Arrest of Chain Growth of Replicon‐Sized Intermediates by Aphidicolin During Rat Fibroblast Cell Chromosome Replication

Abstract

The effect of aphidicolin, a specific inhibitor of DNA polymerase .alpha., on size maturation of nascent DNA intermediates was studied in cultured rat fibroblast cells. Results provided the 1st evidence of DNA synthesis associated with merging of intermediates of larger than replicon size. Aphidicolin at a concentration (1.4 .mu.g/ml) causing 90-95% inhibition of [3H]thymidine incorporation, resulted in accumulation of intermediates of nearly the same size as the replicon (2-5 .times. 107 Da [dalton]); although the synthesis of short nascent fragments (referred to as Okazaki fragments) continued in the presence of aphidicolin, the rate of their elongation to the replicon size was greatly decreased. On removal of aphidicolin, these accumulated intermediates merged into high-MW DNA. This merging of the intermediates was associated with DNA synthesis in gaps between adjacent intermediates, as revealed by photolysis of bromodeoxyuridine-DNA leader with long-wave UV light; when the cells had been pulse-labeled for 5 min with bromodeoxyuridine immediately after removal of the drug, the large DNA arising from aphidicolin-arrested intermediates was cut into fragments of the original size by long-wave UV irradiation. The arrest of chain elongation at the replicon size by aphidicolin might be due to inhibition of this DNA synthesis in gaps, because aphidicolin did not cause degradation of nascent DNA.