Recently replicated simian virus 40 DNA is a preferential template for transcription and replication

Abstract

The template activities for the processes of replication and transcription were compared for recently replicated ("new") and uniformly labeled ("old") simian virus 40 (SV40) DNA in infected monkey cells (line TC7). New SV40 DNA (pulse-labeled for 1 h) served as a template for a second round of replication with a relatively high probability (8% of the DNA replicated per h) for a period of 5 h, after which time its template activity rapidly decreased by severalfold. Old SV40 DNA (labeled for 24 h) functioned as a template for replication with a constant probability (1.8% of the DNA replicated per h) for at least 12 h. The proportion of RNA polymerase with nonreplicated and with recently replicated (bromodeoxyuridine-substituted) viral DNA was determined by an assay that used the Triton-soluble SV40 transcription complex. The proportion of RNA polymerase associated with nonreplicated SV40 DNA decreased very slowly (to 50% in 6 h), strongly suggesting that replicating viral genomes are not required as templates for the initiation of late transcription. This hypothesis was supported by the finding that the RNA synthesized in vitro was associated with covalently closed circular SV40 DNA. Furthermore, after 9 h in bromodeoxyuridine, the recently replicated viral DNA had nearly three times more RNA polymerase per unit of DNA than did the nonreplicated DNA. We thus conclude that recently replicated SV40 DNA is utilized preferentially as a template for transcription and for replication.