The interaction of tumor suppressors and checkpoint pathways with the DNA replication apparatus of human cells is the subject of this application. We have recently identified many of the factors involved in replication initiation and begun to dissect how mis-regulation of these factors is monitored in normal cells and how loss of this surveillance leads to genomic instability. Acute or chronic mis-regulation in levels of replication initiation factors lead to up-regulation of tumor suppressor and checkpoint pathways leading to an arrest of the cell-cycle or of cell-death. Based on this we propose to test the hypothesis that low levels of the replication initiation factor ORC activate checkpoint enzymes like Chk2, Chkl and ATM/ATR and lead to an induction of the cell cycle inhibitor p27. The importance of this checkpoint pathway in maintaining genomic stability will be tested. Geminin interacts with the replication initiation factor Cdtl and inhibits DNA replication and the geminin-Cdtl balance is critical for preventing re-replication. We will test the hypothesis that differences in the checkpoint pathways that are activated following a disturbance in this balance and differences in the ultimate targets of these checkpoint pathways lead to different cell-cycle outcomes when the geminin-Cdtl balance is disturbed. Finally we will test the hypothesis that a geminin dimer has to interact with Cdtl through its coiled-coil domain to inhibit DNA replication. In addition we will test whether additional parts of the geminin molecule have to interact with Cdtl or other proteins to inhibit DNA replication. The last sub-aim will test whether chromatin-modifying activities of Cdtl are critical for its ability to stimulate DNA replication and whether geminin inhibits these activities.
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