Mechanisms of Genome Instability Mediated by Simple DNA Repeats

简单 DNA 重复介导的基因组不稳定性机制

• 批准号: 10116422
• 负责人: SERGEI MIRKIN
• 金额: $ 54.56万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-03-14 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10116422
• 关键词: Achievement; Affect; Aging; Animal Model; Bacteria; Brain; Candidate Disease Gene; Cells; Chronology; DNA; DNA biosynthesis; DNA replication fork; Data; Disease; Disease Progression; Fragile X Syndrome; Friedreich Ataxia; Genes; Genetic; Genetic Counseling; Genomic Instability; Hereditary Disease; Human; Huntington Disease; Inherited; Interphase Cell; Length; Mammalian Cell; Mediating; Medical Genetics; Mitotic; Modeling; Myotonic Dystrophy; Neurodegenerative Disorders; Okazaki fragments; Pathogenesis; Pathway interactions; Proteins; Research; Saccharomyces cerevisiae; Small Interfering RNA; System; Tissues; Yeasts; base; familial amyotrophic lateral sclerosis; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genetic pedigree; genome-wide; intergenerational; knock-down; nervous system disorder; novel; prognostic; repaired; transmission process

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.

项目概要 简单 DNA 重复的扩展与 30 多种遗传性神经系统和疾病有关 人类的神经退行性疾病只需添加数百个致病重复副本即可。 因此，了解大规模的代际传递机制。 重复扩增极其重要，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 DNA 重复序列会阻碍每个细胞的复制叉进程。 研究的实验系统包括细菌、酵母和哺乳动物细胞，这导致我们提出这一点。 当复制叉逃离“重复陷阱”时，可以添加重复序列。 扩张涉及重复DNA链的滑移，通常是小规模的，在DNA复制过程中 根据在人类中观察到的扩增大小，我们认为存在一种独特的机制。 为了证实这个想法，我们开发了一个实验。 该系统在模式生物酿酒酵母中进行大规模重复扩增。 重复扩张的特征与人类谱系中观察到的相似。 当 a 的长度增加时，重复扩展变得明显。 重复序列超过冈崎片段大小，接近人类重复扩展阈值。 大多数参与重复扩增的基因似乎编码复制或复制后的蛋白质。 这些观察使我们概述了大规模重复的两条途径。 基于 DNA 复制过程中的模板转换或断裂诱导的复制的扩展。 利用这些成果，我们计划将我们的研究转向三个新的方向。 正在开发一种新的实验策略来分析非分裂、按时间顺序的重复不稳定性 已知老化的酵母细胞会在有丝分裂后的组织中发生重复扩增，例如大脑和 它们被认为有助于疾病的发病机制，因此，了解遗传控制和 非分裂细胞重复扩增的机制对于理解非分裂细胞的病理生物学非常有价值 其次，我们正在努力建立一个可遗传的系统来分析这些疾病。 然后我们将研究培养的哺乳动物细胞中大规模重复扩增的机制。 在我们的酵母筛选中鉴定出的候选基因在哺乳动物细胞中重复扩增 最后，可扩展重复序列的长度是关键因素。 在确定疾病遗传方面，最近的临床遗传学数据表明存在反式修饰因子， 因此，我们将确定反式扩增的可能性。 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的鉴定。 这种反式修饰因子对于预后目的和遗传咨询可能非常重要。