Catalytic subunit of human DNA polymerase alpha overproduced from baculovirus-infected insect cells. Structural and enzymological characterization.
The human DNA polymerase alpha catalytic polypeptide has been functionally overexpressed by a recombinant baculovirus in insect cells at greater than 1000-fold higher levels than that found in cultured normal human cells. The recombinant polymerase alpha protein is translated from its natural translation start codon under the control of the baculovirus polyhedron promoter producing a protein of 180 kDa, identical in size to that isolated from cultured human cells. This recombinant polymerase alpha is phosphorylated and reactive to a panel of monoclonal antibodies directed against the native polymerase alpha-primase complex and to polyclonal antisera against N- and C-terminal peptides of the polymerase alpha catalytic polypeptide. The recombinant enzyme was immunopurified from insect cells as a single polypeptide. The single subunit recombinant polymerase alpha has no detectable 3'-5' exonuclease activity. The Km for primer-template and dNTP, reactivity to inhibitors, N2-(p-n-butylphenyl)-dGTP (BuPdGTP) and aphidicolin, thermosensitivity, and DNA synthetic processivity and fidelity of the recombinant polymerase alpha are identical to that observed with the four-subunit polymerase alpha-primase complex immunopurified from cultured human cells. These results strongly suggest that the presence of the other subunits, (the p70 and the two primase subunits, p48 and p58), does not influence kinetic parameters of polymerase alpha catalysis, sensitivity to inhibitors, or DNA synthetic fidelity and processivity.
Department of Pathology, Stanford University School of Medicine, California 94305.
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Copeland
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Wang
D000818:Animals; D000911:Antibodies, Monoclonal; D001483:Base Sequence; D002460:Cell Line; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004257:DNA Polymerase II / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D004258:DNA Polymerase III; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D005822:Genetic Vectors; D006367:HeLa Cells / Q000201:enzymology; D006801:Humans; D007313:Insecta; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011499:Protein Processing, Post-Translational; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013379:Substrate Specificity; D013604:T-Phages / Q000201:enzymology; D014162:Transfection
D000911:Antibodies, Monoclonal; D004279:DNA, Viral; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D004257:DNA Polymerase II; D004258:DNA Polymerase III; D004259:DNA-Directed DNA Polymerase