replication. Rescue of DNA replication was evident when active cyclin E/Cdk2 was re-targeted to centrosomes. Taken together, the work presented here reveals novel mechanisms of communication between the centrosome and nuclear replication cycles in which elements of each cycle can directly regulate events in the other.