Mechanisms of Replication-Dependent Microsatellite Instability in Human Disease

人类疾病中复制依赖性微卫星不稳定性的机制

• 批准号: 10004155
• 负责人: Michael LEFFAK
• 金额: $ 30万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-08 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10004155
• 关键词: Address; Affect; Appearance; Binding Proteins; Biochemical; Biochemistry; Biological Assay; Bromodeoxyuridine; CAG repeat; Cell Line; Cells; Chromosomal Breaks; Chromosomal translocation; Chromosomes; Cleaved cell; Clustered Regularly Interspaced Short Palindromic Repeats; Complement; Coupled; DNA; DNA Damage; DNA Double Strand Break; DNA Repair Gene; DNA Repair Pathway; DNA Sequence; DNA Structure; DNA analysis; DNA biosynthesis; DNA replication fork; DNA sequencing; DNA-Directed DNA Polymerase; Dangerousness; Defect; Development; Event; Excision; Fanconi' s Anemia; Flow Cytometry; G-Quartets; Genes; Genetic Diseases; Genetic Recombination; Genome; Genomics; Goals; HSV-Tk Gene; Hela Cells; Human; Human Genome; Inverse Polymerase Chain Reaction; Knock-out; Knowledge; Label; Lead; Location; Maintenance; Malignant Neoplasms; Mass Spectrum Analysis; Measures; Microsatellite Instability; Microsatellite Repeats; Molecular; Mutagenesis; Mutate; Mutation; Nonhomologous DNA End Joining; Pathway interactions; Patients; Polymerase; Protein Deficiency; Proteins; Purines; Pyrimidine; Replication Origin; Role; Signal Transduction; Site; Small Interfering RNA; Source; Stains; Structure; Tertiary Protein Structure; Testing; Therapeutic; Time; Work; base; c-myc Genes; chromatin immunoprecipitation; chromosome mutation; chromothripsis; endonuclease; environmental agent; experimental study; helicase; human disease; inhibitor/antagonist; knock-down; mutant; nuclease; predicting response; prevent; repaired; replication stress; replicator; response; single molecule; targeted treatment; translational impact; triplex DNA

Chromosome breaks are the most dangerous form of DNA damage because they result in multiple types of mutations and gross chromosome rearrangements. DNA is most sensitive to breakage during replication, when hard-to-replicate noncanonical DNA structures cause replication fork stalling. Noncanonical DNA structures are strongly implicated as endogenous sources of chromosome breaks and translocations leading to developmental defects and cancers, however, the mechanisms by which replication fork stalling causes DNA double strand breaks (DSBs) are not known. Despite significant analyses of DNA damage response proteins in global or single molecule studies where the sites of damage are not identified, the molecular mechanisms of replication-dependent DNA strand breakage and repair at specific sites in human cells are incompletely understood. To address this knowledge gap, we will study two types of natural replication barriers (CTG/CAG trinucleotide repeats and asymmetric purine-pyrimidine (Pu/Py) mirror repeats) integrated at an ectopic site in the human genome where their structure and effect on replication can be manipulated. We also examine several endogenous replication fork barriers that induce DSBs during DNA replication. We will use PCR, DNA sequencing, chromatin immunoprecipitation, mass spectrometry and flow cytometry to show (1) how polymerase stalling at noncanonical DNA structures causes DSBs, (2) how DNA repair proteins act to remodel stalled replication forks to restart synthesis, and (3) the mechanisms and genomic consequences of DSB recombination at structure-induced fork barriers. We will test the hypothesis that noncanonical DNA structures induce DSB by blocking the progress of DNA polymerases, promoting nuclease-sensitive fork regression, and inhibiting DNA end processing required for recombination. Conceptual advances from this work will include determination of the molecular mechanisms of DSB formation near specific stalled forks, biochemical analysis of replication fork reversal, and identification of how the processing of structure-induced DSB differs that of nuclease-induced `clean' DSB. Our long-term goal is to define the role of DNA structure-induced g e n o m e instability in human disease. Aim 1 will disclose the relationship between fork stalling and damage signaling, the biochemistry of fork reversal, the function of structure-specific endonucleases at stalled forks, and the impact of DNA secondary structure on fork resection and repair. Aim 2 will build on our demonstration that the Fanconi anemia type J protein (FANCJ) is essential for the maintenance of noncanonical DNA structures across the genome during replication stress, to determine the mechanisms of FANCJ dependent microsatellite stabilization. In Aim 3 we will characterize the genomic consequences of FANCJ deficiency. Our experiments will show how hard-to- replicate DNA sequences cause chromosome breaks and mutations that lead to genetic disease.

染色体断裂是最危险的DNA损伤形式，因为它们会导致多种类型的 突变和总的染色体重排。DNA在复制过程中对断裂最敏感，当 难以复制的非规范DNA结构导致复制分叉停滞。非规范的DNA结构有 与导致染色体断裂和易位的内源性来源密切相关 然而，发育缺陷和癌症，复制分叉停滞导致DNA的机制 双链断裂(DSB)是未知的。 尽管在全球或单分子研究中对DNA损伤反应蛋白进行了重要的分析， 损伤的位置尚未确定，复制依赖的DNA链的分子机制 人类细胞中特定部位的断裂和修复还不完全清楚。要解决这一知识 GAP，我们将研究两种类型的自然复制障碍(CTG/CAG三核苷酸重复和不对称 嘌呤-嘧啶(Pu/Py)镜像重复序列)整合在人类基因组中的一个异位位置 复制的结构和效果是可以操纵的。我们还研究了几个内源复制分叉 在DNA复制过程中导致双链断裂的障碍。我们将使用聚合酶链式反应，DNA测序，染色质 免疫沉淀、质谱学和流式细胞术显示(1)聚合酶如何停滞在 非规范DNA结构导致DSB，(2)DNA修复蛋白如何作用于重塑停滞的复制 分支以重新开始合成，以及(3)DSB重组的机制和基因组后果 结构诱导的叉形障碍。 我们将检验这样一种假设，即非规范的DNA结构通过阻止DNA的前进来诱导DSB 聚合酶，促进核酸酶敏感的分叉回归，并抑制DNA末端处理所需的 重组。这项工作的概念性进展将包括确定 DSB在特定失速叉子附近的形成，复制叉子反转的生化分析，以及 结构诱导的DSB的处理与核酸酶诱导的“干净”DSB的处理有何不同。我们的长期目标 目的是确定DNA结构诱导的基因不稳定性在人类疾病中的作用。 目标1将揭示叉子失速与损伤信号的关系，叉子的生化 逆转，结构特异性内切酶在失速叉子上的功能，以及DNA次级的影响 叉子切除和修复上的结构。目标2将建立在我们的论证的基础上，即J型范可尼贫血 蛋白质(FANCJ)是维持整个基因组的非规范DNA结构所必需的 复制应激，以确定FANCJ依赖的微卫星稳定化的机制。在《目标3》中我们 将表征FANCJ缺乏症的基因组后果。我们的实验将证明- 复制DNA序列会导致染色体断裂和突变，从而导致遗传病。

项目成果

MutSβ promotes trinucleotide repeat expansion by recruiting DNA polymerase β to nascent (CAG)n or (CTG)n hairpins for error-prone DNA synthesis.

• DOI: 10.1038/cr.2016.66
• 发表时间: 2016-07
• 期刊: Cell research
• 影响因子: 44.1

Break-induced replication links microsatellite expansion to complex genome rearrangements.

断裂诱导的复制将微卫星扩展与复杂的基因组重排。

• DOI: 10.1002/bies.201700025
• 发表时间: 2017-08
• 期刊: BioEssays : news and reviews in molecular, cellular and developmental biology
• 作者: Leffak M

Analysis of Trinucleotide Repeat Stability by Integration at a Chromosomal Ectopic Site.

通过染色体异位位点整合分析三核苷酸重复稳定性。

• DOI: 10.1007/978-1-4939-9784-8_8
• 发表时间: 2020
• 期刊: Methods in molecular biology (Clifton, N.J.)
• 作者: Gadgil,RujutaYashodhan;RiderJr,SDean;Lewis,Todd;Barthelemy,Joanna;Leffak,Michael
• 通讯作者: Leffak,Michael