6710 Nucleic Acids Research, 2007, Vol. 35, No. 20 Cell cycle-dependent binding of initiation proteins to fragment B Using synchronized FM3A cells, we demonstrated that an ORC core complex consisting of ORC 2, -4, -5 was stably bound to fragment B throughout the cell cycle, whereas ORC1 exhibited a cell cycle-dependent association. During G1 phase and at the G1/S transition, ORC1, -2, -4 and -5, were bound to fragment B. In S phase, ORC1 dissociated from the initiation complex. Compared to yeast and Drosophila, where ORC remains associated with chromatin throughout the cell cycle (37–39), ORC binding in mammalia is cell cycle- dependent. The association patterns we observed in murine cells are similar to those described for other species. In hamster cells, ORC1 is released from chromatin as cells enter S phase and is re-bound to chromatin during M to G1 transition (6,7). Similarly, we found that murine ORC1 is bound to chromatin in G1 and at the G1/S transition, but not in S phase. Furthermore, we demon- strated that ORC2 remains bound to chromatin through- out the cell cycle. In a recent study, it was shown also that human ORC1 and -2 bind at an origin of DNA replication in G1 phase but in S phase only ORC2 remains bound (5). The pre-RC has an intrinsic sequence-selective DNA-binding activity We have demonstrated that puriﬁed nuclear extracts containing ORC proteins bind ATP dependently and sequence speciﬁcally to fragment 119. The question arises why this sequence is preferred compared to adjacent sequences. A possibility for ORC to ﬁnd its binding site is the presence of ancillary factors promoting chromatin bind- ing. It has been proposed that CDC6 might reduce the aﬃnity of ORC to non-speciﬁc DNA and therefore acts as an essential determinant for origin speciﬁcity in metazoans (40,41). In the case of Epstein-Barr virus, the viral transcription factor EBNA-1 binds ORC and may be involved in the recruitment of ORC to oriP (42). It is highly probable that we copuriﬁed CDC6 or a yet unknown protein, which directs ORC to its binding site and that ORC itself is not responsible for sequence- speciﬁc binding to fragment B. The identiﬁcation of all proteins involved in the formation of the DNA/protein complexes by mass spectrometry would be very interesting and remains to be done. We conﬁned the pre-RC-binding site to 119 bp within fragment B and found that a repetitive 9 bp sequence element, CTCGGGAGA, plays a crucial role in ensuring eﬃcient loading of pre-RC proteins to DNA. In contrast to yeast where the ORC binds to deﬁned sequences called ARS, origins in higher eukaryotes do not appear to contain consensus sequences, although some origins include AT-rich sequences (43). Fragment 119, however, is not particularly AT-rich, since its AT content is only 37% and no AT-rich nucleotide clusters are to be found. By substitutions S1–S4, the AT content is stepwise increased. On S4, the fragment with the highest AT content (44%), nearly no binding of pre-RC proteins was observed. We therefore propose that a higher AT content is not essential for ORC–DNA interaction within frag- ment 119. If the 9 bp elements represent a consensus binding site for pre-RC proteins or for ancilliary factors recruiting pre-RC proteins to origin DNA, these elements are to be found in other origin regions. However, these elements could neither be detected with 100% identity in the origins of the human MCM4/PRKD (U90415), TOP1 (AL035652) and Lamin B2 (M94363) genes nor in the Sciara puﬀ II/9A origin (AF332610). In the TOP1 gene two sequences with 88.8% (CTGGGGAGA) and 77.7% (CTCCGGAGT) and in the MCM4/PRKD gene with 77.7% (CCCGGGAAA) and 66.6% (CCTCCCGGA) identity, respectively, were found. However, these ele- ments are 588 and 620 bp, respectively, apart from each other, and hence are located at a greater distance compared to those in the murine rRNA genes. Interestingly, the distance of 63 bp between both sequence elements corresponds to six helical turns of B DNA (10.5 bp/helical turn). Intrinsically bent or curved DNA molecules result when distinct base sequences or structural motifs are repeated in phase with the DNA helical repeat (44). Signiﬁcantly, substitution 4 within the 9 bp sequence element leads to a slight, but measurable decrease of the relative electrophoretic mobility of the naked DNA, indicating an altered secondary structure as a result of substitution S4. Therefore, the 9 bp sequence element probably does not constitute a consensus binding sequence per se, but rather has eﬀects on the formation of speciﬁc secondary DNA structures, which could allow binding of the pre-RC or its recruitment factor to this region of the murine rDNA. Remarkably, the 9 bp sequence element is to be found a third time within the 119 bp fragment ( 2269 to 2261: CTCCGGAGA) with 88.8% identity and inverted CTCGGGAGA elements are found three times in the murine rRNA genes on the coding (+1794 to +1802: AGAGGGCTC) and non-coding strand (+11238 to +11246: TCTCCCGAG; +12980 to +12988: TCTCCCGAG). It has been described that more active initiation sites exists in the murine rDNA located either between + 2.0 and 8.0 kb or approximately at + 13 kb downstream of the transcription start site (32). Interestingly, an ORC2-binding site was localized by ChIP in the region from +1642 to +2082 including one of the 9 bp sequence elements described above (unpublished data). Also, in the region +13 kb an inverted CTCGGGAGA element was found on the non-coding strand. However, so far this region has not been studied in respect of pre- RC-binding sites. Since pre-RC proteins exhibited rela- tively weak binding to fragment B in vitro, attempts to detect a speciﬁc DNA sequence by DNase I footprint experiments were unsuccessful (data not shown). Despite the sequence-speciﬁc binding of pre-RC proteins to fragment 119 and the evidence that the 9 bp sequence elements play a crucial role in this process, it is possible that the in vivo binding of pre-RCs to origins might be