Structural and functional organization of herpes simplex virus DNA polymerase investigated by limited proteolysis

J Biol Chem. 1994 Sep 9;269(36):22788-96.


The 1235 residue herpes simplex virus DNA polymerase is a prototype alpha-like DNA polymerase and also an antiviral drug target. To investigate its organization, we mapped favored cleavage sites for seven proteases and identified three major classes of stable proteolytic fragments: 70-85-kDa N-terminal fragments, 50-70-kDa fragments that start near residues 600-700, and 12-kDa C-terminal fragments. In coimmunoprecipitation experiments, the first two classes of fragments remained associated; thus, cleavage in the center of the protein did not resolve structurally separate domains. In contrast, the 12-kDa C-terminal fragments did not remain associated with other fragments, suggesting a small separable C-terminal domain. The 70-85-kDa N-terminal fragments contained 3'-5' exonuclease and ribonuclease H activities; however, cleavage at the center of the molecule or near the C terminus appeared to destroy DNA polymerase activity. All three major classes of fragments bound DNA in DNA-cellulose chromatography and Southwestern blot analyses. The C-terminal fragments bound the viral polymerase processivity factor, UL42. The results map activities to regions of herpes simplex virus polymerase and suggest a model for its organization that may be pertinent to other DNA polymerases.

Publication types

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

MeSH terms

  • Animals
  • Blotting, Western
  • Cell Line
  • DNA-Directed DNA Polymerase / chemistry*
  • DNA-Directed DNA Polymerase / isolation & purification
  • DNA-Directed DNA Polymerase / metabolism
  • Exodeoxyribonuclease V
  • Exodeoxyribonucleases / metabolism
  • Herpesvirus 1, Human / enzymology*
  • Immunoblotting
  • Insecta
  • Peptide Fragments / chemistry
  • Peptide Fragments / isolation & purification
  • Peptide Fragments / metabolism
  • Peptide Mapping
  • Ribonuclease H / metabolism
  • Transfection
  • Viral Proteins / chemistry*
  • Viral Proteins / isolation & purification
  • Viral Proteins / metabolism


  • Peptide Fragments
  • Viral Proteins
  • DNA-Directed DNA Polymerase
  • Exodeoxyribonucleases
  • DNA polymerase, Simplexvirus
  • Exodeoxyribonuclease V
  • Ribonuclease H