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Purification and properties of DNA polymerase-beta from guinea pig liver.

Publication ,  Journal Article
Kunkel, TA; Tcheng, JE; Meyer, RR
Published in: Biochim Biophys Acta
September 27, 1978

Deoxyribonucleic acid polymerase-beta (EC 2.7.7.7) has been purified over 100 000-fold from a whole cell extract of guinea pig liver. The enzyme yields a single stainable band when subjected to non-denaturing polyacrylamide gel electrophoresis, and this band corresponds to the DNA polymerase activity when a sister gel is sliced and assayed. The final fraction has a specific activity of 21 000 units/mg; this value can be increased significantly by addition of various components, including glycols, polyamines or any of several protein factors which can be purified from the crude extract. The DNA polymerase-beta lacks detectable exonuclease or endonuclease activity, has an alkaline pH optimum and has a requirement for all four deoxyribonucleoside triphosphates, a divalent cation and a primer-template for maximal activity. While activated DNA is the preferred primer-template, the enzyme is capable of utilizing native and denatured DNA as well as several synthetic polynucleotides as primer-templates. The latter are especially effective when manganese is the divalent cation. Magnesium, at 10 mM, is the preferred divalent cation when activated DNA is used. Manganese, and to a lesser extent cobalt, can substitute for magnesium while zinc and calcium cannot. The beta-polymerase has a half-life of 10 min at 40 degrees C and this is increased in the presence of either DNA or NaCl. The enzyme is stimulated by glycols, polyamines and NaCal or KCl, and is inhibited by several known inhibitors of DNA polymerase activity including o-phenanthroline, heparin, organic solvents and sulfhydryl blocking agents. Guinea pig liver DNA polymerase-beta is remarkably similar to the rat Novikoff hepatoma beta-polymerase with respect to its isoelectric point of 8.4 and its molecular weight of 32 000 as determined by sucrose gradient centrifugation under high or low salt conditions or sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This similarity is further extended to the removal, at the final step in purification, of a protein capable of stimulating the homogeneous enzyme. Removal of this protein could explain the lower molecular weight of the guinea pig and other rodent-derived beta-polymerases, when compared to the beta-polymerases from other systems.

Duke Scholars

Published In

Biochim Biophys Acta

DOI

ISSN

0006-3002

Publication Date

September 27, 1978

Volume

520

Issue

2

Start / End Page

302 / 316

Location

Netherlands

Related Subject Headings

  • Molecular Weight
  • Liver
  • Kinetics
  • Guinea Pigs
  • Drug Stability
  • DNA-Directed DNA Polymerase
  • DNA Polymerase I
  • Animals
  • 51 Physical sciences
  • 31 Biological sciences
 

Citation

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Kunkel, T. A., Tcheng, J. E., & Meyer, R. R. (1978). Purification and properties of DNA polymerase-beta from guinea pig liver. Biochim Biophys Acta, 520(2), 302–316. https://doi.org/10.1016/0005-2787(78)90229-0
Kunkel, T. A., J. E. Tcheng, and R. R. Meyer. “Purification and properties of DNA polymerase-beta from guinea pig liver.Biochim Biophys Acta 520, no. 2 (September 27, 1978): 302–16. https://doi.org/10.1016/0005-2787(78)90229-0.
Kunkel TA, Tcheng JE, Meyer RR. Purification and properties of DNA polymerase-beta from guinea pig liver. Biochim Biophys Acta. 1978 Sep 27;520(2):302–16.
Kunkel, T. A., et al. “Purification and properties of DNA polymerase-beta from guinea pig liver.Biochim Biophys Acta, vol. 520, no. 2, Sept. 1978, pp. 302–16. Pubmed, doi:10.1016/0005-2787(78)90229-0.
Kunkel TA, Tcheng JE, Meyer RR. Purification and properties of DNA polymerase-beta from guinea pig liver. Biochim Biophys Acta. 1978 Sep 27;520(2):302–316.

Published In

Biochim Biophys Acta

DOI

ISSN

0006-3002

Publication Date

September 27, 1978

Volume

520

Issue

2

Start / End Page

302 / 316

Location

Netherlands

Related Subject Headings

  • Molecular Weight
  • Liver
  • Kinetics
  • Guinea Pigs
  • Drug Stability
  • DNA-Directed DNA Polymerase
  • DNA Polymerase I
  • Animals
  • 51 Physical sciences
  • 31 Biological sciences