Developmental regulation of DNA replication during programmed gene ampplification

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DESCRIPTION (provided by applicant): Genome instability, a hallmark of cancer, can be triggered by incomplete replication or partial re-replication of chromosomes. In addition, the spontaneous amplification of proto-oncogenes is commonly associated with tumorigenesis. The model eukaryotes, Drosophila and Tetrahymena, provide unique opportunities to study gene amplification because these events are developmentally programmed. While the DMA sequences requirements for replication initiation are poorly understood in humans, cis-acting determinants have been genetically defined in the Drosophila chorion locus and Tetrahymena ribosomal DMA (rDNA) replicons/amplicons. In both instances, gene amplification is dependent on initiator proteins that normally restrict replication to once per cell cycle. How these amplicons escape cell cycle control is not known. The goal of this proposal is to develop new approaches to understand why specific replication origins re-initiate and amplify DMA when other origins are properly regulated. Prior efforts have focused on the 'amplicons' themselves. The goal of this sabbatical proposal is to develop tools to study the silenced chromosomal origins. The bulk of the research will employ the Tetrahymena model, adapting approaches developed in the mentor's lab to study DNA replication in Drosophila. The applicant will also gain the skills required to use Drosophila as a model organism. He will also receive training in a mammalian replication lab that developed a single molecule approach to study replication initiation. The goal is to master cutting edge technologies that will enhance the investigator's research program at his home institution. Several distinct, but complementary experimental approaches will be pursued to open up new avenues of investigation. The specific aims are: (1) To acquire new skills to study DNA replication during Tetrahymena development, specifically, real-time nascent strand PCR, and single molecule analysis of replicating DNA (SMARD). (2) To develop immunocytological approaches to visualize the sub-nuclear localization of replication proteins during three specific stages of Tetrahymena development- endoreplication phase 1, gene amplification, and endo replication phase 2. (3) To learn and use a real-time PCR chromatin immunoprecipitation assay to examine the temporal association of replication proteins with amplified and non-amplified origins during development. (4) To obtain expertise working with Drosophila to employ it as a new model system. Replication origins that escape cell cycle control have profound destabilizing effects on chromosome stability. Understanding how the safeguards that prevent re-replication are over-ridden in genetically tractable model organisms can enhance our understanding of genome instability in human cancer cells.