Role of regulation of eukaryotic DNA replication in preserving genomic stability

真核 DNA 复制调控在保持基因组稳定性中的作用

• 批准号: 8489302
• 负责人: JOACHIM J LI
• 金额: $ 28.2万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-07-01 至 2015-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8489302
• 关键词: Address; Affect; Aneuploidy; Animal Model; Biological; Biological Assay; Biological Models; Cell Cycle; Cells; Cellular biology; Centromere; Chromosomal Breaks; Chromosome Breakage; Chromosomes; Cis-Acting Sequence; Complex; Cyclin-Dependent Kinases; DNA; DNA Sequence Rearrangement; DNA biosynthesis; Data; Dependency; Diploidy; Elements; Ensure; Eukaryotic Cell; Event; Evolution; Frequencies; Funding; Gene Amplification; Genetic Nondisjunction; Genetic Variation; Genome; Genome Stability; Genomic Instability; Genomics; Goals; Haploidy; Hereditary Disease; Human; Human Genetics; Impairment; Lead; Malignant Neoplasms; Maps; Measures; Mediating; Minor; Modeling; Molecular Evolution; Molecular Genetics; Monitor; Movement; Mutation; Pathway interactions; Proteins; Regulation; Repetitive Sequence; Replication Initiation; Replication Origin; Research; Role; Saccharomyces cerevisiae; Saccharomycetales; Science; Side; Site; Source; Staging; Structure; Techniques; Technology; Temperature; Testing; Time; Yeast Model System; base; cancer cell; cancer genetics; disorder control; genetic analysis; genome analysis; homologous recombination; insight; prevent; promoter; research study; segregation; tool; tumorigenesis

DESCRIPTION (provided by applicant): A central tenet of eukaryotic cell biology is that DNA replication must be tightly controlled so that it occurs only once per cell cycle. It is presumed, but largely untested, that this control is vital for preserving genome integrity. Our long-term goal is to understand how the re-initiation of DNA replication is reliably prevented at the thousands of replication origins scattered throughout eukaryotic genomes, and to discern the effect of disrupting this control on genome stability. We study replication control in budding yeast because this model system offers an exceptional opportunity to dissect the complex, overlapping mechanisms that are required to achieve this control with such extraordinary fidelity. Additionally, the molecular genetic tools available in budding yeast allow us to apply both simple and sophisticated technologies to query the effects of disrupting replication controls. In previous funding periods, we demonstrated that cyclin-dependent kinases (CDKs) use multiple overlapping mechanisms to prevent origins from re-initiating within a single cell cycle. We have also shown that re-replication arising from loss of these controls leads to significant chromosomal breakage and lethality, providing a previously unknown justification for the importance of replication control. More recently, we provided the first evidence that re-replication is a highly efficient means to induce gene amplification (Green et al, Science, in press). Re-replication induced gene amplification (RRIGA) occurred with extraordinary efficiency (roughly 1/20 re-initiation events). This finding supports the compelling hypothesis that even minor impairment of replication control may contribute to genome instability. Ultimately, we hope to demonstrate that re-replication can drive the copy number changes observed in tumorigenesis, human genetic variation, and evolution. Here we propose to expand our understanding of how the loss of replication control leads to genomic instability, both by probing the mechanisms that underlie RRIGA as well as by further investigating the biological consequences and significance of loss of replication control. We propose to (1) define the mechanism and parameters enabling RRIGA; (2) determine how local regulatory factors modulate replication control at origins that are highly susceptible to re-initiation; (3) determine whether RRIGA participates in a model of evolutionary adaptation; and (4) establish whether re-replication can induce chromosome missegregation. These data will significantly enhance our understanding of how re-replication promotes genomic instability, as well as give insight into the biological significance of loss of replication control.

描述（由申请人提供）：真核细胞生物学的中心宗旨是必须严格控制DNA复制，以便每个细胞周期仅发生一次。它假定但在很大程度上未经测试，这种控制对于保留基因组完整性至关重要。我们的长期目标是了解如何在散布在整个真核基因组中的数千个复制起源上可靠地防止DNA复制的重新定位，并辨别破坏这种控制对基因组稳定性的影响。我们研究出现酵母中的复制控制，因为该模型系统提供了一个非凡的机会，可以剖析复杂的重叠机制，这些机制以这种非凡的忠诚度实现了这种控制所需的机会。此外，在萌芽酵母中可用的分子遗传工具使我们能够应用简单和复杂的技术来查询破坏复制控制的效果。在以前的资金期间，我们证明了细胞周期蛋白依赖性激酶（CDKS）使用多种重叠的机制来防止起源在单个细胞周期内重新发射。我们还表明，由于失去这些对照而引起的重新复制会导致显着的染色体断裂和致死性，这为复制控制的重要性提供了以前未知的理由。最近，我们提供了第一个证据，即重新复制是诱导基因扩增的高效手段（Green等，《科学》，印刷中）。重新复制诱导的基因扩增（RRIGA）以非凡的效率（大约1/20重新输入事件）发生。这一发现支持令人信服的假设，即即使是重复控制的轻微损害也可能导致基因组不稳定性。最终，我们希望证明重新复制可以驱动在肿瘤发生，人遗传变异和进化中观察到的拷贝数变化。 在这里，我们建议通过探测RRIGA构成的机制以及进一步研究复制控制损失的生物学后果和意义，从而扩展我们对复制控制丧失如何导致基因组不稳定性的理解。我们建议（1）定义启用Rriga的机制和参数； （2）确定局部调节因素如何调节起源的复制控制，这些因素非常容易受到重新启动； （3）确定RRIGA是否参与进化适应模型； （4）确定重新复制是否可以诱导染色体错误分析。这些数据将显着增强我们对重新复制如何促进基因组不稳定性的理解，并深入了解复制控制丧失的生物学意义。