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; 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DNA损伤最危险的形式，因为它们会导致多种类型 突变和总体染色体重排。当复制过程中，DNA对破裂最敏感，当 难以重复的非规范DNA结构会导致复制叉的停滞。非规范的DNA结构是 强烈暗示是染色体断裂和易位的内源性来源，导致 但是，发育缺陷和癌症，但是，复制叉停滞的机制导致DNA 双链断裂（DSB）尚不清楚。 尽管在全球或单个分子研究中对DNA损伤反应蛋白进行了大量分析，其中 未识别损伤部位，依赖复制依赖性DNA链的分子机制 在人类细胞中特定部位的断裂和修复尚不完全了解。解决这一知识 差距，我们将研究两种类型的自然复制屏障（CTG/CAG三核苷酸重复和不对称 在人类基因组中的异位部位集成的嘌呤 - 吡啶胺（PU/PY）镜面重复序列） 可以操纵对复制的结构和影响。我们还检查了几个内源复制叉 在DNA复制过程中诱导DSB的障碍。我们将使用PCR，DNA测序，染色质 免疫沉淀，质谱和流式细胞术，以显示（1）聚合酶如何停滞 非规范DNA结构会导致DSB，（2）DNA修复蛋白如何作用重塑复制 叉以重新启动合成，（3）DSB重组的机制和基因组后果 结构引起的叉子屏障。 我们将检验以下假设：非规范DNA结构通过阻止DNA的进展诱导DSB 聚合酶，促进核酸酶敏感的叉子回归以及抑制DNA末端加工所需的DNA末端加工 重组。这项工作的概念进步将包括确定分子机制 DSB形成在特定失速叉附近 结构诱导的DSB的处理如何不同，核酸酶诱导的“干净” DSB的处理方式。我们的长期目标 是为了定义DNA结构引起的g e n o m e在人类疾病中的作用。 AIM 1将披露叉车停滞和损坏信号传导之间的关系，叉子的生物化学 逆转，结构特异性核酸内切酶的功能，叉叉和DNA次级的影响 叉切除和修复的结构。 AIM 2将以我们的示威为基础，即Fanconi贫血类型J 蛋白质（FANCJ）对于在整个基因组中维持跨基因组的非规范DNA结构至关重要 复制应力，以确定依赖的微卫星稳定的机制。在目标3中我们 将表征FANCJ缺乏症的基因组后果。我们的实验将表明多么难以 复制DNA序列会导致染色体断裂和突变导致遗传疾病。