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.

项目摘要/摘要 大约一半的人类基因组由重复的DNA序列组成，这些DNA序列被认为控制着 广泛的细胞功能。DNA重复序列在基因组中随处可见，并且在长度上具有多态 由于它们的遗传不稳定。重复元件的突变率是其他部分的101-105倍 基因组，由瞬间不寻常的DNA结构(螺旋外挤出)的形成触发 在DNA新陈代谢过程中。重复不稳定性的有害后果以三胞胎为例。 反复扩张会导致许多神经退行性疾病，如亨廷顿氏病， 弗里德里希共济失调和脆性X相关疾病。三联体重复序列的扩张率与 重复长度和序列同质性。DNA修复机制的进化是为了维持基因组的稳定性， 并保护DNA免受环境因素的破坏。其中一个过程是DNA错配修复 (MMR)，一种高度保守的抗突变途径，通过以下方式维持人类基因组的稳定性 纠正复制错误和防止染色体重排。出乎意料的是，一种诱变的非 MMR的正则功能被认为是三联体重复扩增的原因。MMR功能丧失 减弱三联体重复序列的扩展，尽管这种非规范MMR活性的分子机制是 人们对此知之甚少。然而，MMR的这种突变作用需要蛋白质MutSb和Mutla(可能还需要 MutLg)。FAN1是一种脱氧核糖核酸酶，最初被认为是参与DNA修复的因子 链间交联剂。FAN1功能的丧失加剧了重复扩张，提示FAN1在 通过不了解的机制抑制三联体重复扩增。我们感兴趣的是 MMR和FAN1的这些相反效应之间产生串扰的分子机制 控制三联体重复内的突变产生。我们发现了一种新的FAN1核酸酶激活剂 关于三联体重复挤出，这一发现不仅代表了我们对机制的理解的第一步 FAN1的作用，也是在寻求对三联体重复机制的统一理解 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 用DNA序列/结构和蛋白质的调控效应评价FAN1核酸酶的功能特征 共同因素。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。 按扇形1重复拉伸。在目标3中，我们将使用无偏见的蛋白质组学方法来确定 在细胞环境中促进FAN1核酸酶的功能。这些研究的完成将有助于揭示这些途径 这调节了三联体重复的扩展，并将对基因组的机制产生更广泛的影响 不稳定。