DNA replication in SV40-infected cells: VIII. The distribution of replicating molecules at different stages of replication in SV40-infected cells

Abstract

When 3H-thymidine is added to SV40 infected African green monkey kidney cells little or no mature viral DNA (21 S) is labeled during the first 5–7 min of isotope incorporation. The 3H-thymidine is incorporated instead into replicating viral DNA (25 S DNA). The rate of incorporation of isotope into 25 S plus 21 S DNA is linear with respect to time throughout the entire labeling period. Replicating molecules of SV40 DNA at different stages of replication can be fractionated by ethidium-bromide cesium chloride equilibrium centrifugation. Molecules that had completed 80% of their replication were about 6 times more frequent than molecules having completed 18% replication, 3 times more abundant than molecules that were 32% replicated and 2 times more prevalent than molecules 58% replicated. This unusual distribution of the replicating DNA molecules in the replicative pool may explain the delay in the appearance of mature viral DNA after addition of isotope to infected cells. The sedimentation rate of replicating SV40 DNA can be altered in neutral sucrose gradients by the addition of ethidium-bromide or changes in the ionic strength of the sucrose gradient. The experimental evidence demonstrates that the sense of the superhelix found in replicating and mature SV40 DNA is identical. The implications of these observations are discussed in the context of the available known experimental facts about SV40 DNA replication.