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Nucleic Acids Research Advance Access published online on October 4, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm555
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© 2007 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Molecular Biology

Site-specific interaction of the murine pre-replicative complex with origin DNA: assembly and disassembly during cell cycle transit and differentiation

Elisabeth Zellner, Thomas Herrmann, Carsten Schulz and Friedrich Grummt*

Institute of Biochemistry, Biocenter at the University of Würzburg, Am Hubland, D-97074 Würzburg, Germany

*To whom correspondence should be addressed. Tel: 49 931 888 4042; Fax: 49 931 888 4045; Email: grummt{at}biozentrum.uni-wuerzburg.de

Received April 27, 2007. Revised July 2, 2007. Accepted July 6, 2007.

Eukaryotic DNA replication initiates at origins of replication by the assembly of the highly conserved pre-replicative complex (pre-RC). However, exact sequences for pre-RC binding still remain unknown. By chromatin immunoprecipitation we identified in vivo a pre-RC-binding site within the origin of bidirectional replication in the murine rDNA locus. At this sequence, ORC1, -2, -4 and -5 are bound in G1 phase and at the G1/S transition. During S phase, ORC1 is released. An ATP-dependent and site-specific assembly of the pre-RC at origin DNA was demonstrated in vitro using partially purified murine pre-RC proteins in electrophoretic mobility shift assays. By deletion experiments the sequence required for pre-RC binding was confined to 119 bp. Nucleotide substitutions revealed that two 9 bp sequence elements, CTCGGGAGA, are essential for the binding of pre-RC proteins to origin DNA within the murine rDNA locus. During myogenic differentiation of C2C12 cells, we demonstrated a reduction of ORC1 and ORC2 by immunoblot analyses. ChIP analyses revealed that ORC1 completely disappears from chromatin of terminally differentiated myotubes, whereas ORC2, -4 and -5 still remain associated.

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.


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