Mechanisms of Genome Instability Mediated by Simple DNA Repeats

项目摘要 简单DNA重复序列的扩展与30多种遗传性神经和 人类的神经退行性疾病。数百个使役重复的副本可以在 几代人之间的遗传。因此，了解大规模气候变化的机制 重复扩张是极其重要的，具有广泛的生物医学意义。 我的实验室是第一个证明可扩展的DNA重复阻止复制分叉进展的 所研究的实验体系包括细菌、酵母和哺乳动物细胞。这导致我们提出了 当复制分叉从“重复陷阱”逃脱时，可以添加重复。早期的重复模式 扩张包括在DNA过程中重复的DNA链的滑动，这通常是小规模的 复制。基于在人类身上观察到的扩张的大小，我们认为一种不同的机制 可能会导致重复生物体型的大幅跳跃。为了证实这一想法，我们开发了一种试验性的 在模式生物酿酒酵母中大规模重复扩增的系统。这个系统被揭开了 重复扩张的特征类似于在人类家系中观察到的特征。扩张的速度 随着它们的长度呈指数增长。重复扩展变得很明显，当 Repeat超过Okazaki片段大小，接近人类的Repeat扩展阈值。 大多数参与重复扩增的基因似乎编码复制或后复制的蛋白质. 复制修复机器。这些观察使我们勾勒出了大规模重复的两条途径 基于DNA复制过程中的模板切换或中断诱导的复制的扩展。 利用这些成果，我们计划将我们的研究推向三个新方向。首先，我们 正在开发一种新的实验策略来按时间顺序分析非除法中的重复不稳定性 老化的酵母细胞。已知重复扩张发生在有丝分裂后组织，如大脑，以及 它们被认为与疾病的发病机制有关。因此，了解遗传控制和 在非分裂细胞中重复扩张的机制对于理解非分裂细胞的病理生物学是非常有价值的。 这些疾病。其次，我们正致力于建立一种易于遗传处理的系统来分析 培养的哺乳动物细胞大规模重复扩增的机制。然后我们再来看看效果。 在我们的酵母筛选中发现的候选基因在哺乳动物细胞中的重复扩增 利用siRNA基因敲除。最后，虽然可扩展重复的长度是关键因素 最近的临床遗传学数据表明，决定疾病遗传的反式修饰物的存在 会影响重复扩张和疾病进展的可能性。因此，我们将确定反式- 在我们的酵母实验系统中，在全基因组水平上的重复扩展的修饰物。身份识别 这种反式修饰物对于预测预后和遗传咨询可能是非常重要的。

Project Summary Expansions of simple DNA repeats are implicated in more than thirty hereditary neurological and neurodegenerative disorders in humans. Hundreds of copies of the causative repeat can be added in just a few intergenerational transmissions. Thus, understanding the mechanisms responsible for large-scale repeat expansions is extremely important and has broad biomedical implications. My lab was the first to show that expandable DNA repeats stall replication fork progression in every experimental system studied, including bacteria, yeast and mammalian cells. This led us to propose that repeats can be added while the replication fork escapes from a “repetitive trap”. Early models of repeat expansion involved slippage of repetitive DNA strands, which is normally small-scale, during DNA replication. Based on the size of expansions observed in humans, we believe that a distinct mechanism could cause large jumps in the repeat’s size. To substantiate this idea, we developed an experimental system for large-scale repeat expansions in a model organism, S. cerevisiae. This system uncovered features of repeat expansions similar to that observed in human pedigrees. The rate of expansions increased exponentially with their lengths. Repeat expansions become evident, when the length of a repeat exceeds the Okazaki fragment size, which is close to the repeat expansion threshold in humans. The majority of genes involved in repeat expansions appear to encode proteins of the replication or post- replication repair machineries. These observations led us to outline two pathways for large-scale repeat expansions based on either template-switching during DNA replication, or break-induced replication. Capitalizing on these achievements, we plan to move our research in three new directions. First, we are developing a novel experimental strategy to analyze repeat instability in non-dividing, chronologically aging yeast cells. Repeat expansions are known to occur in post-mitotic tissues, such as the brain, and they are believed to contribute to disease pathogenesis. Thus, understanding the genetic controls and mechanisms of repeat expansions in non-dividing cells is invaluable for understanding the pathobiology of these diseases. Second, we are working on establishing a genetically tractable system to analyze the mechanisms of large-scale repeat expansions in cultured mammalian cells. We will then look at the effect of candidate genes, which were identified in our yeast screens, on repeat expansions in mammalian cells using siRNA gene knockdown. Finally, while the length of an expandable repeat is the key factor determining disease inheritance, recent clinical genetics data point to the existence of trans-modifiers that can affect the likelihood of repeat expansions and disease progression. We will, therefore, identify trans- modifiers of repeat expansion at the genome-wide level in our yeast experimental system. Identification of such trans-modifiers is potentially very important for prognostic purposes and genetic counseling.