Most human tumors arise from multiple genomic changes which gradually transform differentiated and growth-limited cells into highly invasive cells that are unresponsive to growth controls. The genetic evolution of normal cells into cancer cells is largely determined by the fidelity of the DNA replication, repair and segregation. In recent years we have learned that cell cycle arrest in response to DNA damage is an important part of the mechanism used to maintain genome integrity. The control mechanism that restrains the onset of mitosis in response to DNA damage is known as the G2DNA damage checkpoint. The long-term objective of the research described in this proposal is to achieve a comprehensive understanding of the G2 DNA damage checkpoint. These studies will be carried out with the fission yeast Schizosaccharomyces pombe. This organism has served as an outstanding model system for the discovery and analysis of controls regulating the onset of mitosis. The G2-M controls are remarkably conserved between fission yeast and humans. Our limited understanding of checkpoint controls indicate an equally high degree of functional conservation between S. pombe and humans. Therefore the studies described in this application should provide a useful framework for the investigation of human checkpoint mechanisms which have a direct influence on the genomic events leading to cancer.
The aim of the project is to understand at a very basic level how the checkpoint control retains the onset of mitosis. The studies will seek to establish a direct link between checkpoint proteins and central elements of the G2-M control. The studies will involve analysis of Rad3 and Chk1, two checkpoint kinases; Cdc25 and Pyp3, two tyrosine phosphatases that activates Cdc2, the major cyclin-dependent kinase that induces mitosis; and Weel and Mik1, two tyrosine kinases that phosphorylate and thereby inhibit Cdc2. Genetic and biochemical experiments will test the hypothesis that Cdc25 and at least one other regulator of Cdc2 are themselves regulated by Chk1 kinase. Genetic screens will also be carried out to identify novel checkpoint genes. The long-term aim is to achieve a comprehensive understanding of the G2DNA damage checkpoint in fission yeast and to facilitate the translation of these insights into fundamental new information about how genome integrity is maintained in human cells.
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