Nucleic Acids Research, 2007, Vol. 35, No. 20 6707 Figure 4. DNA/protein complex A binds ATP dependently and sequence selectively to fragment B and is supershifted with antibodies against ORC2. (A) EMSAs were performed with proteins of fractions E5 or E11 (300 ng) and g32P-ATP 50 end-labeled fragment B with increasing concentrations of ATP (0.5, 1, 2.5 or 5 mM). (B) For supershifting, binding reactions were performed by incubating g32P-ATP 50 end-labeled fragment B and proteins of fraction E5 in the presence of 5 mM ATP with increasing amounts of antibodies against ORC2 as indicated. (C) EMSAs were performed with proteins of fractions E5 or E11 and fragments A, C and D in the absence or presence of 5 mM ATP. substitutions (Figure 5B, 119.S4) migrates distinctly slower than protein-free WT 119 and 119.S1–119.S3 (lanes 1–4 and 6) suggesting that the secondary structure of the DNA is altered as a result of the S4 nucleotide substitutions. Taken together, we were able to reproduce in vitro by EMSA the sequence-speciﬁc binding of pre-RC proteins to DNA shown in vivo by ChIP experiments. Formation of complex A, which contains ORC2 as conﬁrmed by supershifting, is stimulated by ATP and occurs in a sequence-speciﬁc manner, preferring fragment B over A, C and D. Moreover, mutational analyses of fragment B revealed that (i) the pre-RC-binding site is situated within 119 bp, from 2365 to 2247 upstream the transcription start site and that (ii) two sequence elements, CTCGGGAGA, are crucial for the binding of pre-RC proteins to DNA. Myogenic differentiation of C2C12 cells Recent studies on various organisms have shown that in terminally diﬀerentiated cells or tissues some components of the pre-RC are absent (2). The loss of pre-RC components upon cell cycle exit could be a regulatory mechanism to suppress cell proliferation. To address this issue experimentally, we analyzed protein levels of pre-RC components during myogenic diﬀerentiation and determined whether components of the pre-RC are attached to the OBR in terminally diﬀer- entiated myotubes. For that we used murine C2C12 cells, which can be induced to terminal diﬀerentiation into myotubes by exposure to low mitogen medium for several days. Diﬀerentiation was assessed by morphological examination. After 5 days in diﬀerentiation medium (DM), predominantly elongated myotubes were present in the culture (Figure 6A, upper panel). The percentage of S phase cells considerably decreased during myogenic diﬀerentiation (34.4% in proliferating versus 2.4% in diﬀerentiated cells) concomitantly with a signiﬁcant increase in the percentage of myotubes in the G0/G1 phase of the cell cycle (54.4% in proliferating versus 95.6% in diﬀerentiated cells, middle panel). The cessation