Replication of polyoma DNA in isolated nuclei: II. Evidence for semi-conservative replication

Abstract

Nuclei from polyoma-infected mouse fibroblast 3T6 cells catalysed the incorporation of [ 3H]dTTP into the replicative intermediate of polyoma DNA. The nature of this process was studied by the introduction of a density label into the viral DNA. For this purpose dBrUTP replaced dTTP in the in vitro system and the product formed in the presence of [ 14C]dATP was analysed by equilibrium centrifugation in neutral CsCl or alkaline Cs 2SO 4 gradients. The results suggest that the density label was incorporated into newly synthesized progeny strands and that the incorporation process represented semi-conservative replication. In a second type of experiment the replicative intermediate was first pulselabelled in vivo with [ 3H]thymidine. The isolated nuclei were then incubated in vitro with dBrUTP and [ 14C]dATP. From an analysis of the distribution of the two isotopes in buoyant density gradients we propose that in vitro synthesis represented elongation of essentially all replicative intermediate molecules present in the nuclei at the time of isolation. From the observed density shifts we could calculate that the initial rate of the in vitro elongation process corresponded to a replication time of about 20 minutes and thus may amount to about 20% of the reported in vivo rate.