Suppressors of Eukaryotic Mutator Phenotypes

真核突变表型的抑制因子

• 批准号: 8444937
• 负责人: ALAN J HERR
• 金额: $ 18.68万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-11-12 至 2014-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8444937
• 关键词: Address; Affect; Alleles; Cancer Etiology; Cell Cycle Arrest; Cells; Cessation of life; Coupled; DNA Repair Gene; DNA biosynthesis; DNA-Directed DNA Polymerase; Defect; Development; Environment; Escape Mutant; Essential Genes; Eukaryota; Extinction (Psychology); Foundations; Functional RNA; Future; Gene Expression; Genes; Genetic; Genetic Polymorphism; Genetic Variation; Genome; Genomics; Goals; Growth; Haploidy; Human; Lead; Malignant - descriptor; Malignant Neoplasms; Mediating; Mismatch Repair; Mus; Mutagenesis; Mutation; Pathway interactions; Phenotype; Point Mutation; Polymerase; Property; Research; Role; Site; Suppressor Mutations; Testing; Time; Variant; Yeasts; base; cancer therapy; cost; deletion library; falls; fitness; gain of function; gene discovery; genome analysis; genome sequencing; genome-wide; human disease; insight; loss of function; mutant; next generation sequencing; novel; novel strategies; null mutation; pressure; public health relevance; repaired; research study; sample fixation; screening; senescence; tumor; tumor progression; tumorigenesis

DESCRIPTION (provided by applicant): The overall objective of our research is to understand the role of increased spontaneous mutation (mutator phenotypes) in human disease. This exploratory proposal focuses on an understudied but important property of mutators: their inherent instability. Mutator phenotypes fuel oncogenesis by providing the genetic diversity necessary for emergence of malignant clones. However, deleterious mutations also accumulate in mutator cells. Thus, while mutators accelerate oncogenesis, the fitness cost of increased mutation imposes indirect selection pressure to reduce mutation rates. This counter-selection will occur after adaptation to a stable environment where conditions no longer favor the genetic potential of mutators. One possible mechanism to reduce mutation rates is the acquisition of compensatory alleles at modifier loci that suppress the mutator phenotype. To identify genetic pathways that mediate mutator suppression, we developed a suppressor screen in yeast that exploits the synergistic relationship between DNA polymerase ¿ (Pol ¿) proofreading and mismatch repair (MMR). Double mutants are inevitable, suggesting that extreme mutation rates exceed an error threshold. However, variants that escape this error-induced extinction (eex) rapidly emerge from mutator clones. One-third of the escape mutants result from second- site changes in Pol ¿ that suppress the mutator phenotype, while two-thirds are due to unknown mutator suppressor alleles in the genome. The goal of the proposed exploratory study is to identify these genomic antimutator alleles and the genes that mediate mutator suppression. We will use two complementary genome-wide approaches: 1) an adaptation of synthetic genetic array (SGA) analysis that allows us to screen all non-essential genes in yeast for their roles in lethal mutagenesis, and 2) a pooled linkage strategy coupled with next-generation sequencing that can specifically identify functional suppressor mutations among the many incidental mutations expected in mutator-derived clones. These experiments promise to break new ground in understanding the fate of eukaryotic mutators by identifying novel genetic interactions and pathways that mediate mutagenesis and repair. The genes discovered in our analysis will provide the foundation for future studies in mice and humans assessing the role of antimutator alleles in the etiology of cancer.

描述（由申请人提供）：我们研究的总体目标是了解增加的自发突变（突变表型）在人类疾病中的作用。这个探索性的建议集中在一个未被充分研究但重要的突变体特性：它们固有的不稳定性。突变表型通过为恶性克隆的出现提供必要的遗传多样性来促进肿瘤的发生。然而，有害的突变也会在突变细胞中积累。因此，在突变体加速肿瘤发生的同时，增加突变的适应度成本施加了降低突变率的间接选择压力。这种反选择将在适应稳定的环境后发生，在这种环境中条件不再有利于突变体的遗传潜力。降低突变率的一种可能机制是在抑制突变表型的修饰位点获得代偿等位基因。为了确定介导突变抑制的遗传途径，我们在酵母中开发了一种抑制因子筛选，利用DNA聚合酶¿（Pol¿）校对和错配修复（MMR）之间的协同关系。双突变是不可避免的，这表明极端突变率超过了一个错误阈值。然而，逃避这种错误诱导灭绝（eex）的变体迅速从突变克隆中出现。三分之一的逃逸突变源于抑制突变表型的Pol¿的第二位点变化，而三分之二是由于基因组中未知的突变抑制等位基因。提出探索性研究的目的是鉴定这些基因组抗突变等位基因和介导突变抑制的基因。我们将使用两种互补的全基因组方法：1)合成基因阵列（SGA）分析的适应性，使我们能够筛选酵母中所有非必需基因在致死性突变中的作用；2)结合下一代测序的汇集连锁策略，可以在突变源克隆中预期的许多偶然突变中特异性识别功能性抑制突变。这些实验通过识别新的基因相互作用和介导突变和修复的途径，有望在理解真核突变体的命运方面开辟新的领域。在我们的分析中发现的基因将为未来在小鼠和人类中评估抗突变等位基因在癌症病因学中的作用提供基础。