Contrasting effects of 2′,3′-dideoxyadenosine on two steps in the replication of φX174 DNA in Escherichia coli

Abstract

The nucleoside analogue 2′,3′-dideoxyadenosine inhibited the replication in vivo of both Escherichia coli DNA and bacteriophage X174 replicative form (RF) DNA. However, synthesis of the complement to the infecting single-stranded viral DNA to form the parental RF was not inhibited by the drug. Gap-filling experiments, sedimentation velocity analysis, and treatment of the parental RF synthesized in a polA host with the Neurospora crassa single-strand-specific nuclease together provided evidence for the presence of multiple gaps in the complementary strand; parental RF synthesized in the presence of the analogue had identical characteristics. This is evidence for multiple sites for initiation of synthesis of a complementary strand on φX174 viral strand DNA in vivo. T4 polynucleotide ligase alone was unable to generate covalently closed RF I from these molecules, but in combination with either T4 DNA polymerase or E. coli DNA polymerase I, RF I was efficiently synthesized, suggesting that the parental RF did not have 2′,3′-dideoxyadenosyl residues terminally incorporated in the nascent complementary strand DNA. Possible reasons for the differential effect of 2′,3′-dideoxyadenosine on complementary strand synthesis and RIP replication are suggested.