Replication of polyoma DNA in nuclei isolated from azidocytidine-inhibited fibroblasts.

2'-Deoxy-2'-azidocytidine was earlier shown to inhibit the synthesis of DNA (but not RNA or protein) in polyoma virus-infected 3T6 fibroblasts.The diphosphate of azidocytidine is a known inhibitor of ribonucleotide reductase, but deoxyribonucleotide pools of inhibited cells were only marginally affected.Nuclei isolated from the inhibited cells incorporated dNTPs into polyoma DNA at a fairly normal rate with incorporation occurring mainly around the origin of replication.This led to the earlier suggestion that azidocytidine interferes with origin initiation.We now investigate polyoma DNA replication in nuclei isolated after azidocytidine inhibition of cells from a hydroxyurea-resistant cell line that contains an increased activity of ribonucleotide reductase.The nuclei rapidly incorporated labeled dNTPs into polyoma DNA progeny strands which between 10 s and 15 min grew from an average length of 100 to 1600 nucleotides.Incorporation initially occurred to a large extent around the origin of replication, but a substantial part of isotope was also recovered more distant from the origin.All strands were elongated at similar rates irrespective of their location.With 5-bromodeoxyuridine triphosphate replacing d"P, only 28% of the thymine of progeny strands was substituted by bromouracil after 20 s of incubation.This value increased to 76% after 16 min.These data demonstrate that synthesis in isolated nuclei starts from short primers, preferentially but not exclusively located in the origin region, and suggest that azidocytidine in intact cells inhibits chain elongation and not origin initiation.