The broad long-term goal of this project is to understand mechanisms that restrict DNA replication to once-per- cell cycle. Our preliminary studies have identified checkpoint signaling pathways that prevent DNA re-replication. We have termed these pathways 're-replication checkpoints'. Re-replication checkpoints ensure that only one round of DNA synthesis occurs during S-phase and represent a fundamental mechanism for maintaining genomic stability. The 'Specific Aims' of this project are: (1) To test the hypothesis that the Rad9-Radl-Husl (9-1-1) checkpoint protein complex prevents DNA re-replication. (2) To test the hypothesis that perturbation of re-replication checkpoints promotes gene amplification. We will study the effect of 9-1-1 perturbation on re-replication using several complementary approaches. These include: over-expressing the 9-1-1 antagonist, Husl B, RNA interference experiments targeting Rad9 using a novel adenovirus vector we constructed for gene silencing, and studying cell lines from transgenic Husl-null animals. The putative mechanism by which 9-1-1 prevents re-replication will be determined using biochemical approaches including studying 9-1-1-mediated signaling pathways and putative interactions between 9-1-1 and DNA replication factors. Acquisition of methotrexate resistance will be used as an assay to study amplification of a defined genomic locus (the DHFR gene). We will test the effects of perturbing the re-replication checkpoint on DHFR amplification. Microarray-based Comparative Genomic Hybridization (CGH) will be used to identify re-replicating genomic loci in 9-1-1-deficient cells. Many human oncogenes are over-expressed in cancer cells as a result of gene amplification. Therefore, gene amplification is a hallmark of cancer cells and contributes to tumor progression. Additionally, genes amplified in tumor cells can confer resistance to chemotherapies. Therefore, an understanding of factors that pre-dispose to gene amplification will be of prognostic and therapeutic value in the clinic. If gene amplification in tumors results from loss of re-replication checkpoints, these checkpoints could be exploited using drugs or gene therapies. The results of our studies may provide a novel paradigm for mechanisms of gene amplification and will be directly relevant to tumorigenesis in humans.
