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

doi:10.1074/jbc.271.47.29740

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

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

^¶To whom correspondence should be addressed:
Dept. of Medicine, University of Miami School of Medicine, P. O. Box 016960 (R99), Miami, FL 33101.
Tel.: 305-243-6304; Fax: 305-243-4519.

Abstract

The catalytic subunit of human DNA polymerase δ has been overexpressed in insect cells by a recombinant baculovirus. The recombinant protein has a M_r = ∼125,000 and is recognized by polyclonal antisera against N-terminal and C-terminal peptides of the catalytic subunit of human DNA polymerase δ. 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 δ 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 δ 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 δ is essential for functional interaction with PCNA.

Footnotes

↵* This work was supported by Grant DK26206 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

pol

polymerase

PCNA

proliferating cell nuclear antigen

PCR

polymerase chain reaction

BSA

bovine serum albumin

DTT

dithiothreitol

Bis-Tris

2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-propane-1,3-diol

PAGE

polyacrylamide gel electrophoresis

bp

base pair(s).
↵²J.-Q. Zhou, H. He, C.-K. Tan, K. M. Downey, and A. G. So, manuscript in preparation.
- Received July 15, 1996.
- Revision received September 4, 1996.