Mutation Hotspots in structured DNA

结构化 DNA 中的突变热点

• 批准号: 8666257
• 负责人: SUSAN THOMAS LOVETT
• 金额: $ 32.05万
• 依托单位: BRANDEIS UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8666257
• 关键词: Accounting; Affect; Animal Model; Bacteria; Bacteriophages; Biochemical; Biological Assay; Cells; Complex; DNA; DNA Damage; DNA Polymerase III; DNA Repair; DNA-Binding Proteins; DNA-Directed DNA Polymerase; Direct Repeats; Drosophila melanogaster; Escherichia coli; Eukaryota; Event; Exodeoxyribonuclease I; Exonuclease; Frequencies; Gene Mutation; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Goals; Hereditary Disease; Human; Impairment; Inverted Repeat Sequences; LacZ Genes; Malignant Neoplasms; Measures; Mutagenesis; Mutagens; Mutate; Mutation; Organism; Orthologous Gene; Pathway interactions; Polymerase; Prevalence; Property; Reporter; Reporting; Repression; Role; SS DNA BP; Saccharomyces cerevisiae; Sequence Analysis; Site; Structure; System; Testing; Validation; Variant; Werner Syndrome; Work; Yeasts; age effect; base; follow-up; genome analysis; genome sequencing; neglect; nuclease; repaired; tool; tumor progression; yeast genetics

Mutations associated with imperfect inverted repeat sequences, "quasipalindromes", with potential for DNA secondary structure, are widespread. They have been noted as mutational hotspots in bacteriophage, yeast and humans and mutate by a replication template-switch mechanism. In humans, template-switch mutations contribute to a large set of genetic diseases, structural variation in genomes and to 20% of the mutations that affect p53 in human cancers. Despite the prevalence and importance of this class of mutation, little systematic work has been done to define the parameters that govern the mutagenic mechanism, mutagens specific to this class or cellular factors that influence mutation rate. The long-term goal of this study is a more complete mechanistic understanding of quasipalindrome-associated mutagenesis. Objectives will be to define the structural parameters and cellular pathways that govern mutability. Because all cells mutate and repair DNA in fundamentally similar ways by evolutionarily related pathways, these studies using the model organisms, Escherichia coli, Saccharomyces cerevisiae, and Drosophila melanogaster should reveal mechanisms applicable to repair of DNA in human cells. The first aim of this proposal is to investigate the connection between replication, DNA damage repair and transcription collisions with template-switch mutagenesis in E. coli. The hypothesis that targeting of exonucleases to lagging strand features via singlestrand DNA binding protein and replication clamps accounts for the observed strand bias of mutagenesis will be tested. The impact of replication fork collisions with transcription complexes will be assessed. Biochemical analysis of DNA polymerase III and its interactions with clamp and clamp-loader will be correlated with genetic effects on mutagenesis to clarify its mechanism. Additional template-switch vulnerable sites will be sought by whole-genome sequence analysis. The second aim is to develop the first eukaryotic mutational reporters for template-switch mutagenesis using the URA3 gene of S. cerevisiae. In addition, an existing lacZ reporter for mutagenesis in Drosophila melanogaster will be retrofitted to report specific types of mutations, including template-switching at quasipalindromes or direct repeats, and base substitutions and frameshifts that respond to particular types of polymerase errors or DNA damage. The effects of aging on mutation frequency will be tested using these constructs. This study will significantly advance our understanding of DNA mutagenesis by providing new tools and information about this important and neglected class of mutations.

与不完美反向重复序列（“quasipalindromes”）相关的突变，具有 DNA 潜力 二级结构，分布广泛。它们被认为是噬菌体、酵母菌中的突变热点 和人类并通过复制模板切换机制进行突变。在人类中，模板转换突变 导致大量遗传疾病、基因组结构变异以及 20% 的突变 影响人类癌症中的 p53。尽管这类突变很普遍且很重要，但很少 已经进行了系统的工作来定义控制诱变机制、诱变剂的参数 特定于影响突变率的此类或细胞因素。本研究的长期目标是 对准回数相关诱变的更完整的机制理解。目标将是 定义控制可变性的结构参数和细胞途径。因为所有的细胞都会发生突变 通过进化相关的途径以基本相似的方式修复 DNA，这些研究使用该模型 有机体、大肠杆菌、酿酒酵母和果蝇应该揭示 适用于人体细胞 DNA 修复的机制。该提案的首要目的是调查 复制、DNA 损伤修复和转录碰撞与模板开关之间的联系 大肠杆菌中的诱变。核酸外切酶通过单链靶向滞后链特征的假设 DNA 结合蛋白和复制夹解释了观察到的突变链偏向 被测试。将评估复制叉与转录复合物碰撞的影响。 DNA 聚合酶 III 的生化分析及其与夹具和夹具加载器的相互作用将 与诱变的遗传效应相关联，以阐明其机制。附加模板开关 将通过全基因组序列分析寻找脆弱位点。第二个目标是发展第一个目标 使用酿酒酵母的 URA3 基因进行模板转换诱变的真核突变报告基因。在 此外，现有的用于果蝇诱变的 lacZ 报告基因将进行改造，以报告 特定类型的突变，包括准配体或直接重复的模板转换，以及碱基 响应特定类型聚合酶错误或 DNA 损伤的替换和移码。这 将使用这些结构来测试衰老对突变频率的影响。 这项研究将通过提供新的工具和方法，显着增进我们对 DNA 突变的理解。 有关这一类重要且被忽视的突变的信息。