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Mol. Cell. Biol. doi:10.1128/MCB.01392-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Identification of new human origins of DNA replication by an origin-trapping assay

Department of Gene Vectors, GSF-National Research Center for Environment and Heath, Marchioninistrasse 25, D-81377 Munich

^* To whom correspondence should be addressed. Email: schepers{at}gsf.de.

	Abstract

Metazoan genomes contain thousands of replication origins, but only a limited number have been characterized so far. We developed a two-step origin-trapping assay in which human chromatin fragments associated with origin recognition complex (ORC) in vivo were first enriched by chromatin immunoprecipitation. In a second step, these fragments were screened for transient replication competence in a plasmid-based assay utilizing the Epstein-Barr-Virus latent origin oriP. OriP contains two elements, an origin (DS) and the family of repeats, that when associated with the viral protein EBNA1, facilitates extrachromosomal stability. Insertion of the ORC-binding human DNA fragments in oriP-plasmids in place of DS enabled us to screen functionally for their ability to restore replication. Using the origin-trapping assay, we isolated and characterized five previously unknown human origins. The assay was validated with nascent strand abundance assays that confirm these origins as active initiation sites in their native chromosomal context. Furthermore, ORC and MCM2-7 components localized at these origins in G1 phase of the cell cycle, but were not detected in mitosis. This finding extends the current understanding of origin-ORC dynamics by suggesting that replication origins must be re-established during the early stages of each cell division cycle and that ORC itself participates in this process.

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