Purification and characterization of the catalytic subunit of human DNA polymerase delta expressed in baculovirus-infected insect cells.
The catalytic subunit of human DNA polymerase delta has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a Mr = approximately 125,000 and is recognized by polyclonal antisera against N-terminal and C-terminal peptides of the catalytic subunit of human DNA polymerase delta. The recombinant protein was purified to near homogeneity (approximately 1200-fold) from insect cells by chromatography on DEAE-cellulose, phosphocellulose, heparin-agarose, and single-stranded DNA-cellulose. The purified protein had both DNA polymerase and 3'-5' exonuclease activities. The properties of the recombinant catalytic subunit were compared with those of the native heterodimeric DNA polymerase delta isolated from fetal calf thymus, and the enzymes were found to differ in several respects. Although the native heterodimer is equally active with either Mn2+ or Mg2+ as divalent cation activator, the recombinant catalytic subunit is approximately 5-fold more active in Mn2+ than in Mg2+. The most striking difference between the two proteins is the response to the proliferating cell nuclear antigen (PCNA). The activity and processivity of native DNA polymerase delta are markedly stimulated by PCNA whereas it has no effect on the recombinant catalytic subunit. These results suggest that the small subunit of DNA polymerase delta is essential for functional interaction with PCNA.
Zhou
J Q
JQ
Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101, USA.
Tan
C K
CK
So
A G
AG
Downey
K M
KM
D000818:Animals; D002384:Catalysis; D002417:Cattle; D002848:Chromatography, DEAE-Cellulose; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D017924:Nucleopolyhedroviruses / Q000235:genetics; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013950:Thymus Gland / Q000201:enzymology
D018809:Proliferating Cell Nuclear Antigen; D011994:Recombinant Proteins; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase