Stimulation of the DNA-dependent protein kinase by poly(ADP-ribose) polymerase.
The DNA-dependent protein kinase (DNA-PK) is a heterotrimeric enzyme that binds to double-stranded DNA and is required for the rejoining of double-stranded DNA breaks in mammalian cells. It has been proposed that DNA-PK functions in this DNA repair pathway by binding to the ends of broken DNA molecules and phosphorylating proteins that bind to the damaged DNA ends. Another enzyme that binds to DNA strand breaks and may also function in the cellular response to DNA damage is the poly(ADP-ribose) polymerase (PARP). Here, we show that PARP can be phosphorylated by purified DNA-PK, and the catalytic subunit of DNA-PK is ADP-ribosylated by PARP. The protein kinase activity of DNA-PK can be stimulated by PARP in the presence of NAD+ in a reaction that is blocked by the PARP inhibitor 1, 5-dihydroxyisoquinoline. The stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation occurs independent of the Ku70/80 complex. Taken together, these results show that PARP can modify the activity of DNA-PK in vitro and suggest that these enzymes may function coordinately in vivo in response to DNA damage.
Ruscetti
T
T
Cell and Molecular Biology Group (LS-4), Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA.
Lehnert
B E
BE
Halbrook
J
J
Le Trong
H
H
Hoekstra
M F
MF
Chen
D J
DJ
Peterson
S R
SR
D004249:DNA Damage / Q000502:physiology; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins / Q000378:metabolism; D004789:Enzyme Activation / Q000502:physiology*; D006367:HeLa Cells; D006801:Humans; D007546:Isoquinolines / Q000494:pharmacology; D009243:NAD / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D051716:Replication Protein A
D004268:DNA-Binding Proteins; D007546:Isoquinolines; D009687:Nuclear Proteins; C497167:RPA1 protein, human; D051716:Replication Protein A; D009243:NAD; C095653:1,5-dihydroxyisoquinoline; D011065:Poly(ADP-ribose) Polymerases; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases