Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen

Mol Cell Biol. 1993 Feb;13(2):809-20. doi: 10.1128/mcb.13.2.809-820.1993.

Abstract

Initiation of cell-free simian virus 40 (SV40) DNA replication requires the interaction of DNA polymerase alpha/primase with a preinitiation complex containing the viral T antigen and cellular proteins, replication protein A, and topoisomerase I or II. To further understand the molecular mechanisms of the transition from preinitiation to initiation, the intermolecular interaction between human DNA polymerase alpha and T antigen was investigated. We have demonstrated that the human DNA polymerase alpha catalytic polypeptide is able to associate with SV40 large T antigen directly under physiological conditions. A physical association between these two proteins was detected by coimmunoprecipitation with monoclonal antibodies from insect cells coinfected with recombinant baculoviruses. A domain of human polymerase alpha physically interacting with T antigen was identified within the amino-terminal region from residues 195 to 313. This domain of human polymerase alpha was able to form a nonproductive complex with T antigen, causing inhibition of the SV40 DNA replication in vitro. Kinetics of the inhibition indicated that this polymerase domain can inhibit viral replication only during the preinitiation stage. Extra molecules of T antigen could partially overcome the inhibition only prior to initiation complex formation. The data support the conclusion that initiation of SV40 DNA replication requires the physical interaction of T antigen in the preinitiation complex with the amino-terminal domain of human polymerase alpha from amino acid residues 195 to 313.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, Viral, Tumor / metabolism*
  • Base Sequence
  • Binding Sites
  • Catalysis
  • DNA Polymerase II / metabolism*
  • DNA Replication*
  • DNA, Viral / biosynthesis
  • Enzyme-Linked Immunosorbent Assay
  • Humans
  • Molecular Sequence Data
  • Precipitin Tests
  • Recombinant Fusion Proteins / metabolism
  • Simian virus 40 / genetics*

Substances

  • Antigens, Viral, Tumor
  • DNA, Viral
  • Recombinant Fusion Proteins
  • DNA Polymerase II