Figure 4.

Screening of Top1-binding sites in the Bmal1 promoter region. Top1-binding sites were determined by Top1-mediated cleavage assays. Bmal1 promoter region was amplified by PCR using sense (5′-TAACGGGAAGAGGCAGGTATC-3′) and antisense (5′-TCCAGAAGCTCCCGGGGATGTATC-3′) DNA probes. DNA fragments of the Bmal1 promoter region that had been end-labeled with [³²P] on either sense (Left panel) or antisense (Right panel) strand was reacted with either 50 units of Top1 alone or 50 units of Top1 plus 0.5 mM camptothecin. Purified DNA was resolved on 8% polyacrylamide–urea gels adjacent to a sequencing ladder primed with the same ³²P-labeled oligonucleotide as a position marker. Top1-mediated cleavage sites are indicated by triangles and summarized in map. Open boxes and arrows, RORE and labeled probes, respectively. CPT, camptothecin.