Functional and bioinformatic analysis of unconventional DNA replication programs in Tetrahymena

对模式真核生物——嗜热四膜虫（Tetrahymena thermophila）——一种单细胞纤毛虫的研究，已经为染色体的功能组织及其正确传递的要求提供了丰富的信息。每次细胞分裂，染色体必须复制一次，且只能复制一次，否则后代可能无法存活。然而，染色体上成千上万的位点可能被选择来开始DNA复制，我们对如何选择和调节复制起始位点知之甚少。该项目将利用新的DNA测序技术来确定四膜虫大核中约200条染色体的复制起始位置。该项目将为本科生和研究生提供广泛的生物信息学培训，并为高中生提供通过计算机和湿台式实验室体验研究的机会。真核生物DNA复制领域的一个中心教条是，复制起始位点是由起源识别复合体在染色体上的特定位点（复制起点）的招募决定的。先前对四膜虫的研究表明，ORC在暴露于遗传毒性物质羟基脲后迅速耗尽。已知的起源被绕过，新的备份途径在ORC补充之前复制整个基因组。该项目将通过在正常和orc耗尽的条件下绘制全基因组范围内的复制起始位点来确定DNA复制开始的位置和方式。新生链DNA文库的高通量测序将用于鉴定包含组成和差异调节复制起源的基因组片段。反向遗传方法将使用基于质粒的测定来验证起源活性。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ORC独立机制启动DNA复制。这些研究可能揭示了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。

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.