Functional and Bioinformatic Analysis of Unconventional DNA Replication Programs in Tetrahymena
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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.