MECHANISMS OF RAD9-MEDIATED CHECKPOINTS AND APOPTOSIS: Cell-cycle checkpoints play a critical role in the maintenance of genomic integrity by inhibiting progression through the cell cycle or initiating programmed cell death in the presence of damaged DNA or incomplete DNA replication. Studies in fission yeast implicate that members of the Rad family of checkpoint proteins including Radi, Rad3, Rad9, Radl7, Rad26, and Hus1 play important roles in the activation of DNA damage and replication checkpoints. We have reported that Rad9 can interact with Bcl-2 and Bcl-xL through a BH3-like region located in the amino terminus of the Rad9 protein, and can promote apoptosis in mammalian cells. DNA damage enhances Rad9 phosphorylation and induces Rad9 to move to the nuclear envelope, where it colocalizes with Bcl-2. In addition, our preliminary data indicate that c-Abl-mediated phosphorylation of Rad9 on Y28 induces increased association of Rad9 with BcI-xL and enhances the effect of Rad9 on apoptosis induction. Interestingly, the Rad9 protein is hyperphosphorylated and some form appears to be cell cycle-dependent. Moreover, we have recently found that Hus1 forms a protein complex with PCNA in human skin Flow2000 fibroblasts when DNA is damaged or replication is inhibited. Exposure of Flow2000 cells to 8 Gy ionizing radiation (that induces G2/M-arrest but not apoptosis) or hydroxyurea triggered translocation of Husi from the cytosol to the nucleus, where it colocalized with PCNA and Rad9. This nuclear translocation and the complex formation of Husl with PCNA correlate closely with changes in cell cycle distribution in response to radiation exposure. The goal of this proposal is to test the hypothesis that Rad9 is an important modulator of the DNA integrity checkpoint pathway determining whether a cell should transiently delay cell-cycle progression or die after DNA damage and that damage induced complex formation with a discrete set of cellular proteins plays a critical role in Rad9 function.
