Altered minisatellite DNA tracts have been linked to many human diseases, including HRAS1-related cancers, progressive myoclonus epilepsy, insulin-dependent diabetes mellitus, asthma, ulcerative colitis and even ADHD. Unfortunately very little is known about the factors, both environmental and genetic, that regulate minisatellite stability. Progress in identifying the factors that cause alterations in repetitive minisatellite DNA tracts has been slowed by the lack of strong assay systems. We recently developed a robust assay system in the budding yeast S. cerevisiae that readily detects tract alterations as changes in colony color and morphology, allowing for simple, rapid screening. Importantly, we used our assay to demonstrate that minisatellite tract alterations result from perturbations in the level of zinc, demonstrating that our novel assay is an ideal means to determine the effect of exposure to environmental conditions and compounds on minisatellite stability. Equally importantly, disruption of zinc homeostasis causes minisatellite rearrangements, but only when the cells are in a post-mitotic, quiescent state. Our understanding of the factors inducing genetic rearrangement in post-mitotic cells is extremely limited, unlike the situation with replicating cells, where a large amount of effort has been expended on determining the mechanisms that alter the genome during a typical cell cycle. The majority of human cells are quiescent, spending most of their lifespan in that state. Importantly, the initial oncogenic mutations that generate a cancer cell occur in these quiescent cells. We hypothesize that these mutations result from a failure of repair factors to identify or repair DNA damage in the quiescent cell. However, the nature of the mutation-inducing agents and the DNA repair systems these agents activate have not been identified in quiescent cells. Identification of these environmental and genetic factors in quiescent cells is vital to our understanding of early oncogenic events. Therefore, to address all of these significant knowledge gaps, we will identify compounds and environmental conditions that influence minisatellite repeat stability in quiescent cells, using our well-characterized assay system in combination with rapid high- throughput microbiological techniques and whole-genome analysis. Importantly, our assay system uniquely allows us to differentiate between events occurring in actively dividing cells and in post-mitotic cells, guaranteeing that we are surveying the complete spectrum of possible alterations. Once we have identified conditions or compounds that affect minisatellite repeat stability, we will screen the entire yeast genome to identify all of the genes required for the effect. Therefore, the experiments described in this project allow us to identify environmental conditions and compounds that affect one of the most common repetitive DNA types and to determine the genes mediating the effects, data that will significantly impact our understanding of such diverse diseases as cancers, especially initial oncogenic events, epilepsy, and diabetes.
Repetitive DNA tracts are a primary source of genome instability;alterations in repetitive minisatellite tracts have been associated with the onset of many human diseases, including cancers, epilepsy and diabetes. We recently constructed a novel and unique assay for minisatellite instability that detects alterations occurring in both actively-growing yeast cells and cells that have ceased growing and entered stationary phase (the state for most human cells). We will use this assay to identify all of the environmental factors that influence minisatellite stability in growing and stationary cells, and identify the genes that control the effect.