PhD Graduate: Wei Theng POH
under the A*STAR-Dundee Partnership Programme (ADP)
Thesis Title: The role of Cdc7 and cyclin-dependent kinases in DNA replication and S phase
In the cell cycle, DNA replication takes place during S phase to faithfully duplicate a cell’s genetic material. In eukaryotes, S phase onset involves the initiation of numerous licensed replication origins across the genome. Initiation of DNA replication from licensed origins requires two protein kinases: cyclin-dependent kinase (Cdk) and Cdc7. Although S phase-Cdk activity has been intensively studied, relatively little is known about how Cdc7 is regulated. PHA-767491, a small molecule inhibitor of Cdc7, was first characterised and used to dissect the role of Cdc7 in regulating S phase progression in Xenopus egg extracts. Cdc7 is not rate limiting for the progression of the replication timing programme once its essential function has been executed, unlike Cdk whose activity is required throughout S phase. Protein Phosphatase 1 (PP1) was identified as a modulator of Cdc7 activity in egg extracts. It rapidly reverses Cdc7-dependent phosphorylation of chromatin-bound Mcm4 and likely functionally lowers Cdc7 activity during an etoposide-induced checkpoint response. This provides a novel mechanism for regulating Cdc7 by counteracting its activity on essential replication substrates in the event of replicative stress. In the second part of this work, S phase entry and progression was examined in mouse embryonic fibroblasts lacking both Cdk1 and Cdk2, which provide S phase-Cdk activity in metazoans. Contrary to expectations, Cdk1/Cdk2 double knockout cells can enter S phase in the absence of detectable S phase-Cdk activity. S phase progression, however, was inefficient. Cdc6 and cyclin E1 proteins were found to accumulate in high levels in these cells, although the exact function(s) and mechanism(s) for these observations remain to be elucidated.