Suppressors of Eukaryotic Mutator Phenotypes

描述(申请人提供)：我们研究的总体目标是了解增加的自发突变(突变子表型)在人类疾病中的作用。这项探索性的建议侧重于突变子一个未被充分研究但却很重要的特性：它们固有的不稳定性。突变体表型通过提供出现恶性克隆所需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，虽然突变者加速了肿瘤的发生，但突变增加的适应成本施加了间接选择压力，以降低突变率。这种反选择将在适应稳定的环境后发生，在稳定的环境中，条件不再有利于突变体的遗传潜力。降低突变率的一个可能机制是在抑制突变子表型的修饰基因座上获得补偿等位基因。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物筛选，利用DNA聚合酶(POL)校对和错配修复(MMR)之间的协同关系。双重突变是不可避免的，这表明极端的突变率超过了错误阈值。然而，从突变子克隆中迅速出现了逃脱这种错误诱导灭绝(EEX)的变种。三分之一的逃逸突变是由Pol？的第二位点变化导致的，这种变化抑制了突变子的表型，而三分之二的突变是由于基因组中未知的突变子抑制等位基因造成的。这项拟议的探索性研究的目标是确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)适应合成遗传阵列(SGA)分析，使我们能够筛选酵母中所有非必需基因在致死突变中的作用；2)池连锁策略与下一代测序相结合，可以在突变子衍生的克隆中的许多偶然突变中专门识别功能抑制突变。这些实验有望通过识别新的基因相互作用和介导突变和修复的途径，在理解真核突变体的命运方面开辟新的天地。在我们的分析中发现的基因将为未来在老鼠和人类身上评估抗突变等位基因在癌症病因学中的作用的研究提供基础。

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.