Mechanisms of Genome Instability Mediated by Simple DNA Repeats

项目摘要 简单DNA重复序列的扩增与30多种遗传性神经系统和 人类的神经退行性疾病。几百个使役重复的拷贝可以在短短的时间里被添加进去 几代人之间的遗传因此，了解负责大规模 重复扩增是极其重要的，并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重复序列在每个细胞中都能阻止复制叉的进展的实验室。 实验系统研究，包括细菌，酵母和哺乳动物细胞。这使我们提出， 当复制叉从“重复陷阱”中逃脱时，可以添加重复。早期的重复模型 扩增涉及重复DNA链的滑动，这通常是小规模的，在DNA扩增过程中， 复制的根据在人类中观察到的扩张大小，我们认为有一种独特的机制 可能会导致重复序列大小的大幅跳跃。为了证实这个想法，我们开发了一个实验性的 系统的大规模重复扩增的模式生物，S。啤酒。该系统揭示了 重复扩增的特征与在人类谱系中观察到的相似。膨胀率 随着它们的长度呈指数增长重复扩张变得明显，当一个 重复超过冈崎片段的大小，这是接近人类的重复扩增阈值。 参与重复扩增的大多数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大规模重复的两条途径 基于DNA复制期间的模板转换或断裂诱导的复制的扩增。 利用这些成就，我们计划将我们的研究推向三个新的方向。一是 正在开发一种新的实验策略来分析非分裂，按时间顺序重复的不稳定性， 老化的酵母细胞已知重复扩增发生在有丝分裂后的组织中，例如脑，并且 它们被认为有助于疾病的发病机理。因此，了解遗传控制和 在非分裂细胞中重复扩增的机制对于理解 这些疾病。第二，我们正在建立一个遗传上易于处理的系统来分析 在培养的哺乳动物细胞中的大规模重复扩增的机制。然后我们再看看 候选基因，这是在我们的酵母筛选中确定的，在哺乳动物细胞中重复扩增 使用siRNA基因敲除。最后，虽然可扩展重复序列的长度是关键因素， 确定疾病遗传，最近的临床遗传学数据指出存在反式修饰物， 会影响重复扩张和疾病进展的可能性。因此，我们将确定跨- 在我们的酵母实验系统中，在全基因组水平上重复扩增的修饰剂。鉴定 这种反式修饰物对于预后目的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.