Causes and Consequences of Genomic Instability at Fragile Sites

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DESCRIPTION (provided by applicant): 6. PROJECT SUMMARY Chromosomes in cancer cells contain changes that cause improper cell growth. When a cell can't correctly repair breaks in its DNA or has difficulty during the process of copying its DNA, genetic changes can result. When tumor cells with these changes divide and reproduce, they pass these genetic changes on to their daughter cells, eventually creating a metastasized tumor. Some regions of chromosomes are more prone to genetic changes than others. Many types of tumors have been shown to contain abnormalities at regions called "fragile sites." Fragile sites in human cells are normally stable, but when the process of copying DNA is slowed or stalled, these sites are hotspots for breaks. Yeast cells also contain fragile sites, and we will use the powerful genetic tools of the yeast model system to design experiments that would be technically difficult in human cells. In the first aim of this proposal, we will study the fidelity and extent of DNA replication that occurs when breaks are repaired during mitosis by homologous recombination. Abnormal cell growth in tumors is often caused by amplification or deletion of genes involved in cell growth and DNA repair. Thus, in the second aim we will study variations in copy number stimulated by breaks at a yeast fragile site, using a system that allows us to select for yeast that have lost or gained copies of genes that enable it to grow on media containing copper and formaldehyde. Because yeast fragile sites have a simpler structure than human ones, in the third aim, we will study the process of DNA replication in human fragile site sequences that are carried on yeast chromosomes. The experiments in this aim will test hypotheses about the models that have been proposed to explain why breaks form in human fragile sites. These experiments to study DNA damage at fragile sites and repair at stalled replication forks will help us understand the situations and environments that promote genetic changes in tumor cells.