Poly(ADP-ribose) Polymerase Stimulates DNA Polymerase Alpha by Physical Association

J Biol Chem. 1993 Jan 5;268(1):93-9.

Abstract

The direct effect of the eukaryotic nuclear DNA-binding protein poly(ADP-ribose) polymerase on the activity of DNA polymerase alpha was investigated. Homogenously purified poly(ADP-ribose) polymerase (5 to 10 micrograms/ml) stimulated the activity of immunoaffinity-purified calf or human DNA polymerase alpha by about 6 to 60-fold in a dose-dependent manner. It had no effect on the activities of DNA polymerase beta, DNA polymerase gamma, and primase, indicating that its effect is specific for DNA polymerase alpha. Apparently, poly(ADP-ribosyl)ation of DNA polymerase alpha 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 Vmax 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 co-immunoprecipitated with DNA polymerase alpha, using anti-DNA polymerase alpha antibody, clearly showing that poly(ADP-ribose) polymerase may be physically associated with DNA polymerase alpha. 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 alpha, suggesting the existence in vivo of a complex of these two enzymes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Antibodies
  • Cattle
  • Chromatography, Affinity
  • Chromatography, Gel
  • DNA Polymerase II / isolation & purification
  • DNA Polymerase II / metabolism*
  • DNA Primase
  • Enzyme Activation
  • Humans
  • Kinetics
  • Poly(ADP-ribose) Polymerases / isolation & purification
  • Poly(ADP-ribose) Polymerases / metabolism*
  • Protein Binding
  • RNA Nucleotidyltransferases / isolation & purification
  • Thymine Nucleotides / metabolism
  • Thymus Gland / enzymology*

Substances

  • Antibodies
  • Thymine Nucleotides
  • Poly(ADP-ribose) Polymerases
  • DNA Polymerase II
  • DNA Primase
  • RNA Nucleotidyltransferases
  • thymidine 5'-triphosphate