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Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replicationin vitro

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Summary

The role of DNA polymerases in the replication of SV40 DNA was studied using a T-antigen-dependent assay supplemented with a human KB cell extract. Inhibition of DNA polymerase α by addition of aphidicolin or monoclonal antibodies prevented DNA synthesis, confirming the requirement for this enzyme in replication. The replication process was unaffected by ddTTP at a concentration (5 µM) inhibitory to DNA polymerases β and γ, however, higher concentrations of ddTTP (200 µM) caused an apparent accumulation of relaxed circular plasmid with a concomitant decrease in DNA synthesis. An analysis of this replication intermediate indicated that it was formed during the replication reaction and that the replicative cycle was nearly complete. A kinetic study of ddTTP inhibition strongly suggested DNA polymerase ε (PCNA-independent DNA polymerase δ) was the target of the inhibitor and that this enzyme functions during the final stages of DNA replication.

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  1. Department of Chemistry and Biochemistry, University of Guelph, Guelph, Ontario, Canada

    Peter Zahradka

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Zahradka, P. Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replicationin vitro . Mol Cell Biochem 110, 65–73 (1992). https://doi.org/10.1007/BF02385007

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