Uncoupling of SV40 tsA replicon activation from DNA chain elongation by temperature shifts and aphidicolin arrest

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Abstract
To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 40°, to complete ongoing replication and returned to 32°, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 40° without the drug, 3H-dT was incorporated into SV40 FI DNA, almost to the extent seen with cells recovered in the absence of the drug. To determine whether DNA synthesis would begin from the origin, following the temperature-shifts-aphidicolin regimen, chains subsequently pulse-labeled with (α-32P in dGTP isolated nuclei were analyzed for size distribution and genomic location. These chains reached up to 300–400 nucleotides in size, unlike the control which featured comparable amounts of label in long chains and Okazaki pieces. The nascent DNA of the drug-treated system could be chased into longer chains, indicating that it was a replicative intermediate; and it hybridized preferentially to an origin proximal fragment of AtuI restricted SV4O DNA, demonstrating partial replicon synchronization.