A novel cell cycle checkpoint in 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 and cancer development is frequent ly 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 delayed by UV light in a process that depends on the Gcn2 kinase. Therefore, we wish to identify all proteins that interact with Gcn2, both upstream and downstream. This kinase is known to phosphorylate the translation initiation factor eIF2a in response to nutrient limitation. We will find out whether eIF2a phosphorylation is a part of the UV-induced checkpoint response. The strategy for identification of interaction targets for the different 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. We shall also mo dify the Gcn2 kinase so that interaction partners can be more readily identified.