Mechanisms linking replication stress to genome instability in fission yeast

 描述（由申请人提供）：遗传不稳定性与突变率、染色体重排和非整倍性增加有关。这是癌症以及其他重大健康挑战（包括发育缺陷、神经系统疾病和衰老）的标志。来自多个系统的数据表明 DNA 复制压力是基因组不稳定的关键因素。稳定复制叉、防止异常分裂和促进 DNA 修复的分子机制是疾病的主要屏障；因此，了解它们的功能与人类健康直接相关。该提案采用已建立的模型遗传系统来识别和表征维持基因组稳定性的分子途径。该项目在裂殖酵母中使用遗传学、分子生物学和新颖的细胞生物学方法来检查细胞在正常营养细胞周期和减数分裂期间对复制应激的反应。粟酒裂殖酵母是一种成熟的染色体生物学模型，与人类细胞具有许多相同的特征。该提案研究了这样一个假设，即对复制应激的反应动态决定了细胞是否会阻止细胞周期，或者是否会逃避正常检查点并继续异常分裂，从而产生染色体重排和增加突变率。复制压力可能因基因组而异，该研究的一个重要组成部分是将着丝粒周围作为模型脆弱位点进行分析。另一个新颖的部分是分析减数分裂期间的复制应激，作为与出生缺陷和不孕症相关的染色体重排的一个因素。一项重大创新是活细胞谱系的新系统 分析与定量分析相结合，研究对损坏和检查点规避的动态响应。这种活体全细胞分析可以识别不同的细胞亚群，这些亚群经历不同的结果，从而形成与多种疾病相关的不稳定循环。通过将这种新的细胞生物学方法与卓越的酵母基因发现工具相结合，并识别导致异常分裂和进一步应激的分子事件，该项目将解决当前理解中的一个关键差距。导致对压力及其相关病理的不同反应的途径是什么？它们之间有何不同，从而产生不同的结果，例如聚集突变、CNV、缺失和重复以及染色体重排？总之，这些研究提供了保守蛋白如何相互作用以维持真核细胞基因组稳定性的整体图景，从而识别人类疾病的标记和危险因素。

 DESCRIPTION (provided by applicant): Genetic instability is associated with increased rates of mutation, chromosome rearrangement, and aneuploidy. This is a hallmark of cancer, as well as other significant health challenges including developmental defects, neurological disorders, and aging. Data from multiple systems suggests that DNA replication stress is a key contributor to genome instability. Molecular mechanisms that stabilize replication forks, prevent abnormal divisions, and promote DNA repair are a primary barrier to disease; therefore, understanding their function has direct relevance to human health. This proposal employs an established model genetic system to identify and characterize molecular pathways that maintain genome stability. The project uses genetics, molecular biology, and novel cell biology methods in the fission yeast S. pombe to examine the response of cells to replication stress during the normal vegetative cell cycle, and during meiosis. S. pombe is a well-established model for chromosome biology that shares many features with human cells. The proposal investigates the hypothesis that the dynamics of the response to replication stress determines whether cells arrest the cell cycle, or whether they evade normal checkpoints and go on to divide abnormally, generating chromosome rearrangements and increased rates of mutation. Replication stress may vary across the genome and a significant component of the study is the analysis of the pericentromere as a model fragile site. An additional novel component is the analysis of replication stress during meiosis as a contributor to chromosome rearrangements associated with birth defects and infertility. A significant innovation is a new system for live cell pedigree analysis coupled with quantitative analysis to investigate the dynamic response to damage and checkpoint evasion. This live whole-cell analysis allows the identification of distinct sub-populations of cells that undergo different outcomes creating a cycle of instability that is associated with multiple diseases By combining this new cell biological approach with superb yeast gene-discovery tools, and identifying the molecular events that lead to abnormal divisions and further stress, this project will tackle a critical gap in current understanding. What are the pathways that contribute to different responses to stress and their associated pathologies? How do they differ from one another to generate distinct outcomes such as clustered mutations, CNVs, deletions and duplications, and chromosome rearrangements? Together, these studies provide a holistic picture of how conserved proteins interact to maintain genome stability in a eukaryotic cell, identifying markers and risk factors for human disease.