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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 (dyad symmetry element [DS]) and the family of repeats, that when associated with the viral protein EBNA1 facilitate extrachromosomal stability. Insertion of the ORC-binding human DNA fragments in oriP plasmids in place of DS enabled us to screen functionally for their abilities 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 contexts. Furthermore, ORC and MCM2-7 components localized at these origins during G1 phase of the cell cycle but were not detected during mitosis. This finding extends the current understanding of origin-ORC dynamics by suggesting that replication origins must be reestablished during the early stages of each cell division cycle and that ORC itself participates in this process.
Supplemental material for this article may be found at http://mcb.asm.org/.
We thank W. Hammerschmidt for continuous support and discussion and D. Schaarschmidt for help with ChIP experiments and for critically reading the manuscript. We thank Natalie Grober for technical assistance and are grateful to M. Leffak for sharing the nascent strand abundance protocol. We thank A. Thomae and J. Baltin for discussion and comments on the manuscript.
This work was supported by grants from the Deutsche Forschungsgemeinschaft (Sche 470-4 and SFB 646).