Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus

M. D. Challberg, P. T. Englund

Research output: Contribution to journalArticle

Abstract

The vaccinia virus-induced DNA polymerase has been purified about 500-fold from a cytoplasmic extract of vaccinia-infected HeLa cells. Analysis of the purified fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveals a single polypeptide of 110,000 daltons, which is greater than 95% pure. This polypeptide co-sediments with polymerase activity through a glycerol gradient. The sedimentation coefficient of the enzyme is 6.3 S, and its Stokes radius is 4.6 nm. The molecular weight of the native enzyme derived from these values is 115,000 Vaccinia polymerase is therefore a single large polypeptide of 110,000 to 115,000 daltons. The purified fraction has no significant endonuclease activity, but a strong exonuclease activity co-purifies with polymerase activity through every step in the isolation. The polymerase and exonuclease activities are inactivated at 45°C at the same rate. It is likely, therefore, that both activities are catalyzed by the same polypeptide. The exonuclease hydrolyzes DNA predominantly in the 3' → 5' direction, to produce 5' mononucleotides. The exonuclease degrades single-stranded DNA more rapidly than duplex DNA, and the rate of digestion of both single-stranded and double-stranded DNA increases as the size of the substrate decreases. Single-stranded circular DNA is a potent inhibitor of the exonuclease activity, but duplex circular DNA has no significant effect on its activity.

Original languageEnglish (US)
Pages (from-to)7812-7819
Number of pages8
JournalJournal of Biological Chemistry
Volume254
Issue number16
StatePublished - 1979

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Exonucleases
Vaccinia virus
Viruses
Purification
Vaccinia
Circular DNA
Peptides
Single-Stranded DNA
DNA
Exodeoxyribonucleases
Endonucleases
DNA-Directed DNA Polymerase
Enzymes
Electrophoresis
HeLa Cells
Sedimentation
Sodium Dodecyl Sulfate
Glycerol
Polyacrylamide Gel Electrophoresis
Digestion

ASJC Scopus subject areas

  • Biochemistry

Cite this

Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus. / Challberg, M. D.; Englund, P. T.

In: Journal of Biological Chemistry, Vol. 254, No. 16, 1979, p. 7812-7819.

Research output: Contribution to journalArticle

Challberg, M. D. ; Englund, P. T. / Purification and properties of the deoxyribonucleic acid polymerase induced by vaccinia virus. In: Journal of Biological Chemistry. 1979 ; Vol. 254, No. 16. pp. 7812-7819.
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