DNA replication is an essential and regulated cycle event required for the inheritance of genetic information. Every cell cycle, thousands of replication start sites (origins) must be selected and activated to ensure that the genome is copied exactly once within the confines of S-phase. Failure to properly regulate the DNA replication program may lead to catastrophic genomic instability. Our work in D. melanogaster and S. cerevisiae has identified chromatin architecture and ATP-dependent chromatin remodeling activities as important determinants of origin selection. We are proposing genetic, genomic and biochemical approaches to understand how the local chromatin architecture and ATP-dependent chromatin remodeling contribute to the selection and regulation of replication origins in yeast and Drosophila. Specifically, we will assess the interplay between chromatin dynamics (replication independent histone H3/H4 exchange) and origin selection and activation. We will also test the role of candidate ATP- dependent chromatin remodeling activities on origin selection and activation using comprehensive genomic approaches. We have recently developed a genome-wide 'footprinting'assay that will be used to address fundamental questions regarding origin architecture and the distribution of key replication initiation factors t individual origins. As part of our modENCODE studies, we identified multiple components of the NURF chromatin remodeling complex as having strong predictive value for ORC localization. We will test the role of NURF in specifying Drosophila origins of replication. By exploiting the experimental strengths of both yeast and Drosophila, we will generate important mechanistic insights into how the chromatin architecture specifies eukaryotic origins of replication.
Genomic instabilities commonly associated with tumorigenesis including chromosomal fragile sites, deletions, translocations, gene amplifications and regions of copy number variation are all likely intricately linked to the DNA replication program. We will identify chromatin-dependent mechanisms and factors that mediate the selection and regulation of individual origins of replication along the chromosome. An increased understanding of how individual origins are selected and regulated to maintain genomic stability will have important implications for developmental, stem cell and cancer biology.
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