Human origins of DNA replication : identification, analysis and application

While replication origins, cis-acting sequences directing the initiation of DNA synthesis, have been well-characterized in many model organisms, the multiple sequence and protein components present at the chromosomal origins of higher eukaryotic organisms have not yet been fully defined. Genetic assays that identify origin function in cloned DNA fragments would provide a useful approach for the isolation and analysis of mammalian DNA replication origins.In this thesis, (1) cloned fragments from a known mammalian origin, the ori$ beta$ of the hamster 3$ sp prime$ DHFR region, are demonstrated to replicate autonomously, both following transfection into human cells, and when used as templates in an in vitro replication system based on human cell extracts; (2) larger scale versions of these two assay methodologies are used to isolate over 40 novel putative origins of DNA replication from anticruciform purified human genomic DNA libraries; (3) transfection and in vitro autonomous replication assays are applied to demonstrate the potential origin function of a mitochondrial DNA sequence implicated in the insertional mutagenesis of a human genomic locus; (4) an origin mapping strategy based on the in vitro assay is used to provide evidence for the existence of a replication origin in a cloned and sequenced portion of the human 15q11q13 chromosomal subdomain, a region associated with allele-specific replication timing, genomic imprinting, and genetic disease; and (5) some of these autonomously replicating origins are cloned into a selectable YAC vector and are shown to permit the long term episomal maintenance, in human cells, of the transfected plasmid constructs.These results consistently demonstrate that short mammalian genomic DNA fragments can replicate autonomously, supporting the applicability of the replicon model in humans, and could be extended to the search for an origin core consensus element, to the investigation of higher order organization and temporal control of human DNA replication origins, and to the construction of a complete human artificial chromosome