The precise inheritance of genetic material in eukaryotes requires that initiation at each of the hundreds to thousands of replication origins be subject to exquisite regulation so that the DNA is duplicated exactly once per cell cycle. When replication control mechanisms go awry, genomic instability is predicted to occur, but the precise molecular consequences of deregulated replication for genome integrity are completely unknown. Additionally, the full battery of regulatory strategies that govern replication has not been defined. Cyclin dependent kinases (CDKs) are key molecular regulators that both stimulate initiation and inhibit re- initiation of DNA replication. To understand the molecular basis of genome integrity, it is essential to develop a more sophisticated understanding of these CDK-dependent regulatory events. Additionally, it is critical to analyze how disrupting this regulation affects faithful inheritance of the genome. To this end, we have developed several innovative tools that allow us to study the genesis and consequences of re-replication in the budding yeast Saccharomyces cerevisiae.
Our Specific Aims are as follows: (1) We have discovered that CDKs target polymerase alpha primase to block re-replication, which challenges the prevailing paradigm that the only strategy used by CDKs to inhibit re-replication is to prevent reassembly of a pre-replicative complex. Thus in this aim, we will characterize a novel replication control mechanism by investigating how CDKs inhibit polymerase alpha-primase to prevent re-replication. (2) We will continue to uncover new strategies for replication control by completing our ongoing screen to identify new CDK targets involved in either triggering initiation or preventing re-replication;this screen has already successfully identified five such targets, including polymerase alpha primase. (3) Using a robust copy number assay, we have obtained the first evidence that re-replication causes a heritable genetic change, namely a gene duplication event that represents an early step of gene amplification. We will exploit this unprecedented opportunity to examine the mechanisms by which re-replication promotes gene amplification. Because gene amplification is a primary means of activating oncogenes in cancer cells, these studies will shed light on the molecular triggers of tumorigenesis, and potentially identify targets of therapeutic significance.
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