Environmental Factors Influencing Minisatellite Stability in Yeast

描述（由申请人提供）：改变的小卫星DNA片段与许多人类疾病有关，包括HRAS 1相关癌症、进行性肌阵挛癫痫、胰岛素依赖型糖尿病、哮喘、溃疡性结肠炎甚至ADHD。不幸的是，我们对调节小卫星稳定性的环境和遗传因素知之甚少。由于缺乏强有力的分析系统，在确定重复小卫星DNA片段中引起改变的因素方面的进展已经放缓。我们最近在芽殖酵母S.酿酒酵母，容易检测道的变化，如菌落颜色和形态的变化，允许简单，快速筛选。重要的是，我们使用我们的测定来证明小卫星道的改变是由锌水平的扰动引起的，这表明我们的新测定是确定暴露于环境条件和化合物对小卫星稳定性的影响的理想手段。同样重要的是，锌稳态的破坏导致小卫星重排，但只有当细胞处于有丝分裂后的静止状态时。我们对有丝分裂后细胞中诱导基因重排的因素的理解是非常有限的，不像复制细胞的情况，其中大量的努力已经花费在确定在典型的细胞周期期间改变基因组的机制上。大多数人体细胞是静止的，它们的大部分寿命都处于这种状态。重要的是，产生癌细胞的初始致癌突变发生在这些静止细胞中。我们假设这些突变是由于修复因子未能识别或修复静止细胞中的DNA损伤所致。然而，突变诱导剂和DNA修复系统，这些代理激活的性质尚未确定在静止细胞。在静止细胞中鉴定这些环境和遗传因素对我们理解早期致癌事件是至关重要的。因此，为了解决所有这些重大的知识差距，我们将使用我们充分表征的测定系统结合快速高通量微生物学技术和全基因组分析来鉴定影响静止细胞中小卫星重复稳定性的化合物和环境条件。重要的是，我们的检测系统独特地允许我们区分活跃分裂细胞和有丝分裂后细胞中发生的事件，保证我们正在调查可能改变的完整谱。一旦我们确定了影响小卫星重复稳定性的条件或化合物，我们将筛选整个酵母基因组，以确定影响所需的所有基因。因此，该项目中描述的实验使我们能够识别影响最常见的重复DNA类型之一的环境条件和化合物，并确定介导效应的基因，这些数据将显著影响我们对癌症等多种疾病的理解，特别是初始致癌事件，癫痫和糖尿病。 公共卫生相关性：重复DNA片段是基因组不稳定性的主要来源;重复小卫星片段的改变与许多人类疾病的发生有关，包括癌症、癫痫和糖尿病。我们最近构建了一种新颖独特的小卫星不稳定性检测方法，该方法可检测活跃生长的酵母细胞和已停止生长并进入静止期（大多数人类细胞的状态）的细胞中发生的变化。我们将使用这种检测来确定所有的环境因素，影响小卫星的稳定性，在生长和静止的细胞，并确定基因控制的效果。

DESCRIPTION (provided by applicant): 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. PUBLIC HEALTH RELEVANCE: 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.