The role of the Cdc7 kinase in DNA replication and in the DNA damage response

The Cdc7 protein kinase plays a central role in coordinating DNA replication and the damage response. Cdc7 is activated by its regulatory subunits Dbf4 or Drf1, and phosphorylates chromatin-bound Mcm2-7, which is required for replication forks to initiate 1. There is also evidence that Cdc7 plays an important role in the cellular response to DNA damage, possibly acting as a control point for shutting down replication when DNA damage is detected 2,3. There is evidence that Cdc7 is misregulated in cancer cells, and that Cdc7 inhibitors have good potential as anti-cancer agents 4. The aim of the PhD project is to address how Cdc7 is regulated. In particular, we will address when Cdc7 is activated in the cell cycle and when it acts on individual replication origins. We will also examine how Cdc7 activity is changed in response to DNA damage and how this affects the process of DNA replication. This knowledge would then be used to explore the differences in Cdc7 activity between normal and cancer cells. The project would initially use biochemical analysis in Xenopus egg extracts 1,5. Results obtained in the Xenopus system will subsequently be transferred to mammalian cells and or genetic mouse models.

1. Characterisation of pyrrolopyridinone as a Cdc7 inhibitor

The first known inhibitor of Cdc7, pyrrolopyridinone, has been reported 6. We have synthesised this compound and it shows very good activity in inhibiting DNA replication in Xenopus extracts. We first need to carefully characterise the inhibition and determine whether it gives results consistent with previous reports of Cdc7 knockdown in Xenopus extracts 1,7-9.

2. Execution point of Cdc7 in an undisturbed S phase

Relatively little is known about the execution point of Cdc7 function. Experiments in yeast suggest that Cdc7 function is required during S phase, not just at the G1/S transition 10,11.  However, results in Xenopus suggest that a significant degree of replication can be supported if Cdc7 function is blocked prior to entry into S phase 2,9.

3. Cdc7 and replicative stress

The way that Cdc7 plays a role in checkpoint responses is controversial, particularly in animal cells 2,4,12,13. We would investigate the role that Cdc7 plays in responding to replicative stress.

This is an existing collaboration that has already resulted in a publication [Poh, W. T., Chadha, G. S., Gillespie, P. J., Kaldis, P. & Blow, J. J. Open Biol 4, 130138 (2014)]

References
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6 Vanotti, E. et al. J Med Chem 51, 487-501 (2008).
7 Jares, P., Luciani, M. G. & Blow, J. J. BMC Mol Biol 5, 5 (2004).
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12 Costanzo, V. et al. Mol Cell 11, 203-213 (2003).
13 Tenca, P. et al. J Biol Chem 282, 208-215 (2007).

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