Single Stranded DNA: The Genome's Achilles Heel

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DESCRIPTION (provided by applicant): Chromosomal DNA replication is a highly choreographed process that must take place in an orderly and timely fashion to secure the accurate transmission of genetic information. My general objectives are to understand the mechanisms by which defects in chromosomal DNA replication give rise to genomic instability and disease development. This research plan is centered around exploring the hypothesis that single-stranded DNA (ssDNA) production upon replication fork stalling and destabilization in replication checkpoint-deficient mutants is a detrimental event that prevents complete synthesis of the genome, causes chromosomal breakage and genomic instability. I base this hypothesis on the following observations: (1) Saccharomyces cerevisiae replication checkpoint-deficient rad53 mutant cells contain stalled replication forks and accumulate large regions of ssDNA upon treatment with the genotoxic agent hydroxyurea (HU); (2) the ability of rad53 cells to recover from transient exposure to HU decreases as the duration of exposure increases; (3) viability of these mutant cells after transient exposure to HU cannot be rescued by simply delaying mitosis; (4) rad53 cells, upon removal of HU, are able to synthesize much of their genomic DNA, but certain regions of the genome are under-replicated. In order to test this hypothesis, I devised three specific aims to: (1) Investigate the consequence of replication fork instability using rad53 cells in HU through analyzing replication dynamics, ssDNA production, and chromosomal integrity by microarray-based density transfer, microarray-based genomic ssDNA labeling, and pulse field gel electrophoresis, respectively. (2) Elucidate the mechanism and the consequence of replication fork instability by conducting a genetic screen to isolate multi-copy suppressors of rad53 lethality after transient exposure to HU. (3) Adapt a genomic ssDNA mapping technique that I previously developed in yeast to (a) map origins of replication in human cells; (b) identify chromosomal fragile sites and map potential sites of genomic rearrangement that may lead to disease development in human cells. This comprehensive research plan will enable us to gain broader understanding of how defects in eukaryotic DNA replication give rise to genomic instability and human diseases such as cancer. In the long term, I hope that this research will aid in promoting the development of diagnostic and treatment tools for human diseases.