Cancer therapies frequently rely on drugs or agents that trigger genomic instability by taking advantage of the fact that cancer cells have defects in the response to DNA damage and/or in the coordination of centrosome duplication with DNA replication. Checkpoints are biochemical pathways that provide the cell with mechanisms to detect DNA damage and respond by arresting the cell cycle to allow DNA repair. Thus, checkpoint pathways are being tested for their value as therapeutic targets. The conserved checkpoint kinase Chk1 regulates mitotic progression in response to ionizing radiation, other forms of DNA damage and replication interference. Although it has been postulated that the checkpoint pathways play a role in DNA repair, the role of checkpoint proteins, in DNA metabolism remains unknown. Chk1 is phosphorylated following a checkpoint signal, a process that is regulated by the kinases Atr and Atm. We have shown that Chk1, like Atr, is associated with chromatin in cycling cells and that the chromatin-associated Chkl is phosphorylated in the absence of exogenous DNA damage. The phosphorylated form of Chk1 localizes to chromatin, centrosomes and sites of DNA replication. Our findings support the hypothesis that Chk1 has a role(s) in an intrinsic checkpoint at every cell cycle. Furthermore, Chk1 interacts both in vitro and in vivo with two enzymes involved in DNA replication, repair and chromosome topology and with two proteins that localize to centrosomes. Chk1 phosphorylated three of these proteins in vitro. These findings provide evidence for a connection between the checkpoint pathways and DNA replication and/or repair and are consistent with a role for Chk1 in the checkpoint that monitors integrity of replication forks. The central hypothesis underlying the work proposed here is that phosphorylation and interactions between Chk1 and chromatin- or centrosome-associated proteins play a role in DNA repair and/or chromosome stability. Thus, one of the long-term goals of these studies is to pinpoint the region of these proteins that is involved in the interaction with Chk1. This information could be used to identify agents that block the interaction with the checkpoint and that enhance the effect of therapeutic drugs that cause damage to DNA. The studies proposed in aims I and II will address the physiological role of Chk1 in monitoring or regulating DNA replication, repair, decatenation and chromosome segregation.
In aim III we will address the physiological significance of both the interaction of Chk1 with centrosomal proteins and the localization of Chk1 to centrosomes.
