Elucidating Mechanisms of Loss of Heterozygosity in Diploid Cells

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Project Summary Abstract Loss of heterozygosity (LOH) is the loss of the functional copy of a tumor suppressor gene in heterozygous individuals, leaving only the mutant copy, thereby contributing to cancer initiation and progression. Understanding how LOH arises in healthy cells is a vital component of cancer research, as it could lead to opportunities to prevent cancer in patients with increased early screening for individuals with mutations putting them at high risk for LOH, or with identification and targeting of cells that have undergone dangerous LOH. LOH can develop through deletions of whole chromosome arms or smaller regions of the genome, nondisjunction incidents, or homologous recombination events between homologous chromosomes (i.e., interhomolog homologous recombination, IH-HR). Despite decades of research, a critical need remains to identify the causes of LOH in normal or precancerous cells. Detecting these mechanisms in already established tumors is often difficult due to high levels of aneuploidy, ongoing chromosome instability, and DNA damage. We have recently developed a high throughput flow cytometry-based system which is sensitive for detecting LOH in normal diploid cells. Our objective is to exploit this system to identify and quantify mechanisms of LOH that arise from DNA double-strand breaks (DSBs), and the factors that modulate these events. We hypothesize that mechanisms of LOH which occur as a result of DSBs include copy number neutral LOH, as from IH-HR or non-disjunction associated with chromosome duplication and furthermore, that these events are modulated by the location of the DSB and DNA repair proteins, including proteins that act on recombination intermediates. We will address this hypothesis through the following specific aims: In Aim 1, we will investigate how proteins that act on recombination intermediates impact LOH arising from IH-HR, utilizing our flow-cytometry based system. We will assay the frequency of LOH that arises from IH-HR in the presence or absence of proteins that both prevent and contribute to crossover events, which can lead to long-range LOH. In Aim 2, we will take an unbiased approach to explore additional mechanisms of LOH arising from a DSB. We will determine how LOH mechanism is affected by the location of a DSB along a chromosome and by loss of different DSB repair pathways. This work will allow us to identify the factors that can contribute to or even prevent LOH, and give a better understanding of how these initiating events in cancer occur.