Expression of the catalytic subunit of human DNA polymerase delta in mammalian cells using a vaccinia virus vector system.
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.
Zhang
P
P
Department of Medicine, University of Miami School of Medicine, Florida 33101, USA.
Frugulhetti
I
I
Jiang
Y
Y
Holt
G L
GL
Condit
R C
RC
Lee
M Y
MY
D000818:Animals; D001483:Base Sequence; D002384:Catalysis; D002460:Cell Line; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004258:DNA Polymerase III; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase / Q000235:genetics* / Q000302:isolation & purification; D005822:Genetic Vectors; D006801:Humans; D008969:Molecular Sequence Data; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D014616:Vaccinia virus / Q000235:genetics*
D017931:DNA Primers; D011994:Recombinant Proteins; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase