Identification of DNA Polymerase δ in CV-1 Cells: Studies Implicating both DNA Polymerase δ and DNA Polymerase a in DNA Replication

Russell A. Hammond, John J. Byrnes, Michael R. Miller

Research output: Contribution to journalArticle

60 Scopus citations

Abstract

DNA polymerases δ and α were purified from CV-1 cells, and their sensitivities to the inhibitors aphidicolin, (p-n-butylphenyl)deoxyguanosine triphosphate (BuPdGTP), and monoclonal antibodies directed against DNA polymerase α were determined. The effects of these inhibitors on DNA replication in permeabilized CV-1 cells were studied to investigate the potential roles of polymerases δ and α in DNA replication. Aphidicolin was shown to be a more potent inhibitor of DNA replication than of DNA polymerase α or δ activity. Inhibition of DNA replication by various concentrations of BuPdGTP was intermediate between inhibition of purified polymerase α or δ activity. Concentrations of BuPdGTP which totally abolished DNA polymerase α activity were much less effective in reducing DNA replication, as well as the activity of DNA polymerase δ. Monoclonal antibodies which specifically inhibited polymerase α activity reduced, but did not abolish, DNA replication in permeable cells. BuPdGTP, as well as anti-polymerase α antibodies, inhibited DNA replication in a nonlinear manner as a function of time. Depending upon the initial or final rates of inhibition of replication by BuPdGTP and anti-α antibodies, as little as 50%, or as much as 80%, of the replication activity can be attributed to polymerase α. The remaining replication activity (20-50%) is tentatively attributed to polymerase δ, because it was aphidicolin sensitive and resistant to both anti-polymerase α antibodies and low concentrations of BuPdGTP. A concentration of BuPdGTP which abolished polymerase α activity reduced, but did not abolish, both the synthesis and maturation of nascent DNA fragments. This information suggests that polymerases α and δ are involved in both the synthesis and maturation of nascent DNA. This is the first report to present evidence suggesting that both polymerases α and δ play a significant role in mammalian DNA replication.

Original languageEnglish (US)
Pages (from-to)6817-6824
Number of pages8
JournalBiochemistry
Volume26
Issue number21
DOIs
StatePublished - Jan 1 1987

ASJC Scopus subject areas

  • Biochemistry

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