Stimulation of the DNA-dependent Protein Kinase by Poly(ADP-Ribose) Polymerase*
- Tracy Ruscetti‡,
- Bruce E. Lehnert‡,
- James Halbrook§,
- Hai Le Trong§,
- Merl F. Hoekstra§,
- David J. Chen¶ and
- Scott R. Peterson‡‖
- From the ‡Cell and Molecular Biology Group (LS-4),¶DNA Damage and Repair Group (LS-6), Life Sciences Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545 and§ICOS Corporation, Bothell, Washington 98021
Abstract
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.
Footnotes
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↵* This work was supported by Los Alamos National Laboratory LDRD grants, a grant from the U. S. Department of Energy, and National Institutes of Health Grant CA50519.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵‖ To whom correspondence should be addressed: Mail Stop M888, Los Alamos, NM 87545. Tel.: 505-667-9690; Fax: 505-665-3024; E-mail:spete{at}telomere.lanl.gov.
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↵1 The abbreviations used are: DSBs, double-strand breaks; DNA-PK, DNA-dependent protein kinase; DNA-PKcs, catalytic subunit of DNA-PK; PARP, poly(ADP-ribose) polymerase; PAGE, polyacrylamide gel electrophoresis; hnRNP-U, human heterogeneous nuclear ribonucleoprotein-U; DHQ, 1,5-dihydroxyisoquinoline; RPA, replication protein A.
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↵2 S. R. Peterson, unpublished observations.
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- Received August 27, 1997.
- Revision received March 31, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
