Figure 4. The DS-element is not required for ORC-dependent replication during S phase.

(A) HCT116 cells were stably transfected with the wild-type oriP p2908 and the DS-deletion ΔDS(p2910). The episomal status of single cell clones was determined by Gardella gel analysis. To estimate the copy number of the mini-EBV genomes, the designated number of cells was analyzed per lane. Raji was used for standardization [55]. (B) The ΔDS-LCL ΔDS(p2910) replicates once per cell cycle during S phase. Both cell lines were synchronized in mitosis with nocodazole. After release the DNA was labeled with BrdU for 18 h and 24 h, when cells were in late S and mitosis respectively. Total DNA of 1×10⁷ cells was separated on a CsCl gradient. The relative concentration of unsubstituted (LL) and hemisubstituted (HL) DNA respectively was determined by real time PCR using mini-EBV (red lines) and the bulk chromosomal DNA (blue lines) and plotted against the CsCl-density. The densities of LL and HL DNA 1.70 and 1.73 g/cm³ are indicated on the right of the graphs.