Published online 4 October 2007 Nucleic Acids Research, 2007, Vol. 35, No. 20 6701–6713 doi:10.1093/nar/gkm555 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 W u¨rzburg, Am Hubland, D-97074 Wu¨ rzburg, Germany Received April 27, 2007; Revised July 2, 2007; Accepted July 6, 2007 ABSTRACT 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 differen- tiation 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 myo- tubes, whereas ORC2, -4 and -5 still remain associated. INTRODUCTION The initiation of DNA replication in eukaryotic cells is strictly coordinated within the cell cycle and starts from discrete sites deﬁned as origins of replication. In the budding yeast Saccharomyces cerevisiae the hexameric origin recognition complex (ORC) binds to the origin in an ATP-dependent manner and serves as a recruitment factor for sequential loading of CDC6, CDT1 and minichromosome maintenance (MCM) proteins 2–7 to form the pre-replicative complex (pre-RC) in the early G1 phase of the cell cycle. Transition to S phase is initiated by activation of speciﬁc protein kinases and is marked by the conversion of pre-RCs to initiation complexes (1,2). Although most of what we know about initiation of DNA replication derives from studies using S. cerevisiae, the basic composition of core initiation proteins has been conserved among all eukaryotic organisms examined thus far (1,3,4). In yeast, the ORC remains bound to origin DNA throughout the cell cycle. In higher eukaryotes, however, the association of the ORC with chromatin seems to be cell cycle regulated. In human cells, ORC1 dissociates from chromatin in S phase, whereas ORC2 remains bound (5). Likewise, hamster ORC1 is released from chromatin as cells enter S phase and is re-bound to chromatin during M to G1 transition (6,7). CDC6 is bound to chromatin in G1 phase in yeast and mammalian cells. As cells enter S phase, CDC6 is ubiquitinated and degraded in yeast (8–10) whereas mammalian CDC6 is phosphorylated, translocated to the cytoplasm and degraded in mitosis in an ubiquitin-dependent manner (11,12). The activity of CDT1 is essential for CDC6-dependent recruitment of the MCM proteins (13,14). Hence, CDT1 accumulates during G1 phase and is inactivated after onset of S phase to prevent re-replication (15–17). Mammalian MCM proteins associate with chromatin during G1 phase (11,18–20), after the G1/S transition human MCM proteins are progressively released (11). During vertebrate ontogenesis, cells can exit the cell cycle early in G1 phase and enter a quiescent (G0 phase) or terminally diﬀerentiated state (21). It has been proposed that the reduction of proteins involved in DNA replication might contribute to this process (22). For instance, MCM2-7 are depleted both in G0 phase and permanently arrested cells. CDC6 also seems to be reduced in these cells. However, levels of ORC proteins appear to remain high during progression into quiescence (2). In contrast to the conserved mechanism of initiation, the process that determines origin selection is highly divergent. *To whom correspondence should be addressed. Tel: 49 931 888 4042; Fax: 49 931 888 4045; Email: grummt@biozentrum.uni-wuerzburg.de The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. 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.