Nucleic Acids Research, 2007, Vol. 35, No. 20 6705 Figure 1. Pre-RC proteins are associated with region B in the origin of bidirectional replication (OBR). (A) Scheme of the origin of replication in murine rDNA. The transcription start site is marked as +1. OBR designates the origin of bidirectional replication mapped by nascent strand determination analysis (32). DNA fragments used in ChIP and EMSA assays are denoted according to the nucleotide position at the 50 and at the 30 ends relative to the transcription start site. The 119 bp fragment used in EMSA experiments is marked by arrows. The conspicuous 9 bp repetitive sequence elements are underlined. (B) Cross-linked chromatin was immunoprecipitated with antibodies against ORC2 (aORC2) and preimmune serum (control). Isolated DNA was analyzed with primer pairs ﬂanking regions A, B, C and D, respectively. PCR products of the corresponding chromatin samples before immunoprecipitation are shown (input). components to fragment B or whether they are due to the interaction with unknown proteins. Next, the eﬀect of ATP on the formation of complexes A and B was examined. The presence of two ATP-binding subunits in eukaryotic ORCs (27,34–36) lead us to expect an ATP-dependent increase in DNA binding if complex A or B contain these pre-RC components. The addition of increasing amounts of ATP to fraction E5 results in a dose-dependent increase of the formation of complex A, not however, on the formation of complex B (Figure 4A). This indicates that complex A, but not complex B, is probably caused by the binding of pre-RC proteins to origin DNA. To prove an involvement of pre-RC proteins in the formation of complex A more directly, we performed supershift experiments using increasing amounts of antibodies against ORC2, as a representative for pre-RC components. Including ORC2 antibodies into the DNA/ protein-binding reaction led to a further retardation of the mobility of complex A (Figure 4B), indicating that ORC2 is part of complex A. In contrast, antibodies against ORC2 had no eﬀect on the mobility of complex B (data not shown), again hinting to an ORC-independent complex formation in the latter case. Sequence specificity of pre-RC binding To investigate whether proteins in fraction E5 and E11 bind to fragment B in a sequence-speciﬁc manner, EMSA studies were carried out comparing binding to DNA fragments A, C and D (Figure 1A), both in the presence and absence of ATP. Neither proteins in fraction E5 nor