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: 8107243
• 负责人: JOACHIM J LI
• 金额: $ 29.51万
• 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/8107243
• 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; 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. PUBLIC HEALTH RELEVANCE: Abnormal genetic changes underlie the formation of cancer cells and can lead to human genetic diseases, but the sources of these changes is poorly understood. Our research in a yeast model organism is establishing important connections between these diseases and the control of DNA replication, which normally ensures that every segment of DNA is replicated exactly once each time a cell divides. Our proposed project will investigate why inappropriate re-replication arising from the loss of replication control is so amazingly efficient at inducing the types of genetic changes observed in cancer and genetic disorders. These findings should provide strong impetus for cancer biologists and human geneticists to investigate the role of replication dysregulation in their fields.

描述（由申请人提供）：真核细胞生物学的中心原则是DNA复制必须受到严格控制，以便每个细胞周期仅发生一次。据推测，但在很大程度上未经测试，这种控制是至关重要的，以保持基因组的完整性。我们的长期目标是了解如何在分散在真核基因组中的数千个复制起点处可靠地阻止DNA复制的重新启动，并辨别破坏这种控制对基因组稳定性的影响。我们研究芽殖酵母的复制控制，因为这个模型系统提供了一个特殊的机会，解剖复杂的，重叠的机制，需要实现这种控制与这种非凡的保真度。此外，芽殖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影响。在以前的资助期间，我们证明了细胞周期蛋白依赖性激酶（CDK）使用多种重叠机制来防止起源在单个细胞周期内重新启动。我们还表明，这些控制的损失所产生的再复制导致显着的染色体断裂和致死性，提供了一个以前未知的理由复制控制的重要性。最近，我们提供了第一个证据，即再复制是诱导基因扩增的高效手段（绿色等人，科学，出版中）。再复制诱导的基因扩增（RRIGA）以非凡的效率发生（大约1/20的再起始事件）。这一发现支持了一个令人信服的假设，即使是轻微的损害复制控制可能有助于基因组的不稳定性。最终，我们希望证明重新复制可以驱动在肿瘤发生，人类遗传变异和进化中观察到的拷贝数变化。 在这里，我们建议扩大我们的理解如何复制控制的损失导致基因组的不稳定性，无论是通过探测RRIGA的基础机制，以及通过进一步调查的生物学后果和复制控制的损失的意义。我们建议：（1）定义机制和参数，使RRIGA;（2）确定本地调节因子如何调节复制控制的起点，是高度敏感的重新启动;（3）确定是否RRIGA参与了一个模型的进化适应;（4）建立是否重新复制可以诱导染色体错误分离。这些数据将显著增强我们对再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。 公共卫生相关性：异常的遗传变化是癌细胞形成的基础，并可能导致人类遗传疾病，但这些变化的来源知之甚少。我们在酵母模式生物中的研究正在这些疾病与DNA复制控制之间建立重要联系，这通常确保细胞每次分裂时DNA的每个片段都精确复制一次。我们提出的项目将研究为什么由复制控制的丧失引起的不适当的再复制在诱导癌症和遗传疾病中观察到的遗传变化类型方面如此惊人地有效。这些发现应该为癌症生物学家和人类遗传学家提供强大的动力，以研究复制失调在其领域中的作用。