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Molecular and Cellular Biology, October 2006, p. 7731-7746, Vol. 26, No. 20
0270-7306/06/$08.00+0     doi:10.1128/MCB.01392-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Identification of New Human Origins of DNA Replication by an Origin-Trapping Assay{triangledown} ,{dagger}

Jeannine Gerhardt, Samira Jafar, Mark-Peter Spindler, Elisabeth Ott, and Aloys Schepers*

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

Received 28 July 2006/ Accepted 4 August 2006

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.

* Corresponding author. Mailing address: Department of Gene Vectors, GSF-National Research Center for Environment and Health, Marchioninistrasse 25, D-81377 Munich, Germany. Phone: 0049 89 7099509. Fax: 0049 89 7099225. E-mail: schepers{at}gsf.de.

{triangledown} Published ahead of print on 5 September 2006.

{dagger} Supplemental material for this article may be found at http://mcb.asm.org/.

Molecular and Cellular Biology, October 2006, p. 7731-7746, Vol. 26, No. 20
0270-7306/06/$08.00+0     doi:10.1128/MCB.01392-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Pich, D., Humme, S., Spindler, M.-P., Schepers, A., Hammerschmidt, W. (2008). Conditional gene vectors regulated in cis. Nucleic Acids Res 36: e83-e83 [Abstract] [Full Text]  
  • Siddiqui, K., Stillman, B. (2007). ATP-dependent Assembly of the Human Origin Recognition Complex. J. Biol. Chem. 282: 32370-32383 [Abstract] [Full Text]  


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