SV40 T antigen binds directly to the large subunit of purified DNA polymerase alpha

EMBO J. 1990 Oct;9(10):3329-36.

Abstract

Purified SV40 large T antigen and purified DNA polymerase alpha-primase form a complex detectable by ELISA and by a modified immunoblotting technique. The interaction is specific for the large catalytic subunit of polymerase alpha. The amino terminal 83 amino acids of T antigen are both necessary and sufficient for binding to the polymerase. However, antibody epitopes located in the carboxy terminal ATPase domain of T antigen are masked in the polymerase-T antigen complex, and complex formation is inhibited by an antibody directed against the carboxy terminus of T antigen, suggesting that this region of T antigen, though not required for binding, is in close proximity to the bound polymerase. The affinity of human DNA polymerase alpha for T antigen is approximately 10-fold greater than that of polymerase alpha from calf thymus, consistent with the interpretation that polymerase alpha is at least in part responsible for the primate-specific replication of SV40 DNA in vivo and in vitro. The results suggest that specific protein-protein interaction between DNA polymerase alpha and T antigen plays an important role in viral DNA replication.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antigens, Polyomavirus Transforming*
  • Binding Sites
  • Cattle
  • Chromatography, Affinity
  • Chromatography, Ion Exchange
  • DNA Polymerase II / immunology
  • DNA Polymerase II / isolation & purification
  • DNA Polymerase II / metabolism*
  • DNA Primase
  • Enzyme-Linked Immunosorbent Assay
  • Epitopes / analysis
  • Immunoblotting
  • Kinetics
  • Macromolecular Substances
  • RNA Nucleotidyltransferases / immunology
  • RNA Nucleotidyltransferases / isolation & purification
  • RNA Nucleotidyltransferases / metabolism*
  • Recombinant Proteins / metabolism
  • Simian virus 40 / immunology*
  • Simian virus 40 / metabolism
  • Thymus Gland / enzymology

Substances

  • Antigens, Polyomavirus Transforming
  • Epitopes
  • Macromolecular Substances
  • Recombinant Proteins
  • DNA Primase
  • RNA Nucleotidyltransferases
  • DNA Polymerase II