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.
Abnormal cell growth in cancer cells results from genetic changes, such as amplifications, deletions, and mutations, that alter gene function. These genetic changes can be stimulated by stress during replication, the process of copying a cell's DNA. Replication stress, in which the copying process is slowed or stalled, causes breaks at particular hotspots known as fragile sites, and many tumors have genetic changes at fragile sites. This proposal uses the yeast model system to investigate the fidelity of repair processes at fragile sites, analyze the contribution of fragile site instability to amplifications and deletions, and tet hypotheses about why fragile sites are unstable during replication stress. The results of these studies will help us understand how and why genetic changes arise in tumor cells.