Expression of the catalytic subunit of human DNA polymerase delta in mammalian cells using a vaccinia virus vector system

J Biol Chem. 1995 Apr 7;270(14):7993-8. doi: 10.1074/jbc.270.14.7993.


The catalytic polypeptide of human DNA polymerase delta was overexpressed in BSC-40 cells (African green monkey kidney cell line) using the vaccinia virus/pTM1 system. The recombinant human DNA polymerase delta was purified to homogeneity in two steps using an immunoaffinity column and a single-stranded DNA-cellulose column. Levels of expression were about 1% of soluble cytosolic protein. The recombinant catalytic subunit was fully active and exhibited enzymatic properties similar to that of the native two-subunit enzyme including the possession of an associated 3' to 5' exonuclease activity. Recombinant pol delta was stimulated by proliferating cell nuclear antigen (PCNA); however, the degree of stimulation was lower than that of the native human enzyme. Analysis of a double mutant of the catalytic subunit, H142R/F144S, showed that it had a greatly reduced sensitivity to PCNA, suggesting that the PCNA binding site of pol delta may be located in this region of the N terminus.

Publication types

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

MeSH terms

  • Animals
  • Base Sequence
  • Catalysis
  • Cell Line
  • Chlorocebus aethiops
  • Cloning, Molecular
  • DNA Polymerase III
  • DNA Primers
  • DNA-Directed DNA Polymerase / genetics*
  • DNA-Directed DNA Polymerase / isolation & purification
  • Genetic Vectors*
  • Humans
  • Molecular Sequence Data
  • Mutation
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Vaccinia virus / genetics*


  • DNA Primers
  • Recombinant Proteins
  • DNA Polymerase III
  • DNA-Directed DNA Polymerase