Gene replication in the presence of aphidicolin

DNA replication in the nucleus of eukaryotic cells is restricted to the S phase of the cell cycle, and different genes are duplicated at specific times, according to a well-defined temporal order. We have investigated whether activation of initiation sites, in proximity to genes that are replicated in different portions of the S phase, could be detected when synchronized 10T1/2 cells were maintained in aphidicolin (APC), an inhibitor of DNA polymerases alpha and delta. Cells released from confluence arrest into medium containing 2 micrograms/mL APC progressed into the S phase, and nascent DNA accumulated during incubations of 24 and 32 h. Exposure to APC for 40 or 48 h resulted in growth of the radiolabeled DNA into larger molecules. Replicating DNA was isolated in CsCl gradients and probed with 32P-labeled gene probes for early-replicating genes (e.g., Ha-ras, mos, and myc) and a late-replicating gene (VH Ig). DNA replicated during the 24-h incubation in APC was enriched in Ha-ras gene sequences. The VH Ig gene did not replicate in cells incubated for as long as 56 h with APC. The myc and the mos genes were detected after 32 and 40 h in APC, respectively. The myc gene is replicated in 10T1/2 cells after Ha-ras but before mos. Therefore, the order of activation of these genes was conserved in the presence of APC. The delay in replication of myc and mos correlated well with the slowing of DNA replication by APC.