Mechanisms linking replication stress to genome instability in fission yeast

 描述(由申请人提供)：基因不稳定与突变率、染色体重排和非整倍体增加有关。这是癌症的标志，也是其他重大健康挑战的标志，包括发育缺陷、神经障碍和衰老。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障碍；因此，了解它们的功能直接关系到人类健康。这一建议使用了一个已建立的模型遗传系统来识别和描述维持基因组稳定的分子途径。该项目使用遗传学、分子生物学和分裂酵母S.pombe中的新细胞生物学方法来检测细胞在正常营养细胞周期和减数分裂期间对复制压力的反应。S.pombe是一个成熟的染色体生物学模型，它与人类细胞有许多共同的特征。该提案调查了一种假设，即对复制应激的反应动态决定了细胞是否阻止了细胞周期，或者它们是否避开了正常的检查点，继续进行异常分裂，产生了染色体重排和更高的突变率。复制压力可能在整个基因组中有所不同，这项研究的一个重要组成部分是将着丝粒周围作为一个模型脆弱部位进行分析。另一个新的组成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不育相关的染色体重排的贡献者。一项重大的创新是活细胞谱系的新系统 分析与定量分析相结合，以研究对损坏和检查点逃避的动态响应。这种活的全细胞分析允许识别经历不同结果的不同细胞亚群，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，创造与多种疾病相关的不稳定循环，并识别导致异常分裂和进一步压力的分子事件，该项目将解决当前理解中的一个关键差距。导致对压力的不同反应及其相关病理反应的途径是什么？它们之间有什么不同，以产生不同的结果，如簇状突变、CNV、缺失和复制，以及染色体重排？总而言之，这些研究提供了一幅关于保守蛋白质如何相互作用以维持真核细胞基因组稳定的整体图景，确定了人类疾病的标记和风险因素。

 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.