6706 Nucleic Acids Research, 2007, Vol. 35, No. 20 Figure 2. Sequence-dependent association of pre-RC proteins with the OBR throughout the cell cycle in vivo. (A) FACS analyses showing cell cycle arrest of FM3A cells in G1 phase (82.6%), at the G1/S boundary (82.8%) and in S phase (66.8%). (B) Synchronized FM3A cells were treated with formaldehyde, cell lysates were prepared and subjected to immunoprecipitation using antibodies against ORC1, -2, -4 and -5 or using preimmune sera (control). Representative results of three experiments are shown. E11 bound fragment A (Figure 4C, left panel). Proteins of fraction E5 showed no signiﬁcant binding activity to fragments C or D as well (Figure 4C, middle and right panel). Thus, proteins of fraction E5 bind preferentially to fragment B, suggesting that complex A is formed sequence selectively. In contrast, complex B is formed on fragments B, C and D indicating sequence-unspeciﬁc DNA binding of proteins in fraction E11. To further conﬁne the pre-RC-binding site within fragment B, this fragment was shortened by 100 and 200 bp, respectively, either at the 30 or at the 50 end, (Figure 1; 3. 100; 3. 200; 5. 100; 5. 200). As shown in Figure 5A, lane 2 and 3, complex A is formed with fragments truncated 100 bp at their 30 or 50 ends. However, if 200 bp are deleted, formation of the protein/DNA complex is abrogated in both cases (Figure 5A, lanes 4 and 5). This indicates that a DNA sequence in the middle of fragment B is essential for the formation of complex A. EMSA experiments with a 119 bp fragment located in the middle of fragment B ( 2365 to 2247, see Figure 1) showed that complex A is still formed in an ATP- dependent manner (Figure 5A, lanes 7 and 8). Thus, this 119 bp DNA fragment is suﬃcient for pre-RC/DNA complex formation. Inspecting this central 119 bp region closely, two conspicuous 9 bp sequence elements, CTCGGGAGA, were observed which are repeated at intervals of 63 bp ( 2343 to 2335; 2280 to 2272) and which are completely or partially disrupted by the 200 bp deletions. To examine whether these sequence elements play a role in formation of complex A, we altered the sequence by introducing C to A, T to G and vice versa substitutions Figure 3. Puriﬁcation and analysis of proteins from murine nuclear extracts. (A) Nuclear extracts from FM3A cells were puriﬁed twice on cationic exchange columns and once by gel ﬁltration on a SuperdexTM200 10/300 GL column, calibrated with thyroglobulin and ferritin. To test for DNA-binding activity aliquots of fractions E4 to E15 were subjected to EMSA with 10 fmol g32P-ATP 50 end-labeled fragment B in the presence of 5 mM ATP and 250 ng poly dI+dC. Protein/DNA complexes A and B are indicated. (B) Immunoblot analysis of fractions E5 and E11 with antibodies raised against ORC1, -2, -4 and -5, respectively. using site-speciﬁc mutagenesis. Four successive substitu- tions of 4 bp each were constructed, whereby the obtained constructs were used as template DNA for the subsequent mutagenesis (119.S1, 119.S2, 119.S3 and 119.S4; for sequences, see Table 1). EMSA experiments carried out with the mutated 119 bp DNA fragments showed that substitutions 1 (119.S1; lanes 9 and 10), 2 (119.S2, lanes 11 and 12) and 3 (119.S3, lanes 13 and 14) did not aﬀect formation of the DNA/protein complexes. However, if 8 out of 9 bp in both sequence elements (119.S4) are substituted, formation of complex A is signiﬁcantly reduced (lanes 15 and 16) indicating that the CTCGGGGAGA sequences might inﬂuence binding of pre-RC proteins to DNA in the murine rDNA locus. Interestingly, a naked DNA fragment containing all four