Mechanisms of Genome Instability Mediated by Simple DNA Repeats

家长资助奖摘要 简单DNA重复序列的扩展与30多个遗传性神经疾病有关 以及人类的神经退行性疾病。数百个致因重复的副本可以是 只增加了几个代际传输。因此，理解这些机制 负责大规模重复扩增是极其重要的，具有广泛的生物医学意义 这意味着什么。我的实验室是第一个证明可扩展DNA重复阻止复制分叉的实验室 研究的每个实验系统的进展，包括细菌、酵母和哺乳动物 细胞。这导致我们建议在复制分叉从 “重复陷阱”。早期的重复扩张模式包括重复DNA链的滑动， 这通常是小规模的，在DNA复制期间。基于扩展的大小 在人类身上观察到的，我们认为一种不同的机制可能会导致 重复的大小。为了证实这一想法，我们开发了一个大规模的 在模式生物酿酒酵母中重复扩增。该系统揭示了 重复扩张，类似于在人类谱系中观察到的。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，当长度 超过Okazaki片段大小，接近重复扩展阈值 在人类身上。参与重复扩增的大多数基因似乎编码 复制或复制后修复机器。这些观察结果让我们勾勒出两个要点 基于DNA过程中模板切换的大规模重复扩增途径 复制，或中断诱导的复制。利用这些成就，我们计划 我们的研究有三个新的方向。首先，我们正在开发一种新的实验策略来 分析未分裂、按时间顺序老化的酵母细胞中的重复序列不稳定性。重复扩展 已知存在于有丝分裂后组织中，如大脑，据信它们对 对疾病发病机制的研究。因此，了解重复的遗传控制和机制 在非分裂细胞中的扩张对于理解这些疾病的病理生物学是非常宝贵的。 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。然后我们来看一下 在我们的酵母筛选中发现的候选基因对重复扩增的影响 在哺乳动物细胞中使用siRNA基因敲除。最后，虽然可扩展的 重复是决定疾病遗传的关键因素，最近的临床遗传学数据表明 反式修饰物的存在会影响重复扩张和疾病的可能性 进步。因此，我们将在全基因组范围内识别重复扩张的反式修饰物 在我们的酵母实验系统中。这种反式修饰物的鉴定可能非常 对于预测和遗传咨询很重要。

Abstract of the Parent Funded Award 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.