Replication of non-repetitive DNA during early, middle and late S phase: The general class and the subset transcribed

Abstract

Synchronized cultures of Chinese hamster ovary cells were pulse-labelled with 5-bromodeoxyuridine (BrdU) during early (0–2.0 h), middle (2.5–4.0 h) and late (4.5–6.0 h) S phase in two successive cell cycles. In each case, the DNA containing BrdU in both strands was duplicated at the same time in both cycles and was isolated for further characterization by centrifugation in CsCl gradients. These DNAs were then radiolabelled by nick-translation and used in either DNA-DNA or RNA-DNA hybridization experiments. In the DNA-DNA experiments, advantage was taken of the substantial rate increases attainable in high concentrations of dextran sulfate to obtain complete reassociation curves with relatively small amounts of material. Assuming that no unresolved low repetition frequency components exist, renaturation kinetics suggest that early replicating DNA contains a greater proportion of non-repetitive sequences than DNA synthesized at later times, the order being early>middle>late. However, in terms of complexity the non-repeated DNA duplicated early had only 74% of the diverse sequences present in logphase cells, whereas that replicated in middle and late S phase had 82 and 79.5%, respectively. It therefore appears that while DNA synthesized at different times in S phase may contain varying proportions of non-repetitive sequences, when their diversity is taken into account very few of these sequences (25% or less) exhibit temporal control of replication. Finally, measurements with total cell RNA indicated that the transcribed fraction of non-repeated DNA showed a slight preference for replication in early S phase.