Characterization of the human DNA polymerase delta catalytic subunit expressed by a recombinant baculovirus.
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.
Suzuki
S
S
Medical Biological Institute, Nagoya, Japan.
Suzuki
M
M
Yoshida
S
S
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002417:Cattle; D002460:Cell Line; D004258:DNA Polymerase III / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D018076:DNA, Complementary / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D021122:Protein Subunits; D011994:Recombinant Proteins; D004258:DNA Polymerase III