Contrasting effects of 2',3'-dideoxyadenosine on two steps in the replication of phi X 174 DNA in Escherichia coli

McFadden, G.; Denhardt, D.T.

Virology 76(2): 870-875


ISSN/ISBN: 0042-6822
PMID: 320756
DOI: 10.1016/0042-6822(77)90268-9
Accession: 068526209

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The nucleoside analog 2',3'-dideoxyadenosine inhibited the replication in vivo of E. coli DNA and bacteriophage .vphi.X174 replicative form (RF) DNA. 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 indicated the presence of multiple gaps in the complementary strand; parental RF synthesized in the presence of the analog had identical characteristics. Multiple sites for initiation of synthesis of a complementary strand on .vphi.X174 viral strand DNA in vivo were indicated. T4 polynucleotide ligase alone was unable to generate covalently closed RF I from these molecules, but in combination with 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 RF replication are suggested.