Stimulation of the DNA-dependent Protein Kinase by Poly(ADP-Ribose) Polymerase*

Tracy RuscettiDagger , Bruce E. LehnertDagger , James Halbrook§, Hai Le Trong§, Merl F. Hoekstra§, David J. Chen, and Scott R. PetersonDagger parallel

From the Dagger  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
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Abstract
Introduction
Procedures
Results
Discussion
References

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.

    INTRODUCTION
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Abstract
Introduction
Procedures
Results
Discussion
References

Biochemical pathways that function in the recognition and repair of DNA damage are critical for maintaining genomic integrity. Potentially devastating DNA damage in the form of double-strand breaks (DSBs)1 can occur when cells are exposed to ionizing radiation, oxidative stress and radiomimetic drugs. The DNA-dependent protein kinase (DNA-PK) is a key component of DNA DSB rejoining pathways in mammalian cells. DNA-PK is a heterotrimeric enzyme complex comprised of a 460-kDa catalytic subunit (1) and a regulatory component consisting of the Ku70 and Ku80 proteins (2, 3). The Ku70 and Ku80 proteins form a heterodimeric complex that binds to the ends of double-stranded DNA with high affinity (4-8). The catalytic subunit of DNA-PK (DNA-PKcs) binds to the Ku70/80 complex in the presence of double-stranded DNA (9) and phosphorylates a wide variety of protein substrates in vitro on serine and threonine residues (10, 11). The protein kinase activity of DNA-PK is autoregulatory; in the absence of a phosphorylation substrate, DNA-PKcs autophosphorylates and dissociates from the Ku70/80-DNA complex (12).

Evidence that DNA-PK plays an integral role in the repair of DNA DSB has been provided through the characterization of rodent cell lines that have mutations that disrupt the expression of the Ku80 (13-19) or DNA-PKcs (20-24). Although it is clear that DNA-PK is an important component of mammalian DNA DSB repair pathways, it is not known how the enzyme participates in these processes. In vitro, DNA-PK preferentially phosphorylates protein substrates that co-localize on the same DNA molecule (3, 25). This suggests that the specificity of the phosphorylation reaction may be regulated, in part, via the co-localization of the enzyme and substrate target on DNA. Based on these data, it has been proposed that DNA-PK could participate in the DNA rejoining reaction by phosphorylating DNA repair factors that co-localize with it on broken DNA ends (26-28). This modification might regulate the catalytic activities or DNA binding affinities of these proteins. In addition, other proteins that bind to DNA strand-break sites could function coordinately with DNA-PK by modifying the kinase activity or assembly of the holoenzyme.

In this report we describe experiments that show a functional interaction between DNA-PK and another DNA-activated enzyme, the poly(ADP-ribose) polymerase (PARP). Like DNA-PK, PARP can bind to DNA ends (29, 30) and has been proposed to function in the cellular response to DNA damage (31-32). PARP can be phosphorylated by DNA-PK in vitro, and PARP, in turn, ADP-ribosylates the DNA-PKcs. Furthermore, the addition of PARP and its substrate, NAD+, increased the catalytic activity of DNA-PK. The activation of DNA-PK by PARP is independent of the Ku70/80 complex, and 1,5-dihydroxyisoquinoline, a specific inhibitor of PARP, blocks the ability of PARP to stimulate DNA-PK. In addition, the stimulation of DNA-PK by PARP-mediated protein ADP-ribosylation was directly correlated with the ADP-ribosylation of the DNA-PKcs. These results show that the activities of these two enzymes have the capacity to function in a coordinated manner in vitro and suggest that PARP may facilitate DNA DSB repair by stimulating the protein kinase activity of DNA-PK.

    EXPERIMENTAL PROCEDURES
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Abstract
Introduction
Procedures
Results
Discussion
References

Cell Growth and Extract Preparation-- HeLa cells were grown in suspension at 37 °C using RPMI 1640 medium (Life Technologies, Inc.) containing 10% fetal calf serum, 20 µg/ml penicillin and streptomycin, and 10 µg/ml ciprofloxacin (Miles). HeLa cell nuclear extracts were prepared as described previously (22).

Protein Purification-- DNA-PK was purified from HeLa cell nuclear extract as described previously (33). DNA-PK complexes were isolated from HeLa cell nuclear extracts using a Ku80 immunoaffinity matrix as follows: monoclonal antibodies specific for Ku80 (34) were conjugated to protein G agarose beads using dimethylpimelimidate HCl to achieve a final concentration of 1 mg of antibody/ml of agarose beads. 50 ml of HeLa cell nuclear extract (10 mg/ml) was incubated with 2 ml of the anti-Ku80 matrix for 4 h at 4 °C in the presence of 100 µg/ml sheared salmon sperm DNA. The resin was then packed into a 5-ml disposal column (Bio-Rad) and washed with 5 column volumes of HEPES chromatography buffer (50 mM HEPES, pH 7.5, 2 mM EDTA, 0.01% Nonidet P-40, 1 mM dithiothreitol, 20 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, pepstatin A, and leupeptin) containing 0.1 M KCl. Proteins that were bound to the column matrix were eluted sequentially with 5 column volumes each of HEPES chromatography buffer containing 0.2 and 0.5 M KCl. The antibody-bound Ku70/80 complexes were then eluted from the washed column matrix using 5 column volumes of 50 mM Tris-HCl, pH 7.9, 50% ethylene glycol, 1.75 M MgCl2, 1 mM dithiothreitol, 20 µg/ml phenylmethylsulfonyl fluoride.

Proteins found in the 0.5 M KCl anti-Ku80 column fractions were dialyzed against HEPES chromatography buffer containing 0.1 M KCl and further purified by heparin-agarose column chromatography using 1 ml of Hi-Trap heparin-agarose column (Amersham Pharmacia Biotech) equilibrated with HEPES chromatography buffer containing 0.1 M KCl. The column was washed with 5 ml of HEPES chromatography buffer containing 0.1 M KCl, and the remaining proteins were eluted from the heparin-agarose column sequentially with 5-ml step gradients of HEPES chromatography buffer containing 0.2, 0.3, 0.5, and 1.0 M KCl. Fractions enriched for DNA-PKcs and Ku70/80 eluted from the column with 0.3 M KCl. Two proteins with molecular masses of approximately 120 kDa eluted from the column with 0.5 M KCl. To purify further the two 120-kDa proteins the 0.5 M KCl fraction was diluted with HEPES chromatography buffer to achieve a KCl concentration of 0.1 M and applied to a 1-ml Hi-Trap SP-Sepharose (Amersham Pharmacia Biotech) column. The SP-Sepharose column resolved the two 120-kDa proteins into separate protein fractions, p120a, which eluted from the SP-Sepharose column at 0.3 M KCl and p120b, which eluted at 0.5 M KCl. Each of the p120 fractions were diluted with HEPES chromatography buffer to achieve a KCl concentration of 0.1 M and loaded individually on a 1 ml DNA-agarose column. The DNA-agarose column was washed with HEPES chromatography buffer containing 0.1 M KCl, and the proteins were eluted sequentially with HEPES chromatography buffer containing 0.2, 0.3, 0.5, and 1.0 M KCl. Both p120a and p120b eluted from the DNA-agarose with 0.5 M KCl. Peak fractions from the DNA-agarose column were pooled individually for p120a and p120b and dialyzed against TM buffer (50 mM Tris-HCl, pH 7.9, 12.5 mM MgCl2, 1 mM EDTA, 20% glycerol, 1 mM dithiothreitol, 20 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, pepstatin A, and leupeptin) containing 0.1 M KCl. The protein concentrations of the dialyzed samples were determined by Bradford analysis using bovine serum albumin as a standard, and the samples were stored in 100-µl aliquots at -70 °C.

Recombinant Ku70/80 was purified from extracts prepared from Spodoptera frugiperda Sf9 cells that were co-infected with baculoviral expression constructs containing the human Ku70 and Ku80 genes. The Ku70/80 complex was purified by heparin-agarose (Amersham Pharmacia Biotech), Q-Sepharose, SP-Sepharose, and DNA-agarose chromatography as described (33). Recombinant p53 protein was expressed as a hexahistidine-tagged fusion protein. The sequences encoding the full-length p53 gene were amplified by polymerase chain reaction using the wild-type human cDNA as template DNA. The primers were designed with restriction enzyme sites that allowed direct cloning into the pBlueBacHis2 vector (Invitrogen). The recombinant baculovirus was used to infect S. frugiperda Sf9 cells, and the cells were harvested 3 days postinfection. The protein was purified from cell extracts using TALON chelating resin (CLONTECH) and eluted with 50 mM imidizole. Recombinant p53 was dialyzed into HEPES chromatography buffer containing 0.1 M KCl, 10 mM MgCl2, 1 mM dithiothreitol, and 20 µg/ml phenylmethylsulfonyl fluoride. The protein concentration of p53 was determined by Bradford analysis and stored in aliquots at -70 °C. Recombinant human RPA was purified as described previously (24).

Protein Microsequence Analysis-- Protein microsequence information for the p120a and p120b samples was obtained by analyzing HPLC-purified peptides derived from tryptic digest of gel-purified samples using an applied Biosystems 473A protein/peptide sequencer. The N-terminal sequence of three tryptic peptides from the p120a sample showed 100% identity with the human PARP. The peptide sequences are as follows: SKLPKPVQDLIK, corresponding to amino acids 663-674 of PARP; KFYPLFIDYGQDEEAVK, corresponding to amino acids 637-653 of PARP; and VGTVIGSNK, corresponding to amino acids 592-600 of PARP. Mass spectroscopic analysis of an additional 13 tryptic peptides using a Perspective Biosystems MALDI-TOF Voyager-DE mass spectrometer confirmed the identity of the p120a protein as PARP. The N-terminal sequence of four tryptic peptides from the p120b sample showed a 100% identity with the human heterogeneous nuclear ribonucleoprotein-U (hnRNP-U). The peptide sequences are as follows: SSGPTSLFAVT, corresponding to amino acids 187-197 of hnRNP-U; GYFEYIEENK, corresponding to amino acids 237-246 of hnRNP-U; EKPYFPIPEEYTFIQ, corresponding to amino acids 443-458 of hnRNP-U; and NFILDQTNVSAA, corresponding to amino acids 557-568 or hnRNP-U.

Protein Kinase Assays-- Standard DNA-PK reactions contained 30 ng of purified DNA-PKcs (5 µg/ml), 20 ng of recombinant Ku70/80 (10 µg/ml), and 0.57 ng of a gel-purified 43-base pair DNA fragment prepared by digesting pGEM3Zf+ (Promega) with PstI and EcoRI. DNA-PKcs and Ku were preincubated with the DNA at 25 °C for 20 min individually or in the presence of 60 ng of PARP (30 ng/µl) in a final reaction volume of 40 µl as indicated in the appropriate figure legends. Protein kinase reactions were initiated by addition of [gamma -32P]ATP to achieve a final concentration of 12.5 µM (specific activity 20 Ci/mmol) and, where indicated, 75 ng of purified hnRNP-U as a protein kinase substrate. Standard protein kinase reactions were incubated for 30 min at 30 °C and terminated by boiling in SDS-PAGE sample buffer. Reaction products were resolved by SDS-PAGE and visualized by autoradiography of the dried gel. Data were quantified by calculating the optical densities of the autoradiographic bands using 1-Dimension Whole Band software (BioImage).

Protein and Immunoblot Analysis-- Immunoblot analysis was performed as described previously (22). Immobilized proteins were probed with a rabbit polyclonal antibody specific for the N-terminal 20 amino acids of PARP (PARP-N20, Santa Cruz Biochemical, Santa Cruz, CA), antibody 42-26 and the anti-Ku80 antibody LL1 and visualized using a chemiluminescent detection system (ECL, Amersham Pharmacia Biotech).

ADP-ribosylation Assays-- ADP-ribosylation reactions were performed by addition of nicotinamide adenine dinucleotide (NAD+) to the protein kinase reactions to achieve a final concentration of 200 µM, as indicated in the figure legends. Reactions were incubated for 30 min at 30 °C prior to addition of ATP and protein kinase substrate. To examine the ADP-ribosylation of DNA-PK (Fig. 5A), 60 ng of purified PARP was incubated with 5.7 ng of the same 43-base pair DNA fragment used in the protein kinase reactions and, where indicated, 100 ng of recombinant Ku70/80 or 150 ng of DNA-PKcs. Reactions were incubated for 30 min at 30 °C with 200 nM NAD containing 5 µCi of [32P]adenylate NAD+ and terminated by boiling in SDS sample buffer. Radiolabeled products were resolved by SDS-PAGE and visualized by autoradiography of the dried gel.

    RESULTS
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Abstract
Introduction
Procedures
Results
Discussion
References

To identify proteins that may co-localize with DNA-PK on damaged DNA we used a Ku80 antibody affinity matrix to purify DNA-PK complexes from HeLa cell nuclear extract. Under our experimental conditions, we found that the majority of DNA-PKcs eluted from the anti-Ku80 column in the 0.5 M KCl fraction (Fig. 1A, lane c). In addition to DNA-PKcs, the 70- and 80-kDa subunits of Ku and two other proteins with apparent masses of approximately 120 kDa were also found in the 0.5 M fraction. Residual Ku70/80 complexes bound directly to the Ku80 antibody were also eluted from the column matrix (Fig. 1A, lane d).


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Fig. 1.   Immunoaffinity isolation of DNA-PK complexes. A, large scale immunoaffinity purification of DNA-PK complexes. Proteins bound to an anti-Ku80 immunoaffinity matrix were eluted with buffer containing 0.2 M (lane b) and 0.5 M KCl (lane c) and 1.5 M MgCl2, 50% ethylene glycol (lane d) as described under "Experimental Procedures." Proteins were resolved by 7.5% SDS-PAGE and stained with Coomassie Brilliant Blue. Arrows indicate the position of Ku70, Ku80, DNA-PKcs and two proteins migrating with an apparent molecular masses of 120 kDa that co-purified with the DNA-PK complexes. Molecular mass markers (lane a) are as follows: myosin, 200 kDa; beta -galactosidase, 116 kDa; phosphorylase b, 97.4 kDa; serum albumin, 66 kDa; and ovalbumin, 45 kDa. B, small scale affinity purification of DNA-PK complexes. HeLa cell nuclear extract (lane b) was incubated with CL4B agarose beads (lane c), DNA-agarose beads (lane d), anti-Ku80 antibody beads (lane e), and anti-p21 antibody beads (lane f). Beads were washed with buffer containing 0.2 M KCl and bound proteins eluted with buffer containing 0.5 M KCl. Residual proteins bound to anti-Ku80 (lane g) and anti-p21 (lane h) agarose beads were solubilized by boiling in SDS-PAGE sample buffer. Proteins were resolved by 7.5% SDS-PAGE and stained by Coomassie Brilliant Blue. Arrows indicate the position of Ku70, Ku80, DNA-PKcs, and unidentified proteins migrating with an apparent molecular mass of 120 kDa. Molecular mass markers (lane a) were the same used in A. C, determination of protein kinase activity in the anti-Ku80 0.5 M KCl fraction. Proteins found in the 0.5 M KCl fractions from the anti-p21 and anti-Ku80 columns were dialyzed into buffer containing 0.1 M KCl and incubated with [gamma -32P]ATP to measure in situ protein kinase activity. Arrows identify the major phosphorylated protein species, including the DNA-PKcs, unidentified proteins migrating with an apparent molecular mass of 120 kDa and Ku70. The position of prestained molecular mass markers is indicated in kilodaltons.

We were particularly interested in the two 120-kDa proteins that co-eluted with the DNA-PKcs and Ku70/80 because they appeared to be nearly as abundant as the Ku70/80 and DNA-PKcs subunits. To determine whether these proteins were unique to the anti-Ku80 column or were nonspecifically associating with the agarose beads or immunoglobulin chains, we compared the pattern of proteins eluting from anti-Ku80 agarose beads to those eluting from unconjugated agarose beads, DNA-conjugated agarose beads, and protein G-agarose beads coupled to a monoclonal antibody specific for the Cdk inhibitor p21. Small scale reactions were performed with these reagents using the same conditions employed in the large scale affinity purification. Proteins from the 0.5 M KCl fraction from each of the reactions were analyzed by SDS-PAGE (Fig. 1B). Proteins eluting from the unconjugated beads and the p21 antibody beads were not detectable (Fig. 1B, lanes c and f) with Coomassie staining of the gel. However, several proteins, including DNA-PK and the two p120 proteins, eluted from the DNA-conjugated beads (Fig. 1B, lane d) and the Ku80 antibody beads (Fig. 1B, lane e). As a control, the p21 and Ku80 antibody beads were boiled in SDS sample buffer and analyzed to ensure equivalent amounts of antibody were bound to the protein G-agarose beads (Fig. 1B, lanes g and h). These results indicated that the two 120-kDa proteins were not binding in a nonspecific fashion to the column resin, protein G, or immunoglobulin. Interestingly, the results obtained using the DNA-agarose beads suggested that the relative amounts of the two 120-kDa proteins were in approximately equal stoichiometry with the Ku70, Ku80, and DNA-PKcs proteins.

One of the objectives of the anti-Ku80 affinity purification was to identify proteins that may associate with and become phosphorylated by DNA-PK. To determine whether any of the proteins bound to the anti-Ku80 affinity matrix could be phosphorylated, we performed protein kinase assays using the 0.5 M KCl fractions from the anti-p21 and anti-Ku80 affinity columns (Fig. 1C). As expected, very little protein labeling was detected in the anti-p21 control reactions (Fig. 1C, lane a). However, labeling reactions that were performed with the anti-Ku80 fraction showed radioactive phosphate incorporation in several proteins. The most significant labeling occurred in proteins with SDS-PAGE mobilities consistent with the DNA-PKcs, Ku70 and in two proteins with masses of approximately 120 kDa (Fig. 1C, lane b).

The data presented in Fig. 1 suggested to us that the two abundant 120-kDa proteins that co-purified with the DNA-PK complexes may also be phosphorylated by DNA-PK. However, because the reactions were not performed with purified components, the phosphate incorporation in either of these proteins could be due to other protein kinases in the reaction or automodification by the p120 proteins themselves. To characterize better these proteins and their ability to be phosphorylated by DNA-PK, we further purified each of the 120-kDa proteins by heparin-agarose, SP-Sepharose, and DNA-agarose chromatography as described under "Experimental Procedures." This purification strategy yielded highly purified preparations of the two 120-kDa proteins, which we preliminarily named p120a and p120b. To determine whether these proteins could be phosphorylated by DNA-PK, we performed in vitro protein kinase assays using purified recombinant Ku70/80 and DNA-PKcs (Fig. 2A, lanes b and c) and each of the p120 proteins (p120a and p120b) (Fig. 2A, lanes d and e). In the absence of DNA-PK, there was no detectable labeling of either of the 120-kDa proteins (Fig. 2B, lanes a and d), indicating that no significant protein kinase activity co-purified with the protein samples. However, in the presence of the Ku70/80 complex and the DNA-PKcs, both p120a and p120b were phosphorylated (Fig. 2B, lanes b and e). The p120b appeared to be a better substrate for DNA-PK under these conditions. Interestingly, both p120a and p120b were efficiently phosphorylated by DNA-PKcs in the absence of the Ku70/80 complex (Fig. 2B, lanes c and f). Control reactions containing DNA-PKcs and Ku70/80 or DNA-PKcs alone showed no labeling of proteins in the 120-kDa range.


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Fig. 2.   Purification and characterization of 120-kDa proteins co-purifying with DNA-PK. A, silver stain analysis of purified proteins. 200 ng of purified recombinant Ku70/80 (lane b) and 100 ng of purified HeLa DNA-PKcs (lane c) were resolved by 10% SDS-PAGE and analyzed by silver staining. The two 120-kDa proteins were purified by ion exchange chromatography as described under "Experimental Procedures." 300 ng of p120a (lane d) and 150 ng of p120b (lane e) were resolved by 10% SDS-PAGE and analyzed by silver staining. Molecular mass markers (lane a) were the same as described in Fig. 1A. B, phosphorylation of p120a and p120b by purified DNA-PK. In vitro protein kinase reactions were performed as described under "Experimental Procedures" with 170 ng of p120a or 170 ng of p120b alone (lanes a and d), with DNA-PKcs (lanes b and e), or with both DNA-PKcs and Ku70/80 (lanes c and f). Control reactions were performed with DNA-PK and Ku70/80 (lane g) and DNA-PK alone (lane h) in the absence of p120a and p120b for reference. Kinase reactions were performed as described under "Experimental Procedures," and proteins were resolved by 7.5% SDS-PAGE. Arrows indicate the position of phosphorylated proteins. The position of prestained molecular mass markers is indicated in kilodaltons.

Based on their apparent molecular mass and the ability of each of these proteins to bind to the DNA-agarose column, we considered that at least one of the 120-kDa proteins may be PARP. Immunoblot analysis of the purified p120a and p120b fractions was performed to test this idea using an antibody specific for PARP. The PARP antibody recognized the p120a, but not the p120b protein (Fig. 3A, compare lanes a and b). We also performed in vitro ADP-ribosylation assays using the p120a and p120b samples (Fig. 3B). The p120a protein became radiolabeled in the presence of [32P]adenylate NAD+ in an automodification reaction that was stimulated by the addition of DNA (Fig. 3B, lanes a and b). Reactions performed with the p120b sample showed very little radiolabel incorporation (Fig. 3B, lanes c and d). Further confirmation that the p120a protein was identical to PARP was obtained by protein microsequence analysis as described under "Experimental Procedures." Three tryptic peptides showed 100% identity with regions of PARP, and mass spectroscopic analysis of 13 additional tryptic peptides confirmed the identify of p120a as PARP. Protein microsequencing of four tryptic peptides from the p120b sample revealed that it was identical to the heterogeneous nuclear ribonucleoprotein-U (hnRNP-U) (35).


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Fig. 3.   Identification of p120a and p120b proteins. A, immunoblot analysis of purified p120a and p120b proteins. 170 ng of p120a (lane a) and 170 ng of p120b (lane b) were resolved by 10% SDS-PAGE, transferred to nitrocellulose, and subjected to immunoblot analysis using a polyclonal anti-poly(ADP-ribose) polymerase antibody as described under "Experimental Procedures." The position of prestained molecular mass markers is indicated in kilodaltons. B, 340 ng of purified p120a (lanes a and b) and 340 ng of p120b (lanes c and d) were incubated with 200 nM [32P]adenylate NAD+ with or without 5 ng of DNA, as indicated. Reactions were terminated by boiling in SDS-PAGE sample buffer and resolved by 7.5% SDS-PAGE. The position of prestained molecular mass markers is indicated in kilodaltons. C, anti-Ku80 antibodies immobilized on protein G-agarose beads were incubated with 300 ng of Ku70/80 and 250 ng of PARP (lanes a and b) or 600 ng of DNA-PKcs (lanes c and d). Reactions were performed in the absence (lanes a and c) or in the presense of 1 µg of salmon sperm DNA (lanes b and d). Immunoprecipitation complexes were boiled in SDS-PAGE sample buffer, resolved by 7% SDS-PAGE, and probed using anti-PARP or anti-DNA-PKcs antibodies as described under "Experimental Procedures."

Since PARP can bind to DNA, we tested whether the association of PARP with the anti-Ku80 immunoprecipitation complexes was DNA-dependent. Reactions were performed using purified preparations of Ku70/80, PARP, and DNA-PKcs and the same anti-Ku80 antibodies and conditions employed in the large scale immunoaffinity purification. We found the association of PARP with Ku70/80 was dependent on the inclusion of DNA in the sample (Fig. 3C, compare lanes a and b). Similar results were seen for DNA-PKcs, which only associated with the immunoprecipitated Ku70/80 in the presence of DNA (Fig. 3C, compare lanes c and d). In addition, PARP did not co-immunoprecipitate with DNA-PKcs either in the presence or absence of DNA (data not shown).

Based on our data we concluded PARP was associated with the anti-Ku80 immunocomplexes by virtue of its association with DNA molecules bound to the Ku70/80 complex. It has been proposed that both PARP and DNA-PK may co-localize to the ends of damaged DNA molecules in vivo (32), perhaps functioning coordinately to facilitate DNA repair or DNA damage signaling. Since PARP has previously been shown to ADP-ribosylate a number of nuclear proteins (36), we performed experiments to determine whether PARP could ADP-ribosylate DNA-PKcs or either member of the Ku complex (Fig. 4A). In the absence of added DNA-PK or Ku70/80, there is a pronounced automodification of PARP (Fig. 4A, lane a), which is consistent with other studies (36). When the purified Ku70/80 complex is added to the reaction, the amount of automodified PARP in the reaction was reduced 2-fold, and under these conditions, there was no apparent ADP-ribosylation of the Ku subunits (Fig. 4A, lane b). When incubated with purified DNA-PKcs the automodification of PARP was reduced 1.5-fold (Fig. 4A, lane c), and we observed the appearance of a radiolabeled band with a mobility consistent with the catalytic subunit of DNA-PK.


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Fig. 4.   Poly(ADP-ribose) polymerase ADP-ribosylates the DNA-PKcs. A, ADP-ribosylation reactions were performed with PARP alone (lane a), with recombinant Ku70/80 (lane b), and with purified DNA-PKcs (lane c). Reactions were initiated by addition of 200 nM [32P]adenylate NAD+ as described under "Experimental Procedures." Reactions were incubated for 60 min at 30 °C and radiolabeled proteins resolved by 6% SDS-PAGE. Radiolabeled bands with molecular masses consistent with DNA-PKcs and automodified PARP are indicated by arrows. The position of prestained protein molecular mass markers is indicated in kilodaltons. B, DNA-PK kinase activity was measured with DNA-PKcs and Ku70/80 that had been preincubated for 30 min at 30 °C alone (lanes a and c) or with PARP (lanes b and d) in the presence or absence of 200 µM NAD+, as indicated. Kinase reactions were initiated by addition of [gamma -32P] ATP and hnRNP-U as described under "Experimental Procedures" and radiolabeled proteins resolved by 10% SDS-PAGE. Arrow indicates the position of radiolabeled hnRNP-U. C, protein ADP-ribosylation reactions were performed using purified PARP alone (lanes a and c) or with DNA-PKcs and Ku70/80 (lanes b and d). Reactions were initiated with the addition of 200 µM [32P]adenylate NAD+ and included 1 mM of the PARP inhibitor 1,5-dihydroxyisoquinoline (DHQ) where indicated. Reactions were performed for 30 min at 30 °C as described under "Experimental Procedures," and radiolabeled proteins were resolved by 7.5% SDS-PAGE. D, phosphorylation reactions were performed as described in B with DNA-PKcs, Ku70/80, and PARP in the absence (lanes a and b) or presence (lanes c and d) of 1 mM 1,5-dihydroxyisoquinoline (DHQ). Radiolabeled proteins were resolved by 7.5% SDS-PAGE. Arrow indicates the position of radiolabeled hnRNP-U.

To determine whether the ADP-ribosylation of the DNA-PKcs might affect its protein kinase activity, we compared the in vitro protein kinase activity of purified DNA-PK that had been preincubated alone or with PARP in the presence of 200 µM NAD+. Because PARP and hnRNP-U co-purified with the DNA-PK complexes, we used the hnRNP-U protein as a phosphorylation substrate in these reactions. The addition of PARP to these kinase reactions reduced DNA-PK activity by 20% (Fig. 4B, compare lanes a and b). However, in the presence of PARP and NAD+, the phosphorylation of hnRNP-U was stimulated 7-fold relative to the control reaction (Fig. 4B, compare lanes c and d).

The observation that the PARP-mediated enhancement of DNA-PK activity required NAD+ suggested that the effect was dependent on the catalytic activity of PARP. To test this directly we compared the ability of PARP to stimulate DNA-PK activity in the presence of the ADP-ribosylation inhibitor 1,5-dihydroxyisoquinoline (DHQ) (37). In the presence of 1 mM DHQ the automodification activity of PARP in the presense of 200 µM NAD+ is nearly completely inhibited (Fig. 4C, compare lanes a and b with c and d). Consistent with this, the addition of 1 mM DHQ inhibited the ability of PARP to stimulate DNA-PK-mediated phosphorylation of hnRNP-U but had no effect on the basal activity of DNA-PK (Fig. 4D, compare lanes a and b with lanes c and d).

To gain insight into how PARP stimulates the catalytic activity of DNA-PK, we compared the phosphorylation of hnRNP-U at different time points with and without the addition of NAD+ (Fig. 5A). Without added NAD+ (Fig. 5A, lanes a-e) the phosphorylation of hnRNP-U appeared to proceed at a slower rate relative to the reactions that included NAD+ (Fig. 5A, lanes f-j). Quantification of the autoradiogram showed the rate of DNA-PKcs autophosphorylation was stimulated 1.2-2-fold and that the phosphorylation of hnRNP-U was stimulated up to 6.7-fold by the addition of NAD+ and PARP (Fig. 5B). To determine whether Ku70/80 was required for the stimulatory effect of PARP and NAD+, we compared the phosphorylation of the hnRNP-U protein by DNA-PKcs alone (Fig. 5C, lanes a and b) and with DNA-PKcs and equimolar Ku70/80 complex (Fig. 5C, lanes c and d). The addition of both NAD+ and PARP to the kinase reactions stimulated the activity of the DNA-PKcs 10-fold relative to the control reactions regardless of whether Ku70/80 was added to the reaction.


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Fig. 5.   Poly(ADP-ribosyl)ation stimulates the activity of the DNA-PKcs. A, kinase reactions were performed with DNA-PKcs, Ku70/80, and PARP as described in Fig. 4B in the absence (lanes a-e) or presence (lanes f-j) of 200 µM NAD+. Kinase reactions were initiated by addition of [gamma -32P]ATP and hnRNP-U and incubated for the indicated times at 30 °C. Reactions were terminated by boiling in SDS-PAGE sample buffer and radiolabeled proteins resolved by 10% SDS-PAGE. Arrow indicates the position of radiolabeled hnRNP-U. B, graph showing the quantification of the phosphorylated products from the time course data shown in A. Phosphorylation product values (vertical axis) are based on the quantified volumes of the autoradiograph bands and are plotted against reaction times (horizontal axis). C, DNA-PK reactions were performed with DNA-PKcs alone (lanes a and b) and with DNA-PKcs and Ku70/80 (lanes c and d) as described in Fig. 4B. NAD+ was added to reactions as indicated, and the arrow indicates the position of phosphorylated hnRNP-U.

To determine whether the ability of PARP to stimulate DNA-PK by protein ADP-ribosylation was specific for the phosphorylation of the hnRNP-U substrate, we performed kinase reactions using recombinant human replication protein A (RPA) and recombinant human p53 as protein kinase substrates (Fig. 6). The addition of both PARP and NAD+ stimulated the phosphorylation of the 34-kDa subunit of RPA 4.3-fold relative to the control reaction (Fig. 6, compare lanes a and b). Similarly, the phosphorylation of recombinant p53 was stimulated 3.3-fold by the addition of both PARP and NAD+ to the reaction (Fig. 6, compare lanes c and d).


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Fig. 6.   DNA-PK ADP-ribosylation stimulates the phosphorylation of human replication protein A (RPA) and p53. Protein kinase reactions were performed as described in Fig. 4B using 500 ng of replication protein A (lanes a and b) and 300 ng of p53 (lanes c and d) as protein kinase substrates. NAD+ was added to reactions as indicated, and arrows indicate the position of phosphorylated RPA p34 and phosphorylated p53 proteins.

Taken together, our data indicated that the stimulation of DNA-PK activity by PARP-mediated protein ADP-ribosylation was correlated with the ADP-ribosylation of the DNA-PKcs. However, since both NAD+ and PARP are present in the protein kinase reaction, we could not rule out the possibility that the stimulation was due to the ADP-ribosylation of the protein kinase substrate. To differentiate between these two possibilities, we performed protein kinase reactions in which either DNA-PK or the substrate was incubated with PARP and NAD+ prior to initiating the phosphorylation reaction. Either DNA-PK or p53 were incubated with PARP alone as a control or with PARP and 200 µM NAD+. Free NAD+ was then removed from the reaction by gel filtration using a spin column. Untreated p53 was then added to the pretreated DNA-PK samples, and conversely, untreated DNA-PK was added to the pretreated p53 samples. Protein kinase activity of both of pretreatment conditions was then assayed by addition of radiolabeled ATP. Preincubation of DNA-PK with PARP and NAD+ stimulated the phosphorylation of the p53 substrate 3.9-fold (Fig. 7, compare lanes a and b). In contrast, preincubation of p53 with PARP and NAD+ only stimulated the phosphorylation reaction 1.4-fold relative to the control (Fig. 7, compare lanes c and d). These results were consistent with the idea that ADP-ribosylation of DNA-PK itself, and not the protein kinase substrate, results in the increase in protein phosphorylation.


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Fig. 7.   PARP stimulates DNA-PK by protein kinase ADP-ribosylation. Either 300 ng of DNA-PKcs and 100 ng of Ku70/80 complex (lanes a and b) or 3 µg of p53 (lanes c and d) were incubated for 30 min at 30 °C with 5.7 ng of DNA, 300 ng of PARP, and, where indicated, 200 µM NAD+. Free NAD+ was removed from the reaction using a Bio-Gel P-6 column equilibrated with 0.5× HEPES chromatography buffer containing 5 mM MgCl2 and 1 mM dithiothreitol. 300 ng of p53 was then added to 20 µl of the pretreated DNA-PK samples (lanes a and b), and 30 ng of the DNA-PKcs and 10 ng Ku70/80 were added to 20 µl of the pretreated p53 samples (lanes c and d). Protein kinase activity was then initiated in the pretreated samples by addition of [gamma -32P]ATP, and the samples were incubated for 30 min at 30 °C. The reactions were then terminated by boiling in SDS-PAGE sample buffer and resolved by 10% SDS-PAGE. Arrow indicates the position of phosphorylated p53 protein.

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

A compelling body of evidence indicates the DNA-dependent protein kinase participates in the repair of DNA DSB (26-28). However, despite this information it is not clear what proteins are targets for DNA-PK-mediated phosphorylation in this biochemical pathway or what function(s) these phosphorylations may have. In an effort to identify proteins that may be targets for phosphorylation by DNA-PK, we have isolated DNA-PK complexes from HeLa cell nuclear extracts using an anti-Ku80 affinity matrix. This strategy is based on the assumption that proteins targeted for phosphorylation by DNA-PK may form complexes with the immunoaffinity purified DNA-PK. One of the proteins that co-purified with DNA-PK using these experimental conditions was identified as the poly(ADP-ribose) polymerase, an enzyme that is also activated by binding to DNA strand breaks and that has been proposed to function in the cellular response to DNA damage.

In addition to PARP, we identified hnRNP-U as a second protein that co-purified with the immunoaffinity isolated DNA-PK complexes that was also phosphorylated by purified DNA-PK. hnRNP-U was initially identified as an RNA-binding phosphoprotein found in association with hnRNP particles (35). More recently hnRNP-U has been shown to be a component of the nuclear matrix in HeLa (38), chicken oviduct (39), and rat brain (40) cells. hnRNP-U binds with high affinity to matrix attachment regions including the MII matrix attachment region of the human topoisomerase-I gene (41) and the chicken lysozyme 5'-matrix attachment region (39).

The association of PARP with the affinity purified DNA-PK complexes appears to be mediated primarily via the assembly of these proteins on the same DNA fragment. Since both PARP and Ku can bind to similar DNA structures in vitro (29, 30, 42), it is possible that these proteins could also co-localize on damaged DNA molecules in vivo. The binding of these enzymes to the same broken DNA molecule would increase the likelihood that DNA-PK could phosphorylate PARP and/or PARP could ADP-ribosylate DNA-PK in response to DNA damage. Interestingly, PARP has been shown previously to be a phosphoprotein (43), and the phosphorylation of PARP by protein kinase C reduces its DNA binding affinity and auto-ADP-ribosylation activity (44). However, preliminary data indicate the phosphorylation of PARP by DNA-PK has no obvious effect on PARP DNA binding or catalytic activity.2

PARP has been shown to ADP-ribosylate a variety of proteins involved in DNA metabolism both in vitro and in vivo, including topoisomerase types I and II, DNA-polymerase alpha , DNA-polymerase beta , and DNA ligase II (45-47). With the exception of DNA polymerase alpha  (48) the effect of PARP-mediated ADP-ribosylation of these enzymes has most often been correlated with a decrease in their catalytic activity. Our results are consistent with the idea that PARP-mediated ADP-ribosylation can enhance the protein kinase activity of DNA-PK. This hypothesis is supported by experiments that show PARP stimulates DNA-PK in the presence of NAD+ and that this stimulatory effect is blocked by the PARP inhibitor 1,5-dihydroxyisoquinoline. The ability of PARP to stimulate DNA-PK occurred in the absence of the Ku70/80 complex. This supports the idea that ADP-ribosylation of the DNA-PKcs by PARP accounts for the increase in kinase activity. The ability of PARP to stimulate DNA-PK was not restricted to the hnRNP-U substrate. We also noted the phosphorylation of the 34-kDa subunit of human RPA, and human p53 was also stimulated by PARP in the presence of NAD+. Furthermore, the ability of PARP to stimulate the phosphorylation of p53 was directly correlated with the ADP-ribosylation of DNA-PK and not p53 itself.

PARP and DNA-PK share several similar biochemical properties, including the ability to bind DNA strand breaks, automodify in response to DNA binding, and dissociate from DNA following automodification. Whereas it has been demonstrated that DNA-PK plays an important role in the repair of DNA DSB, participation of PARP in this DNA repair pathway has not been established. Indeed, evidence for the direct involvement of PARP in any mammalian DNA repair pathway is contradictory. Early studies that used chemical inhibitors of poly(ADP-ribosyl)ation indicated PARP may be a component of the DNA excision repair pathway; however, later experiments suggested that these inhibitors block the rejoining of DNA molecules by preventing the auto-ADP-ribosylation and subsequent release of PARP from DNA strand breaks (49, 50).

More recent studies using mice containing homozygous deletions of the PARP gene shows PARP knock-out mice are sensitive to gamma irradiation (51, 52) and that the knock-out mouse cells are sensitive to N-methyl-N-nitrosourea (51). These results indicate PARP either plays a direct role in DNA repair processes or is critical for cellular survival in response to DNA damage. It has been suggested that PARP may function indirectly in the end-rejoining pathway in mammalian cells by binding to DNA strand breaks and preventing the exchange of homologous DNA sequences (32). This idea is supported by recent experiments that show increased V(D)J recombination in severe combined immunodeficiency PARP knock-out mice (53). Our biochemical data support the genetic interaction between DNA-PK and PARP and suggest that in addition to functioning to repress homologous repair of DNA breaks, PARP could further enhance the DNA DSB repair by stimulating the activity of DNA-PK at the site of DNA damage via ADP-ribosylation of the DNA-PKcs.

    ACKNOWLEDGEMENTS

We thank the members of the LANL Cell and Molecular Biology and DNA Damage and Repair groups for critical evaluation of this work. Special thanks goes to Mary Jo Waltman for technical assistance; to Ellen Peterson for critical reading of the manuscript; to Ning-Hsing Yeh, National Yang-Ming Medical College, for the Ku80 antibody; and to Scott Kaufmann, Division of Oncology Research, Mayo Clinic for reagents and guidance. Peptide sequence analysis of hnRNP-U was performed by Steve Smith of the University of Texas Medical Branch, Galveston, TX.

    FOOTNOTES

* 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.

parallel 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.

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.

2 S. R. Peterson, unpublished observations.

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Discussion
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