Functional and bioinformatic analysis of unconventional DNA replication programs in Tetrahymena

四膜虫非常规 DNA 复制程序的功能和生物信息学分析

• 批准号: 1616311
• 负责人: Geoffrey Kapler
• 金额: $ 75万
• 依托单位: The Texas A&M University System HSC
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-15 至 2020-07-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1616311&HistoricalAwards=false
• 关键词: Functional; bioinformatic; analysis; unconventional; DNA

Research on the model eukaryote, Tetrahymena thermophila, a single-cell ciliate, has provided a wealth of information on the functional organization of chromosomes and the requirements for their proper transmission. Chromosomes must be duplicated once and only once per cell division, otherwise the progeny may not survive. Yet, many thousands of sites on a chromosome may be chosen to start DNA replication, and we do not know much about how replication initiation sites are chosen and regulated. New DNA sequencing technologies will be exploited in this project to determine where replication initiates in the ~200 chromosomes in the Tetrahymena macronucleus. This project will provide extensive training in bioinformatics to undergraduate and graduate researchers, as well as offer opportunities for high school students to get a taste for research, through computer-based and wet bench lab experiences. A central dogma in the eukaryotic DNA replication field is that replication initiation sites are dictated by the recruitment of the Origin Recognition Complex to specific sites in chromosomes (origins of replication). Previous studies in Tetrahymena demonstrated that ORC is rapidly depleted following exposure to the genotoxic agent, hydroxyurea. Known origins are bypassed and novel backup pathways replicate the entire genome prior to ORC replenishment. This project will determine where and how DNA replication initiates by mapping replication initiation sites on a genome-wide scale under normal and ORC-depleted conditions. High throughput sequencing of nascent strand DNA libraries will be used to identify segments of the genome that encompass constitutive and differentially regulated replication origins. Reverse genetic approaches will be used to validate origin activity using plasmid-based assays. Mutational studies will determine which DNA sequences are important and whether they require ORC or initiate DNA replication through an ORC-independent mechanism. These studies may reveal conserved pathways that are shared between protists and higher eukaryotes, such as plants, mice and humans.

对模式真核生物嗜热四膜虫（一种单细胞纤毛虫）的研究提供了大量关于染色体功能组织及其正确传递要求的信息。 染色体必须复制一次，而且每次细胞分裂只能复制一次，否则后代可能无法存活。 然而，染色体上的数千个位点可能被选择来启动DNA复制，我们对复制起始位点是如何选择和调节的知之甚少。 新的DNA测序技术将被利用在这个项目中，以确定在四膜虫大核的200条染色体的复制启动。 该项目将为本科生和研究生研究人员提供生物信息学方面的广泛培训，并为高中生提供机会，通过基于计算机的湿台实验室体验来体验研究。 真核DNA复制领域的中心法则是复制起始位点由起源识别复合物募集到染色体中的特定位点（复制起点）决定。 先前在四膜虫中的研究表明，在暴露于遗传毒性剂羟基脲后，ORC迅速耗尽。 绕过已知的起源，新的备用途径在ORC补充之前复制整个基因组。 该项目将通过在正常和ORC耗尽条件下在全基因组范围内绘制复制起始位点来确定DNA复制在何处以及如何起始。 新生链DNA文库的高通量测序将用于鉴定包含组成型和差异调节的复制起点的基因组区段。 反向遗传方法将用于使用基于质粒的测定法验证来源活性。 突变研究将确定哪些DNA序列是重要的，以及它们是否需要ORC或通过不依赖ORC的机制启动DNA复制。这些研究可能揭示原生生物和高等真核生物，如植物，小鼠和人类之间共享的保守途径。