Poly(ADP-ribose) polymerase stimulates DNA polymerase α by physical association

C. M.G. Simbulan, M. Suzuki, S. Izuta, T. Sakurai, E. Savoysky, K. Kojima, K. Miyahara, Y. Shizuta, S. Yoshida

Research output: Contribution to journalArticle

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Abstract

The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP- ribose) polymerase on the activity of DNA polymerase α was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 μg/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase α by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase β, DNA polymerase γ, and primase, indicating that its effect is specific for DNA polymerase α. Apparently, poly(ADP- ribosyl)ation of DNA polymerase α was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP- ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the V(max) of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is coimmunoprecipitated with DNA polymerase α, using anti-DNA polymerase α antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase α. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase α, suggesting the existence in vivo of a complex of these two enzymes.

Original languageEnglish
Pages (from-to)93-99
Number of pages7
JournalJournal of Biological Chemistry
Volume268
Issue number1
Publication statusPublished - 01-01-1993

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Poly(ADP-ribose) Polymerases
DNA-Directed DNA Polymerase
DNA
DNA Primase
Thymus
DNA Primers
Antinuclear Antibodies
DNA-Binding Proteins
Enzymes
Nuclear Proteins
Complex Mixtures
Adenosine Diphosphate
Thymus Gland
Monoclonal Antibodies

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Simbulan, C. M. G., Suzuki, M., Izuta, S., Sakurai, T., Savoysky, E., Kojima, K., ... Yoshida, S. (1993). Poly(ADP-ribose) polymerase stimulates DNA polymerase α by physical association. Journal of Biological Chemistry, 268(1), 93-99.
Simbulan, C. M.G. ; Suzuki, M. ; Izuta, S. ; Sakurai, T. ; Savoysky, E. ; Kojima, K. ; Miyahara, K. ; Shizuta, Y. ; Yoshida, S. / Poly(ADP-ribose) polymerase stimulates DNA polymerase α by physical association. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 1. pp. 93-99.
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Simbulan, CMG, Suzuki, M, Izuta, S, Sakurai, T, Savoysky, E, Kojima, K, Miyahara, K, Shizuta, Y & Yoshida, S 1993, 'Poly(ADP-ribose) polymerase stimulates DNA polymerase α by physical association', Journal of Biological Chemistry, vol. 268, no. 1, pp. 93-99.

Poly(ADP-ribose) polymerase stimulates DNA polymerase α by physical association. / Simbulan, C. M.G.; Suzuki, M.; Izuta, S.; Sakurai, T.; Savoysky, E.; Kojima, K.; Miyahara, K.; Shizuta, Y.; Yoshida, S.

In: Journal of Biological Chemistry, Vol. 268, No. 1, 01.01.1993, p. 93-99.

Research output: Contribution to journalArticle

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AU - Simbulan, C. M.G.

AU - Suzuki, M.

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AU - Savoysky, E.

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N2 - The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP- ribose) polymerase on the activity of DNA polymerase α was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 μg/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase α by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase β, DNA polymerase γ, and primase, indicating that its effect is specific for DNA polymerase α. Apparently, poly(ADP- ribosyl)ation of DNA polymerase α was not necessary for the stimulation. The stimulatory activity is due to poly(ADP-ribose) polymerase itself since it was immunoprecipitated with a monoclonal antibody directed against poly(ADP- ribose) polymerase. Kinetic analysis showed that, in the presence of poly(ADP-ribose) polymerase, the saturation curve for DNA template primer became sigmoidal; at very low concentrations of DNA, it rather inhibited the reaction in competition with template DNA, while, at higher DNA doses, it greatly stimulated the reaction by increasing the V(max) of the reaction. By the automodification of poly(ADP-ribose) polymerase, however, both the inhibition at low DNA concentration and the stimulation at high DNA doses were largely lost. Furthermore, stimulation by poly(ADP-ribose) polymerase could not be attributed to its DNA-binding function alone since its fragment, containing only the DNA-binding domain, could not exert full stimulatory effect on DNA polymerase, as of the intact enzyme. Poly(ADP-ribose) polymerase is coimmunoprecipitated with DNA polymerase α, using anti-DNA polymerase α antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase α. In a crude extract of calf thymus, a part of poly(ADP-ribose) polymerase activity existed in a 400-kDa, as well as, a larger 700-kDa complex containing DNA polymerase α, suggesting the existence in vivo of a complex of these two enzymes.

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Simbulan CMG, Suzuki M, Izuta S, Sakurai T, Savoysky E, Kojima K et al. Poly(ADP-ribose) polymerase stimulates DNA polymerase α by physical association. Journal of Biological Chemistry. 1993 Jan 1;268(1):93-99.