DNA REPLICATION CHECKPOINT

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Eukaryotic cells use cell cycle checkpoints to ensure that mitosis is delayed until chromosomes are completely replicated and repaired. Checkpoint mutants in yeast and mammalian cells are hypersensitive to agents that inhibit DNA synthesis or damage DNA. Checkpoint mutants in mammalian cells are also prone to rearrangement, fragmentation or loss of chromosomes, events that are often causally associated with cancer and other diseases. The aim of this project is to understand at a very basic level the checkpoint events that are engaged when DNA replication is slowed or halted by the drug hydroxyurea (HU). This replication or HU checkpoint prevents the onset of mitosis. The studies will be carried out with the fission yeast Schizosaccharomyces pombe. This organism has served as an outstanding model system for the investigation of cell cycle and checkpoint controls that are generally conserved amongst most species, including humans. The investigations will focus on Cds1, a protein kinase that appears to play a central role in the replication checkpoint. The project has three specific aims. The first aim is to understand how Cds1 helps to enforce the replication checkpoint. Proteins that regulate the onset of mitosis will be evaluated as targets of Cds1. The second aim is to understand how Cds1 is regulated. Mechanisms that activate Cds1 and cause it to phosphorylate its substrates will be investigated. The third aim is to uncover the mechanism that restricts nuclear localization of Cds1 to the DNA synthesis (S) phase of the cell cycle. These studies will improve the understanding of Cds1 as an important element of checkpoint mechanisms in fission yeast and will provide a framework for investigating the functions of homologous proteins in humans.