Functional and bioinformatic analysis of unconventional DNA replication programs in Tetrahymena

对单细胞纤毛虫模式真核生物嗜热四膜虫的研究为染色体的功能组织及其正常传递提供了丰富的信息。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，一条染色体上的数千个位点可能被选择来启动DNA复制，而我们对复制起始点是如何选择和调控的知之甚少。该项目将利用新的DNA测序技术来确定四膜虫大核~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，并通过基于计算机和湿法实验室的体验，为高中生提供尝试研究的机会。真核DNA复制领域的一个中心教条是，复制起始点是由起源识别复合体招募到染色体上的特定位置(复制起点)决定的。先前对四膜虫的研究表明，在暴露于遗传毒剂羟基脲后，ORC会迅速耗尽。已知的起源被绕过，在ORC补充之前，新的备份路径复制整个基因组。这个项目将通过在正常和ORC耗尽的条件下绘制全基因组范围的复制起始点来确定DNA复制在哪里以及如何开始。新生的链DNA文库的高通量测序将用于识别包含构成和差异调控复制起点的基因组片段。反向遗传方法将被用来通过基于质粒的分析来验证起源活性。突变研究将确定哪些DNA序列是重要的，以及它们是需要ORC还是通过ORC独立的机制启动DNA复制。这些研究可能会揭示原生生物和高等真核生物(如植物、老鼠和人类)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.