A novel checkpoint regulating the cell cycle of fission yeast

In this project we shall further study and characterise a novel mechanism of checkpoint regulation that we have recently discovered (Tvegård et al, Genes Dev, 2007). Checkpoints are important regulators of the cell cycle. Cancer development is frequently associated with the lack of checkpoints and checkpoint proteins represent possible targets for cancer treatment. Our goal is to map the novel pathway, from initiation to the final target molecule(s). We have shown that the onset of DNA replication is dela yed by UV light in a process that depends on the Gcn2 kinase, which means that it represents a novel type of checkpoints. Therefore, we wish to identify all proteins that interact with Gcn2, both upstream and downstream. This kinase is known to phosphoryl ate the translation initiation factor eIF2a in response to nutrient limitation, and we wish to find out whether eIF2a phosphorylation is a part of the checkpoint response. Furthermore, we have recent evidence that the classic checkpoint protein Rad3 (the equivalent of ATR in human cells) is involved in the checkpoint, and we shall verify and study this involvement, since it will (if confirmed) represent a totally new function of Rad3. The strategy for identification of interaction targets for the dif ferent proteins involved in the pathway will involve proteomics. We shall tag the respective proteins (Gcn2, Rad3, eIF2a) and try to identify their interaction partners by immunoprecipitation followed by mass spectrometry. This analysis will also involve the mapping of the actual phosphorylation sites.