Title

Purification and characterization of recombinant human DNA polymerase (delta) expressed in baculovirus-infected insect cells

Date of Award

1997

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Antero G. So, Committee Chair

Abstract

DNA polymerase $\delta$ is usually isolated as a heterodimer composed of a 125-kDa catalytic subunit and a 50-kDa small subunit of unknown function. The enzyme is distributive by itself and requires an accessory protein, the proliferating cell nuclear antigen (PCNA), for highly processive DNA synthesis. Both the catalytic and small subunits of human DNA polymerase $\delta$ have been overexpressed in baculovirus-infected insect cells. The recombinant catalytic and small subunits are approximately 125-kDa and 50-kDa, respectively. The recombinant catalytic subunit was purified to near homogeneity from insect cells by chromatography on DEAE-cellulose, phospho-cellulose, heparin-agarose and single-stranded DNA-cellulose. The purified protein had both DNA polymerase and 3$\sp\prime$ to 5$\sp\prime$ exonuclease activities. Two subunits of human DNA polymerase $\delta$ have also been co-expressed and partially purified. The co-expression of the catalytic and small subunits of human DNA polymerase $\delta$ resulted in the formation of a stable, fully functional heterodimer.The properties of the recombinant catalytic subunit and the recombinant heterodimeric DNA polymerase $\delta$ were compared with those of the native heterodimeric DNA polymerase $\delta$ isolated from calf thymus. The catalytic subunit was found to differ from the native enzyme in several respects, however, the recombinant heterodimeric DNA polymerase $\delta$ was found to be very similar to the native enzyme. The most striking difference between the catalytic subunit and heterodimeric forms of DNA polymerase $\delta$ was that the recombinant catalytic subunit was not responsive to stimulation by PCNA, whereas the recombinant heterodimer, similar to native heterodimer, was markedly stimulated (40- to 50-fold). The increase in activity seen in the presence of PCNA was shown to be the result of an increase in processivity. These data establish that the 50-kDa subunit is essential for functional interaction of DNA polymerase $\delta$ with PCNA and for highly processive DNA synthesis.The recombinant small subunit was purified to near homogeneity from insect cells by ammonium sulfate precipitation and chromatography on heparin-agarose, source-15Q and Sephacryl S-300. The small subunit was shown to increase the binding affinity of the catalytic subunit to the template/primer, resulting in the 2-fold increase in the processivity of heterodimeric enzymes. In addition, both the small subunit and native heterodimeric enzyme exist as dimers in solution, whereas the catalytic subunit is monomeric, implying that the small subunit causes the dimerization of polymerase $\delta$. These data suggest that the small subunit of eukaryotic DNA polymerase $\delta$ is a homologue of the $\tau$ subunit of E. coli DNA polymerase III.

Keywords

Biology, Molecular

Link to Full Text

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