A Study of DNA Replication Origins by Comparative Functional Genomics

DNA复制起源的比较功能基因组学研究

• 批准号: 8035972
• 负责人: Ivan Liachko
• 金额: $ 5.47万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-16 至 2012-04-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8035972
• 关键词: Biological Assay; Cell Cycle; Cell Cycle Regulation; Cells; Cloning; Coupling; DNA; DNA biosynthesis; DNA replication origin; Data; Defect; Disease; Elements; Eukaryota; Evolution; Genes; Genome; Genomic Instability; Genomic Library; Genomics; Goals; Human; Indium; Learning; Light; Link; Location; Malignant Neoplasms; Maps; Microarray Analysis; Molecular; Organism; Pattern; Peptide Initiation Factors; Plasmids; Positioning Attribute; Process; Proteins; Publishing; Recruitment Activity; Replication Initiation; Replication Origin; Saccharomyces cerevisiae; Saccharomycetales; Screening procedure; Series; Structure; Techniques; Time; Work; Yeasts; autonomously replicating sequence; comparative; comparative genomics; computerized tools; design; functional genomics; genome sequencing; genome-wide; high standard; insight; novel; research study; vector

DESCRIPTION (provided by applicant): The activity of replication origins is a major way of regulating DNA replication and the cell cycle. This process has been most well studied in the budding yeast Saccharomyces cerevisiae, where origins can be studied as autonomously replicating sequences (ARSs) on plasmids. However, DNA replication initiation mechanisms in other organisms are not well understeood. My preliminary data as well as published work from other labs suggests that replication initiation mechanisms differ among different budding yeast species. The recent abundance of computational tools and sequenced genomes allows the study of replication origins and their evolution across multiple species. I propose to isolate functional ARS sequences from different yeast species and, using comparative genomics, study the molecular determinants of ARS function. This will provide insight into both the mechanisms of ARS function as well as the evolution of replication initiation mechanisms. Using the well developed ARS assay combined with microarray analysis, I will isolate and map ARSs from different species of budding yeast. I will use comparative genomic approaches to characterize the mechanisms of ARS recognition utilized by the different yeasts. I will also assay the evolution of ARS elements and their genomic locations. To identify the differences in ARS recognition proteins that are responsible for differences in ARS function, I will perform a series of inter-species ARS and protein swapping experiments. Relevance: The initiation of DNA replication is a central player in genome dynamics and cell cycle regulation. It is therefore not surprising that defects in this process have been linked to genomic instability and cancer. The proposed studies will contribute to our understanding ofthe molecular mechanisms of replication initiation by tracking the co-evolution of ARSs and ARS recognition proteins across 200 million years of evolutionary time. In addition, we will learn how proteins from different yeasts interact with different ARS sequences. In the long-term, this information will be useful in understanding how DNA replication initiates in human cells and how defects in this process can contribute to cancer.

描述（申请人提供）：复制起点的活性是调节DNA复制和细胞周期的主要方式。这一过程在芽殖酵母酿酒酵母中得到了最深入的研究，其中的起源可以作为质粒上的自主复制序列（ARS）进行研究。然而，其他生物体中的 DNA 复制起始机制尚不清楚。我的初步数据以及其他实验室发表的工作表明，不同芽殖酵母物种之间的复制启动机制有所不同。最近丰富的计算工具和测序基因组允许研究复制起源及其跨多个物种的进化。我建议从不同酵母物种中分离出功能性 ARS 序列，并利用比较基因组学研究 ARS 功能的分子决定因素。这将深入了解 ARS 功能的机制以及复制启动机制的演变。 使用成熟的 ARS 测定法与微阵列分析相结合，我将从不同种类的芽殖酵母中分离并绘制 ARS 图谱。我将使用比较基因组方法来描述不同酵母所利用的 ARS 识别机制。我还将分析 ARS 元件的进化及其基因组位置。为了确定导致 ARS 功能差异的 ARS 识别蛋白差异，我将进行一系列种间 ARS 和蛋白质交换实验。相关性：DNA 复制的起始是基因组动力学和细胞周期调控的核心角色。因此，这一过程中的缺陷与基因组不稳定和癌症有关也就不足为奇了。拟议的研究将通过追踪 ARS 和 ARS 识别蛋白在 2 亿年进化时间内的共同进化，有助于我们理解复制起始的分子机制。此外，我们还将了解来自不同酵母的蛋白质如何与不同的 ARS 序列相互作用。从长远来看，这些信息将有助于了解 DNA 复制如何在人类细胞中启动以及该过程中的缺陷如何导致癌症。