Crosstalk between DNA repair pathways in repeat instability

重复不稳定性中 DNA 修复途径之间的串扰

• 批准号: 10595243
• 负责人: Anna Pluciennik
• 金额: $ 31.2万
• 依托单位: THOMAS JEFFERSON UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-02-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10595243
• 关键词: Age of Onset; Animal Disease Models; Attenuated; Biochemical; CAG repeat; CGG repeat expansion; Cell Extracts; Cell model; Cell physiology; Chromosomal Rearrangement; Closure by clamp; DNA; DNA Damage; DNA Interstrand Cross-Link Repair; DNA Interstrand Crosslinking; DNA Repair; DNA Repair Pathway; DNA Sequence; DNA Structure; DNA replication fork; Data; Deoxyribonucleases; Dependence; Disease; Dissection; Elements; Enzymes; Equilibrium; Evaluation; Excision; Fragile X Syndrome; Friedreich Ataxia; Gene Expression Regulation; Genetic; Genetic Recombination; Genetic study; Genome; Genome Stability; Genomic Instability; Human; Human Genome; Huntington Disease; Ionic Strengths; Knock-out; Length; MLH1 gene; MSH2 gene; MSH3 gene; Maintenance; Malignant Neoplasms; Mediating; Metabolism; Mismatch Repair; Mismatch Repair Gene Inactivation; Molecular; Molecular Conformation; Musculoskeletal Diseases; Mutation; Myotonic Dystrophy; Neurodegenerative Disorders; Neurologic; Onset of illness; PMS1 gene; PMS2 gene; Pathway interactions; Patients; Physiological; Play; Prevention; Process; Production; Proliferating Cell Nuclear Antigen; Proteins; Proteomics; Replication Error; Role; Sequence Homologs; Slide; Structure; System; Trinucleotide Repeats; Work; causal variant; cofactor; environmental agent; gene repair; genome wide association study; human disease; insight; interest; nervous system disorder; novel; nuclease; prevent; protein protein interaction; protein purification; public health relevance; repair function; repaired; response; structural determinants

Project Summary/Abstract Approximately half the human genome is comprised of repetitive DNA sequences that are thought to control a wide range of cellular functions. DNA repeats are found throughout the genome, and are polymorphic in length due to their genetic instability. Mutation rates of repeat elements are 101-105 fold higher than in other parts of the genome, and is triggered by the formation of transient unusual DNA structures (extrahelical extrusions) during DNA metabolic processes. The detrimental consequences of repeat instability are exemplified by triplet repeat expansions that cause a number of neurodegenerative diseases such as Huntington’s disease, Friedreich’s ataxia and Fragile X related disorders. The rate of expansion of triplet repeats is proportional to repeat length and sequence homogeneity. DNA repair mechanisms have evolved to maintain genomic stability, and protect the DNA from damage caused by environmental agents. One such process is DNA mismatch repair (MMR), a highly conserved antimutagenic pathway that maintains the stability of the human genome by correcting replication errors and preventing chromosomal rearrangements. Unexpectedly, a mutagenic non- canonical function of MMR has been implicated as the cause of triplet repeat expansions. Loss of MMR function attenuates triplet repeat expansion, although the molecular mechanisms of this non-canonical MMR activity are poorly understood. However, this mutagenic action of MMR requires the proteins MutSb and MutLa (and possibly MutLg). FAN1 is a deoxyribonuclease that was originally identified as a factor involved in the repair of DNA interstrand crosslinks. Loss of FAN1 function exacerbates repeat expansion, suggesting a role for FAN1 in suppression of triplet repeat expansion by mechanisms that are not understood. We are interested in the molecular mechanisms responsible for the crosstalk between these opposing effects of MMR and FAN1 in the control of mutation production within triplet repeats. We have discovered a novel activator of the FAN1 nuclease on triplet repeat extrusions, a finding that represents the first step not only in our understanding of the mechanism of FAN1 action, but also in our quest to develop a unified understanding of the mechanism of triplet repeat expansion by integrating biochemical, cellular, and genetic studies. In Aim 1, we will elucidate the molecular features of FAN1 nuclease function by evaluating the modulatory effects of DNA sequence/structure and protein co-factors. In Aim 2, we will dissect the role of protein-DNA and protein-protein interactions in the repair of triplet repeat extrusions by FAN1. In Aim 3, we will use unbiased proteomic approaches to identify co-factors that facilitate FAN1 nuclease function in a cellular milieu. Completion of these studies will shed light on the pathways that modulate triplet repeat expansion, and will have implications more broadly for the mechanisms of genome instability.

项目概要/摘要 大约一半的人类基因组由重复的 DNA 序列组成，这些序列被认为控制着 广泛的细胞功能。 DNA 重复序列存在于整个基因组中，并且长度具有多态性 由于其遗传的不稳定性。重复元件的突变率比其他部分高101-105倍 基因组，并由瞬时异常 DNA 结构的形成（螺旋外挤压）触发 在DNA代谢过程中。重复不稳定的有害后果以三重态为例 重复扩张会导致许多神经退行性疾病，例如亨廷顿病， 弗里德赖希共济失调和脆性 X 相关疾病。三联体重复的扩增速率与 重复长度和序列同质性。 DNA修复机制已经进化以维持基因组稳定性， 并保护 DNA 免受环境因素造成的损害。 DNA 错配修复就是这样的过程之一 (MMR)，一种高度保守的抗突变途径，通过维持人类基因组的稳定性 纠正复制错误并防止染色体重排。出乎意料的是，一种非诱变剂 MMR 的规范功能被认为是三联体重复扩展的原因。 MMR 功能丧失 减弱三联体重复扩增，尽管这种非典型 MMR 活性的分子机制是 不太了解。然而，MMR 的这种诱变作用需要蛋白质 MutSb 和 MutLa（并且可能 穆特Lg）。 FAN1 是一种脱氧核糖核酸酶，最初被鉴定为参与 DNA 修复的因子 链间交联。 FAN1 功能的丧失会加剧重复扩增，这表明 FAN1 在 通过尚不清楚的机制抑制三联体重复扩增。我们感兴趣的是 导致 MMR 和 FAN1 的这些相反作用之间串扰的分子机制 控制三联体重复内的突变产生。我们发现了一种新型 FAN1 核酸酶激活剂 关于三重态重复​​挤压，这一发现不仅代表了我们理解该机制的第一步 FAN1 作用的过程，也是我们寻求对三联体重复机制的统一理解的过程 通过整合生化、细胞和遗传学研究进行扩展。在目标 1 中，我们将阐明分子 通过评估 DNA 序列/结构和蛋白质的调节作用来了解 FAN1 核酸酶功能的特征 辅助因素。在目标 2 中，我们将剖析蛋白质-DNA 和蛋白质-蛋白质相互作用在三联体修复中的作用 通过 FAN1 重复挤压。在目标 3 中，我们将使用无偏见的蛋白质组学方法来识别辅助因子， 促进细胞环境中 FAN1 核酸酶的功能。这些研究的完成将揭示途径 调节三联体重复扩增，并将对基因组机制产生更广泛的影响 不稳定。