Environmental Factors Influencing Minisatellite Stability in Yeast

描述(申请人提供)：改变的小卫星DNA束与许多人类疾病有关，包括HRAS1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，人们对控制小卫星稳定性的环境和遗传因素知之甚少。由于缺乏强大的检测系统，在鉴定引起重复小卫星DNA链改变的因素方面进展缓慢。我们最近在萌芽酵母中开发了一个强大的检测系统，该系统可以很容易地检测菌落颜色和形态的变化，从而实现简单、快速的筛选。重要的是，我们使用我们的检测方法证明了小卫星轨道的改变是由锌水平的扰动引起的，这表明我们的新检测方法是确定环境条件和化合物暴露对小卫星稳定性的影响的理想方法。同样重要的是，锌稳态的破坏会导致小卫星重排，但只有当细胞处于有丝分裂后的静止状态时才会发生。与复制细胞的情况不同，我们对诱导有丝分裂后细胞遗传重排的因素的了解极其有限，在复制细胞的情况下，人们花费了大量的努力来确定在典型的细胞周期中改变基因组的机制。大多数人类细胞处于静止状态，生命的大部分时间都在这种状态下度过。重要的是，产生癌细胞的最初致癌突变发生在这些静止的细胞中。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，在静止的细胞中，突变诱导剂的性质和这些诱变剂激活的DNA修复系统还没有被确定。在静止细胞中识别这些环境和遗传因素对于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，我们将使用我们功能完善的检测系统，结合快速高通量微生物学技术和全基因组分析，识别影响静止细胞中小卫星重复序列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中发生的事件和有丝分裂后细胞中发生的事件，从而确保我们正在调查可能变化的完整光谱。一旦我们确定了影响小卫星重复序列稳定性的条件或化合物，我们将对整个酵母基因组进行筛选，以确定该效应所需的所有基因。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，并确定介导这些影响的基因，这些数据将显著影响我们对癌症等各种疾病的理解，特别是最初的致癌事件、癫痫和糖尿病。 与公共卫生相关：重复的DNA链是基因组不稳定的主要来源；重复的小卫星链的变化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而独特的小卫星不稳定性检测方法，可以检测活跃生长的酵母细胞和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。我们将利用这项测试来确定所有影响小卫星在生长和静止细胞中稳定性的环境因素，并确定控制这种影响的基因。

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.