AbstractThe cell cycle is a highly orchestrated developmental process that eventually leads to the reproduction of a cell. In metazoans, it is driven by the successive activation of cyclin-dependent kinases (Cdk) and proper coordination of cell cycle transitions and processes ensure genomic stability. 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 and requires the activities of two protein kinases, S phase-Cdk and Cdc7. In this thesis, I present work relating to the role of the S phase-promoting kinases in DNA replication and S phase regulation.
Using the cell-free system of Xenopus egg extracts, a small molecule inhibitor of Cdc7, PHA-767491, was characterised. PHA-767491 was then used to demonstrate that Cdc7 executes its activity early in S phase before the Cdk-dependent step. Cdc7 is not rate limiting for the progression of the replication timing programme once its essential function has been executed, unlike S-Cdk whose activity is required throughout S phase. Protein Phosphatase 1 (PP1) was identified as a modulator of Cdc7 activity in egg extracts, which 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 the thesis, the design strategy for generating a Cdc7-conditional knockout mouse (cko) is outlined and results from the screen for a transgenic founder are presented. A Cdc7-cko mouse will be a valuable tool to further dissect Cdc7 function and regulation in mammalian cells. In the final section, S phase entry and progression in mouse embryonic fibroblasts lacking both Cdk1 and Cdk2 was examined. 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. The exact function(s) and mechanism(s) for these observations remain to be discovered. With this work, I hope to provide additional insight into the roles and regulation of S phase kinases in eukaryotic DNA replication.
|Date of Award||2012|
|Sponsors||Agency for Science, Technology and Research|
|Supervisor||Julian Blow (Supervisor) & Philipp Kaldis (Supervisor)|
- DNA replication
- S phase