Identification of DNA polymerase δ in CV-1 cells: studies implicating both DNA polymerase δ and DNA polymerase α in DNA replication

DNA polymerases δ and α were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl) deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase α were determined. The effects of these inhibitors on DNA replication, measured with [3H]TTP, in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases δ and α in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase α or δ activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase α or δ activity. Concentrations of BuPdGTP which totally abolished DNA polymerase α activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase δ. Monoclonal antibodies which specifically inhibited polymerase α activity reduced, but did not abolish, DNA replications in permeable cells. BuPdGTP, as well as anti-polymerase α antibodies, inhibited DNA replication in a nonlinear manner as a function of time. A concentration of BuPdGTP which abolished polymerase α activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments. This information suggests that polymerases α and δ are involved in both the synthesis and maturation of nascent DNA. This is the first report to present evidence suggesting that both polymerases α and δ play a significant role in mammalian DNA replication