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Dideoxynucleoside triphosphates inhibit a late stage of SV40 DNA replication in vitro

P Zahradka1

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

Molecular and Cellular Biochemistry
|March 4, 1992
PubMed

Insights

DNA polymerase alpha is essential for SV40 DNA replication. DNA polymerase epsilon plays a key role in the final stages of replication, as shown by ddTTP inhibition studies.

Area of Science:

  • Molecular Biology
  • Virology

Background:

  • Simian virus 40 (SV40) DNA replication is a model system for understanding eukaryotic DNA replication.
  • DNA polymerases are crucial enzymes involved in DNA synthesis and replication.

Purpose of the Study:

  • To elucidate the specific roles of different DNA polymerases in SV40 DNA replication.
  • To identify the DNA polymerase responsible for the later stages of SV40 replication.

Main Methods:

  • Utilized a T-antigen-dependent assay with human KB cell extract for SV40 DNA replication.
  • Employed aphidicolin and monoclonal antibodies to inhibit DNA polymerase alpha.
  • Used dideoxythymidine triphosphate (ddTTP) at varying concentrations to assess its impact on replication and identify target polymerases.

Main Results:

  • Inhibition of DNA polymerase alpha by aphidicolin or antibodies halted DNA synthesis, confirming its essential role.
  • SV40 replication was unaffected by low ddTTP concentrations (5 µM) that inhibit DNA polymerases beta and gamma.
  • High ddTTP concentrations (200 µM) led to accumulation of relaxed circular plasmid intermediates, indicating near-complete replication cycles.
  • Kinetic analysis suggested DNA polymerase epsilon (PCNA-independent DNA polymerase delta) is inhibited by ddTTP.

Conclusions:

  • DNA polymerase alpha is indispensable for initiating SV40 DNA replication.
  • DNA polymerase epsilon functions in the terminal stages of SV40 DNA replication.

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