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.

与不完美的反复重复序列“准圆柱膜”相关的突变，具有DNA的潜力 次要结构是广泛的。它们被认为是噬菌体中的突变热点 和人类，并通过复制模板切换机制进行突变。在人类中，模板开关突变 有助于大量的遗传疾病，基因组的结构差异和20％的突变 影响人类癌症的p53。尽管这类突变的普遍性和重要性，但很少 已经完成了系统的工作来定义控制诱变机制的参数 特定于该类别或影响突变率的细胞因素。这项研究的长期目标是 对准沉着物相关诱变的更完整的机械理解。目标将是 定义控制突变性的结构参数和细胞途径。因为所有细胞都突变，并且 通过进化相关的途径以根本相似的方式修复DNA，这些研究使用模型 有机体，大肠杆菌，酿酒酵母和果蝇的生物应该揭示 适用于人类细胞中DNA的机制。该提议的第一个目的是调查 复制，DNA损伤修复与转录碰撞与模板开关之间的联系 大肠杆菌中的诱变。通过单层靶向靶向外丝裂至滞后链特征的假设 DNA结合蛋白和复制夹具诱变的观察到的链偏置将是 进行测试。将评估复制叉与转录复合物碰撞的影响。 DNA聚合酶III及其与夹具和夹具载荷的相互作用的生化分析将是 与对诱变的遗传作用相关，以阐明其机制。其他模板开关 全基因组序列分析将寻求脆弱的站点。第二个目的是开发第一个 使用S. cerevisiae的URA3基因进行模板开关诱变的真核突变记者。在 此外，将改装果蝇中诱变的现有LACZ记者以报告 特定类型的突变类型，包括在准立体膜上进行模板切换或直接重复和基础 对特定类型的聚合酶误差或DNA损伤做出反应的取代和移架。这 衰老对突变频率的影响将使用这些构建体测试。 这项研究将通过提供新工具和 有关这种重要和被忽视的突变类别的信息。