Suppression of the poly(ADP-ribose) polymerase activity by DNA-dependent protein kinase in vitro

Oncogene. 1999 Aug 12;18(32):4616-25. doi: 10.1038/sj.onc.1202823.

Abstract

It has been suggested that DNA-dependent protein kinase (DNA-PK) is a central component of DNA double-strand-break repair. The mechanism of DNA-PK action, however, has not been fully understood. Poly(ADP-ribose) polymerase (PARP) is another nuclear enzyme which has high affinity to DNA ends. In this study, we analysed the interaction between these two enzymes. First, DNA-PK was found to suppress the PARP activity and alters the pattern of poly(ADP-ribosyl)ation. Although DNA-PK phosphorylates PARP in a DNA-dependent manner, this modification is unlikely to be responsible for the suppression of PARP activity, since this suppression occurs even in the absence of ATP. Conversely, PARP was found to ADP-ribosylate DNA-PK in vitro. However, the auto-phosphorylation activity of DNA-PK was not influenced by this modification. In a competitive electrophoretic mobility shift assay, Ku 70/80 complex, the DNA binding component of DNA-PK, was found to have higher affinity to a short fragment of DNA than does PARP. Furthermore, co-immunoprecipitation analysis suggested direct or close association between Ku and PARP. Thus, DNA-PK suppresses PARP activity, probably through direct binding and/or sequestration of DNA-ends which serve as an important stimulator for both enzymes.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Antigens, Nuclear*
  • Cell Line, Transformed
  • DNA Helicases*
  • DNA, Viral / metabolism
  • DNA-Activated Protein Kinase
  • DNA-Binding Proteins / metabolism
  • Human T-lymphotropic virus 1 / genetics
  • Humans
  • Ku Autoantigen
  • Nuclear Proteins / metabolism
  • Phosphorylation
  • Poly(ADP-ribose) Polymerase Inhibitors*
  • Poly(ADP-ribose) Polymerases / isolation & purification
  • Poly(ADP-ribose) Polymerases / metabolism
  • Protein-Serine-Threonine Kinases / isolation & purification
  • Protein-Serine-Threonine Kinases / metabolism*

Substances

  • Antigens, Nuclear
  • DNA, Viral
  • DNA-Binding Proteins
  • Nuclear Proteins
  • Poly(ADP-ribose) Polymerase Inhibitors
  • Poly(ADP-ribose) Polymerases
  • DNA-Activated Protein Kinase
  • PRKDC protein, human
  • Protein-Serine-Threonine Kinases
  • DNA Helicases
  • XRCC5 protein, human
  • Xrcc6 protein, human
  • Ku Autoantigen