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

基本信息

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
项目类别：
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

项目摘要

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 DNA 重复序列存在于整个基因组中，并且长度具有多态性。由于其遗传不稳定性，重复元件的突变率比其他地区高 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，重复不稳定的缺点以三联体为例。重复扩张会导致许多神经退行性疾病，例如亨廷顿病， Friedreich 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伤，其中之一就是 DNA 错配修复。 (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。 MMR 的规范功能被认为是三联体重复扩展丧失的原因。减弱三联体重复扩增，尽管这种非经典 MMR 活性的分子机制是然而，人们对 MMR 的这种诱变作用知之甚少。 MutLg)。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。 FAN1 功能的丧失会加剧重复扩增，表明 FAN1 在链间交联中发挥作用。我们对通过未知机制抑制三联体重复扩增感兴趣。导致 MMR 和 FAN1 的这些相反作用之间串扰的分子机制我们发现了一种新的 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、细胞和遗传学研究来进行扩展在目标 1 中，我们将阐明分子生物学。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。在目标 3 中，我们将使用无偏蛋白质组学方法来识别辅助因子。促进 FAN1 核酸酶在细胞环境中的功能，这些研究的完成将揭示这些途径。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。