Characterization of the human DNA polymerase delta catalytic subunit expressed by a recombinant baculovirus

Nagoya J Med Sci. 2000 Nov;63(3-4):99-113.


The catalytic subunit of human DNA polymerase (pol) delta, p125, was expressed in recombinant baculovirus-infected insect cells, separated from a baculovirus-encoded DNA polymerase, and was purified to homogeneity by affinity trapping with a histidine-octapeptide at the C-terminus of p125 as the ligand. Purified p125 showed DNA polymerase activity resembling conventionally purified calf thymus pol delta. However, the two differed in four ways: 1) the specific activity of recombinant p125 was one quarter of the calf thymus pol delta; 2) the recombinant p125 was relatively resistant to aphidicolin; 3) the apparent Km for dTTP of the recombinant p125 was estimated at 33 microM, 15-fold the value for calf thymus pol delta; and 4) the recombinant p125 was not stimulated by recombinant PCNA, while activity of calf thymus pol delta increased 150-fold in response. Furthermore, PCNA did not stimulate either the p125 incubated with p50, a small subunit of pol delta, or co-expressed with p50 in insect cells. The full length recombinant p125 migrated slightly faster than pol delta from human cell lines, Jurkat or HeLa, upon SDS-polyacrylamide gel electrophoresis, suggesting a post-translational modification. The results indicate that in vivo assembly of the fully active complex of pol delta requires factors in addition to p125 and p50 subunits, and/or a post-translational modification of p125.

MeSH terms

  • Animals
  • Baculoviridae / genetics
  • Base Sequence
  • Cattle
  • Cell Line
  • DNA Polymerase III / genetics
  • DNA Polymerase III / isolation & purification*
  • DNA Polymerase III / metabolism
  • DNA Primers / genetics
  • DNA, Complementary / genetics
  • Humans
  • In Vitro Techniques
  • Protein Subunits
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism


  • DNA Primers
  • DNA, Complementary
  • Protein Subunits
  • Recombinant Proteins
  • DNA Polymerase III