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: 10116680
• 负责人: SERGEI MIRKIN
• 金额: $ 7.86万
• 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/10116680
• 关键词: Achievement; Affect; Aging; Animal Model; Award; Bacteria; Brain; Candidate Disease Gene; Cells; Chronology; DNA; DNA biosynthesis; DNA replication fork; Data; Disease; Disease Progression; Familial Amyotrophic Lateral Sclerosis; Fragile X Syndrome; Friedreich Ataxia; Funding; 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; Parents; Pathogenesis; Pathway interactions; Proteins; Research; Saccharomyces cerevisiae; Small Interfering RNA; System; Tissues; Yeasts; base; frontotemporal lobar dementia-amyotrophic lateral sclerosis; genetic pedigree; genome-wide; intergenerational; knock-down; nervous system disorder; novel; prognostic; repaired; transmission process

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.

家长资助奖摘要 简单 DNA 重复的扩展与 30 多种遗传性神经系统疾病有关 以及人类的神经退行性疾病。 添加了一些代际传递，从而了解了其中的机制。 负责大规模重复扩增极其重要并且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 重复序列会阻碍复制叉的实验室。 研究的每个实验系统的进展，包括细菌、酵母和哺乳动物 这使我们提出，当复制叉从细胞中逃脱时，可以添加重复序列。 “重复陷阱”。重复扩张的早期模型涉及重复 DNA 链的滑动， 在 DNA 复制过程中，通常是小规模的，基于扩展的大小。 在人类中观察到，我们相信一种独特的机制可能会导致 为了证实这个想法，我们开发了一个大规模的实验系统。 在模型生物酿酒酵母中重复扩增该系统揭示了 重复扩张与人类谱系中观察到的扩张速率相似。 当长度增加时，重复扩展变得明显。 重复次数超过冈崎片段大小，接近重复扩展阈值 在人类中，大多数参与重复扩增的基因似乎编码以下蛋白质： 复制或复制后修复机制 这些观察使我们概述了两个。 基于 DNA 过程中任一模板转换的大规模重复扩增途径 复制，或中断引起的复制 利用这些成就，我们计划采取行动。 首先，我们正在开发一种新颖的实验策略来研究三个新方向。 分析非分裂、按时间顺序老化的酵母细胞的重复扩增的不稳定性。 已知发生在有丝分裂后组织中，例如大脑，并且它们被认为有助于 因此，了解重复的遗传控制和机制。 非分裂细胞的扩增对于理解这些细胞的病理学非常有价值 其次，我们正在努力建立一个基因可处理的系统来分析疾病。 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对重复扩增的影响 最后在哺乳动物细胞中使用siRNA基因敲除，同时长度可扩展。 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增和疾病的可能性 因此，我们将在全基因组范围内识别重复扩增的反式修饰因子。 在我们的酵母实验系统中鉴定此类反式修饰剂的水平可能非常高。 对于预后目的和遗传咨询很重要。