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 overexpres sed 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 colu mn and a single-stranded DNA-cellulose column. Levels of expression we re about 1% of soluble cytosolic protein. The recombinant catalytic su bunit was fully active and exhibited enzymatic properties similar to t hat of the native two-subunit enzyme including the possession of an as sociated 3' to 5' exonuclease activity. Recombinant pol delta was stim ulated by proliferating cell nuclear antigen (PCNA); however, the degr ee of stimulation was lower than that of the native human enzyme. Anal ysis 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.