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Degradation of the viral strand of phiX174 parental replicative-form DNA in a rep- host

K L Bowman, D S Ray

    Journal of Virology
    |October 1, 1975
    PubMed

    Insights

    Parental viral strand degradation during phiX174 replication in E. coli requires gene A-mediated nicking but not strand elongation. Chloramphenicol inhibits degradation and elongation, revealing key steps in viral DNA replication.

    Area of Science:

    • Molecular Biology
    • Virology
    • Bacteriology

    Background:

    • phiX174 replication involves parental viral strand degradation and formation of replicative intermediates.
    • Replicative intermediates sediment at 21s and contain elongated viral strands.

    Purpose of the Study:

    • To investigate the role of gene A-mediated nicking and viral strand elongation in parental viral strand degradation during phiX174 replication.
    • To elucidate the mechanism of replicative intermediate formation.

    Main Methods:

    • Infection of Rep- Escherichia coli with 32P-labeled phiX174.
    • Analysis of DNA sedimentation in neutral and alkaline sucrose gradients.
    • Treatment with chloramphenicol at different concentrations and time points.

    Main Results:

    • Parental viral strand degradation occurred rapidly, with minimal label remaining in the replicative form (RF) by 47 minutes.
    • Degradation was concomitant with the formation of denaturable 21s replicative intermediates containing elongated viral strands.
    • Low-dose chloramphenicol reduced elongation but not degradation; high-dose chloramphenicol prevented both degradation and elongation.

    Conclusions:

    • Gene A-mediated nicking is necessary for parental viral strand degradation.
    • Viral strand elongation is not required for degradation.
    • Chloramphenicol's effect suggests a link between nicking, degradation, and elongation processes.

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