JBC INTERFERin siRNA transfection reagent

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Volume 271, Number 47, Issue of November 22, 1996 pp. 29740-29745
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

Purification and Characterization of the Catalytic Subunit of Human DNA Polymerase delta  Expressed in Baculovirus-infected Insect Cells

(Received for publication, July 15, 1996, and in revised form, September 4, 1996)

Jin-Qiu Zhou Dagger , Cheng-Keat Tan § , Antero G. So Dagger § and Kathleen M. Downey Dagger §

From the Dagger  Department of Biochemistry and Molecular Biology and the § Department of Medicine, University of Miami School of Medicine, Miami, Florida 33101

The catalytic subunit of human DNA polymerase delta  has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a Mr = ~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.


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