Photoaffinity Labeling of Mouse Fibroblast Enzymes by a Base Excision Repair Intermediate

EVIDENCE FOR THE ROLE OF POLY(ADP-RIBOSE) POLYMERASE-1 IN DNA REPAIR*

Olga I. LavrikDagger §, Rajendra PrasadDagger , Robert W. SobolDagger , Julie K. HortonDagger , Eric J. Ackerman, and Samuel H. WilsonDagger ||

From the Dagger  Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, the § Novosibirsk Institute of Bioorganic Chemistry, Siberian Division of Russian Academy of Sciences, 630090 Novosibirsk, Russia, and the  Pacific Northwest National Laboratory, Richland, Washington 99352

Received for publication, March 9, 2001, and in revised form, May 2, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To examine the interaction of mammalian base excision repair (BER) enzymes with DNA intermediates formed during BER, we used a novel photoaffinity labeling probe and mouse embryonic fibroblast cellular extracts. The probe was formed in situ, using an end-labeled oligonucleotide containing a synthetic abasic site; this site was incised by apurinic/apyrimidinic endonuclease creating a nick with 3'-hydroxyl and 5'-reduced sugar phosphate groups at the margins, and then a dNMP carrying a photoreactive adduct was added to the 3'-hydroxyl group. With near-UV light (312 nm) exposure of the extract/probe mixture, six proteins were strongly labeled. Four of these include poly(ADP-ribose) polymerase-1 (PARP-1) and the BER participants flap endonuclease-1, DNA polymerase beta , and apurinic/apyrimidinic endonuclease. The amount of the probe cross-linked to PARP-1 was greater than that cross-linked to the other proteins. The specificity of PARP-1 labeling was examined using various competitor oligonucleotides and DNA probes with alternate structures. PARP-1 labeling was stronger with a DNA representing a BER intermediate than with a nick in double-stranded DNA. These results indicate that proteins interacting preferentially with a photoreactive BER intermediate can be selected from the crude cellular extract.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Base excision repair (BER)1 is one of the main strategies of the cell for defense against exogenous and endogenous genotoxic stress leading to single-base lesions in DNA (1, 2). Mouse embryonic fibroblasts (MEFs) rendered deficient in BER by gene deletion of a BER enzyme are hypersensitive to monofunctional DNA-methylating agents among other stressors, illustrating the importance of this repair system (3-5). The current and generally accepted working model for mammalian BER involves two sub-pathways, each proceeding as a sequential process with several DNA and/or DNA-enzyme intermediates (6-9). In each of the BER sub-pathways, repair can be initiated by spontaneous base loss or DNA glycosylase action to produce an abasic site and in some cases by coupled glycosylase base removal and strand cleavage (1, 10, 11). When the intact abasic site is an intermediate, DNA strand cleavage on the 5' side of the sugar is by the abundant nuclear enzyme apurinic/apyrimidinic endonuclease (APE), and cleavage on the 3' side of the sugar is by the deoxyribose phosphate (dRP) lyase activity of DNA polymerase beta  (beta -pol) (12, 13). The single nucleotide gap is filled by beta -pol, and then the nick is eventually sealed by a DNA ligase, thus completing the "short patch" or "single nucleotide" BER sub-pathway (7, 14, 15). In mammalian cells, repair of methylated bases, oxidized bases, and abasic sites appears to occur predominantly by this sub-pathway (16, 17). In other cases, for example where the sugar of the abasic site is not removed efficiently, the "long patch" BER sub-pathway mediates repair (18). This sub-pathway involves limited strand displacement and DNA synthesis to replace between 2 and as many as 15 nucleotides in the damaged strand (5, 19-21). Finally, this displaced damaged strand is excised by the structure-specific flap endonuclease-1 (FEN-1), and the resulting nick is sealed by a DNA ligase (14, 17, 20). The DNA synthesis step of long patch BER can be conducted by beta -pol or other DNA polymerases (6, 18, 20, 22-26). The long patch BER sub-pathway appears to account for only a small fraction of overall BER in cell extracts that have proficient single nucleotide BER (23, 25).

The various steps in single nucleotide and long patch BER may be coordinated via protein-protein and DNA-protein interactions, and bimolecular complexes have been observed for x-ray cross-complementing factor 1 (XRCC1) and DNA ligase III (7, 27) and for beta -pol and the following: APE (28), PARP-1 (29), and DNA ligase I (14). Yet the mechanism and regulation for each of these interactions are either completely obscure or only beginning to be revealed, and the functional implications of the interactions concerning the efficiency and accuracy of BER are unknown. In the experiments to be described in this report, we examined the interaction between PARP-1 and BER intermediates.

PARP-1 is an abundant nuclear enzyme that is known to bind to DNA nicks and becomes activated to enzymatically poly(ADP-ribosyl)ate many nuclear proteins, including itself, using NAD+ as substrate. Self poly(ADP-ribosyl)ation of PARP-1 is known to cause it to release from its DNA-binding site (reviewed in Ref. 30). PARP-1 in mammalian cells exists in several isoforms (31-34). Recently, Dantzer et al. (29) examined the role of PARP-1 in single nucleotide and long patch BER using biochemical studies of "knock out" MEF cell lines for the beta -pol and/or PARP-1 genes. Among other points, they found that extracts from PARP-1 null MEF were moderately reduced in single nucleotide BER activity and strongly reduced in long patch BER activity pointing to a role of PARP-1 in both sub-pathways and a requirement for PARP-1 in long patch BER (29). A role for PARP-1 in BER had been suggested much earlier, because of the DNA binding specificity of PARP-1 for nicks in DNA (2, 35), and the physical interactions of PARP-1 with the known BER proteins XRCC1 and beta -pol (27, 29, 36). In addition, PARP-1 null MEF cells are known to be hypersensitive to DNA-damaging agents that produce lesions repaired by BER (37, 38). It was also found that PARP-1 can activate DNA ligase during repair in vitro (39), and recently it was shown that PARP-1, beta -pol, and DNA ligase III/XRCC1 form a BER complex that can support the ATP requirement for DNA ligation of the nick (40). Therefore, it is considered well documented that PARP-1 has a role in BER, yet the molecular mechanism of its participation has remained obscure.

In this study, we examined the question of whether proteins in MEF crude extracts can be selectively labeled by a photoaffinity DNA probe representing an early intermediate in long patch BER. We found that only six proteins in the crude extract were strongly labeled by this BER probe, including several well known BER enzymes. The results described here reveal several important features of molecular interactions between BER enzymes and BER intermediates.

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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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Materials-- Rainbow colored molecular mass markers and [gamma -32P]ATP were from Amersham Pharmacia Biotech. Synthetic oligodeoxynucleotides were obtained from Oligos Etc. (Wilsonville, OR) and from GENSET (La Jolla, CA). The photoreactive dCTP analog, exo-N-[beta -(p-azidotetrafluorobenzamido)-ethyl]-deoxycytidine 5'-triphosphate (FAB-dCTP), was synthesized as described previously (41). Dulbecco's modified Eagle's medium (DMEM), GlutaMAX-1, L-glutamine, Hanks' balanced salt solution, and G418 were from Life Technologies, Inc. Methyl methanesulfonate (MMS) and 3-aminobenzamide (3-AB) were from Sigma; 4-amino-1,8-naphthalimide (4-AN) was from Acros Organics/Fisher, and cisplatin (Platinol-AQ) was from Bristol-Myers Squibb Co. Fetal bovine serum (FBS) was obtained from Summit Biotechnology (Ft. Collins, CO) and hygromycin from Roche Molecular Biochemicals.

Cells and Extracts-- The wild-type MEF cell line MB16tsA, clone 1B5, and the beta -pol null cell line MB19tsA, clone 2B2, have been described previously (4) and are available from ATCC (CRL 2307 and CRL 2308, respectively). These transformed fibroblast cell lines were maintained in DMEM containing 10% FBS, GlutaMAX-1, and 80 µg/ml hygromycin in a 10% CO2 incubator at 34 °C. The beta -pol null cells expressing a FLAG epitope-tagged beta -pol were prepared as described previously (42) and were maintained in DMEM, 10% FBS, penicillin/streptomycin, GlutaMAX-1, and 600 µg/ml G418 in a 10% CO2 incubator at 34 °C. These cells were used here in cross-linking experiments and were the MEF cell system used, unless otherwise indicated. Wild-type and PARP-1 null spontaneously immortalized MEFs (43) were obtained from Dr. Josianne Ménissier-de Murcia (CNRS, Illkirch-Graffenstaden, France). The cells were cultured at 37 °C in a 10% CO2 incubator in DMEM containing L-glutamine and 10% FBS. All cells were routinely tested and found to be free of mycoplasma contamination.

Extracts were prepared by suspending 5 × 106 cells in 100 µl of Buffer I (10 mM Tris-Cl, pH 7.8, 200 mM KCl) followed by addition of an equal volume of Buffer II (10 mM Tris-Cl, pH 7.8, 200 mM KCl, 2 mM EDTA, 40% glycerol, 0.2% Nonidet P-40, 2 mM dithiothreitol, 0.5 mM phenylmethylsulfonyl fluoride, 10 mg/ml aprotinin, 5 mg/ml leupeptin, 1 mg/ml pepstatin A). The suspension was rotated at 4 °C for 1 h and centrifuged to pellet cellular debris at 14,000 rpm for 10 min.

Cytotoxicity Assays-- Cytotoxicity was determined by growth inhibition assays as described previously (25). Wild-type and beta -pol null cells or wild-type and PARP-1 null cells were seeded at a density of 40,000 cells/well in 6-well dishes. The following day they were exposed for 1 h to a range of concentrations of MMS or cisplatin in growth medium. Both agents were added directly to the medium at the time of the experiment. After 1 h, cells were washed with Hanks' balanced salt solution, and fresh medium was added. Dishes were incubated for 4-5 days until control untreated cells were ~80% confluent. Cells (triplicate wells for each drug concentration) were counted by a cell lysis procedure (44). Experiments were also conducted in the presence of PARP inhibitors 3-AB and 4-AN. Stock solutions of both inhibitors were prepared in dimethyl sulfoxide and then diluted in growth medium to the highest non-toxic concentration (10 mM for 3-AB and 10 µM for 4-AN). Dilutions of MMS or cisplatin were prepared in medium containing inhibitor, and the cells were dosed as described above. Following the 1-h MMS or cisplatin exposure, cells were washed and then incubated in PARP inhibitor-containing medium for a further 23 h.

Proteins and Antibodies-- Human beta -pol, FEN-1, and APE were purified as described (18, 45, 46). Recombinant PARP-1 was purified by conventional chromatographic methods employing anion and cation exchange according to an adapted procedure (47). Human DNA polymerase alpha  was provided by Dr. William C. Copeland (NIEHS, National Institutes of Health). Human DNA ligase I and DNA ligase III were provided by Dr. Alan E. Tomkinson (University of Texas Health Science Center, San Antonio, TX). Human replication protein A (RPA) was isolated as described (48). Monoclonal antibodies against PARP-1 were purchased from PharMingen (San Diego, CA). Antibodies against DNA ligase I and RPA were purchased from NeoMarkers (Freemont, CA). Antibodies against FEN-1 were provided by Dr. Vilhelm A. Bohr (NIA, National Institutes of Health), and FLAG antibody-specific agarose was purchased from Sigma.

Primer-Template Used in the Cross-linking Experiments-- Primer-templates used in the study are shown in Table I.

Radioactive Labeling of Oligodeoxynucleotide Primers-- Oligodeoxynucleotides were 5'-32P-phosphorylated with T4 polynucleotide kinase as described (49). Unreacted [gamma -32P]ATP was separated by passing the mixture over a Nensorb-20 column using the manufacturer's suggested protocol.

Primer-Template Annealing-- Lyophilized oligodeoxynucleotides were resuspended in 10 mM Tris-HCl, pH 7.4, and 1 mM EDTA, and the optical density was measured. Complementary oligodeoxynucleotides were annealed by heating a solution of equimolar concentrations to 90 °C for 3 min, followed by slow cooling to room temperature.

Photoaffinity Labeling-- The photoaffinity labeling reaction mixture (10 or 20 µl) contained 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 4 or 8 µl of 1.6 mg/ml cellular extract (prepared from beta -pol null MEF expressing a FLAG epitope-tagged beta -pol unless otherwise indicated), 0.4 µM 32P-labeled DNA containing either a reduced abasic site (DNA1), a nicked abasic site (DNA3), or a uracil residue (DNA8) (see Table I), and 20 µM FAB-dCTP. The reconstituted reaction mixture (10 or 20 µl) contained 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 50 mM KCl, 0.4 µM 32P-labeled DNA (DNA1), 20 µM FAB-dCTP, 0.2 µM PARP-1, 0.2 µM FEN-1, 1 µM beta -pol, and 0.7 µM APE. The reaction mixtures were incubated at 25 °C for 30 min to allow incorporation of the photoreactive probe exo-N-[beta -(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine 5'-monophosphate (FAB-dCMP). Then the mixtures were spotted onto Parafilm, placed on ice, and irradiated with UV light (lambda max = 312 nm, 5-7 mJ) with a UV Stratalinker (Stratagene, La Jolla, CA). The photoaffinity labeled proteins were separated by 10% SDS-PAGE. Gels were dried and subjected to autoradiography.

Introduction of Photoreactive FAB-dCMP Probe into DNA-- The reaction conditions for evaluating DNA synthesis with photoreactive FAB-dCTP or dCTP were identical to those used for the cross-linking experiments. After the initial DNA synthesis for 30 min at 25 °C, the reaction was terminated by adding 10 µl of 90% formamide, 50 mM EDTA, and 0.1% bromphenol blue. The mixture was heated for 3 min at 80 °C, and products were analyzed by electrophoresis followed by autoradiography.

Unlabeled DNA Competition for PARP-1 Labeling by Photoreactive DNA-- PARP-1 binding to DNA5, DNA6, and DNA7 (Table I) was evaluated for competition with PARP-1 labeling 32P-labeled photoreactive DNA3. Unlabeled DNA5, DNA6, or DNA7 was used in a 1:1 molar ratio with the probe DNA. Reaction mixtures (10 µl) contained 50 mM Tris-HCl, pH 7.4, 10 mM MgCl2, 4 µl of 1.6 mg/ml cellular extract, 0.4 µM 32P-labeled DNA3, 20 µM FAB-dCTP. Reconstituted system reaction mixtures (10 µl) contained 50 mM Tris-HCl, pH 7.8, 10 mM MgCl2, 50 mM KCl, 0.4 µM 32P-labeled DNA3, 20 µM FAB-dCTP, 0.2 µM APE, and 0.2 µM beta -pol. The reaction mixture was incubated for 30 min at 25 °C with 0.4 µM of unlabeled competitor DNA, DNA5, DNA6, or DNA7. The competitor DNA was added to the reaction mixture before UV irradiation. For the reconstituted system, 0.3 µM PARP-1, 0.2 µM APE, and 0.2 µM beta -pol were used. UV light (lambda max = 312 nm, 3 mJ) irradiation was with a UV Stratalinker. Cross-linked proteins were separated by 10% SDS-PAGE and visualized by autoradiography.

Immunoprecipitation Experiments with Antibodies against DNA Repair Proteins-- Immunoprecipitation was as described (42) using M2-FLAG antibody conjugated to agarose. Briefly, the photoaffinity labeled reaction mixture (200 µl) was mixed with 5 µg of anti-FLAG M2 antibody conjugated to agarose, incubated with rotation for 4 h at 4 °C, and pelleted by centrifugation at 14,000 rpm (15 s). The supernatant was discarded, and the protein-bound agarose complex was washed 4 times with extract buffer. After a final wash, the buffer was removed, and the protein agarose suspension was mixed with 50 µl of SDS-gel loading buffer and incubated in a boiling water bath for 5 min to release the antibody-bound proteins.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Effect of PARP Inhibitors on MMS-induced Cytotoxicity-- To probe for enzymes recognizing BER intermediates, we used crude extracts of paired MEF cell lines that were wild type or deleted in the genes for either beta -pol or PARP-1. The respective wild-type cell lines appeared to be proficient in BER as revealed by their resistance to MMS, a prototype agent for inducing DNA damage repaired by BER (Fig. 1, A and B). The beta -pol null cell line exhibits hypersensitivity to MMS (Fig. 1A), as is well known from previous work (4). Cellular resistance to MMS in these fibroblasts also appeared to depend on PARP-1, since the wild-type cell line was rendered extremely hypersensitive to MMS in the presence of PARP inhibitors 3-AB or 4-AN (Fig. 1A). The extreme hypersensitivity imposed by the PARP inhibitors suggests that PARP is involved in both of the BER sub-pathways, since our previous results indicated that each BER sub-pathway results in greater resistance to MMS than that seen in the presence of 4-AN (25). The PARP-1 null cells were also hypersensitive to MMS (Fig. 1B), and with PARP inhibitors the wild-type cells became even more sensitive than the PARP-1 null cells suggesting a role in BER for other proteins with PARP activity in addition to PARP-1. Therefore, in both of the MEF cell line systems used here, it appears that PARP-1 plays a role in repair of MMS-induced lesions, and we inferred that this reflects a role of PARP-1 in BER. As a control for the cellular response to a "non-BER" genotoxicant, no such hypersensitivity was observed when the beta -pol null cell line was exposed to cisplatin, and 4-AN did not render the wild-type cells hypersensitive (Fig. 1C).


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Fig. 1.   Effect of PARP inhibitors on MMS- and cisplatin-induced cytotoxicity. Survival curves were produced as described under "Experimental Procedures." Wild-type (closed symbols) and beta -pol null (open symbols) mouse fibroblasts (A and C) or PARP-1 wild-type (closed symbols) and null (open symbols) mouse fibroblasts (B) were exposed for 1 h to MMS (A and B) or cisplatin (C) in the absence (, open circle ) or presence of 3-AB (10 mM for 24 h) (black-triangle), or 4-AN (10 µM for 24 h) (black-square). Data are from representative experiments; values represent the mean of triplicate determinations.

Photoaffinity Labeling with a BER Intermediate-- The structure of the photoaffinity labeling probe is illustrated in Fig. 2. The photoreactive dCTP analogue, FAB-dCTP, and a 32P-5'-end-labeled 34-base pair oligonucleotide containing a synthetic abasic site (3-hydroxy-2-hydroxymethyltetrahydrofuran (THF) 5'-phosphate) (i.e. DNA1, Table I), were used to produce the photoreactive BER intermediate in situ (i.e. DNA2, Table I). The arylazido moiety in the photoreactive group (Fig. 2A) can be activated with near (312 nm)-UV light and is therefore a more selective cross-linking group than those with azido moieties requiring higher energy UV-light exposure such as 254 nm. The abasic site-containing DNA was cleaved by APE in the MEF extract, and the photoreactive FAB-dCMP residue was introduced at the resulting 3'-hydroxyl group by a DNA polymerase in the extract. As illustrated in Fig. 3, this process resulted in a photoreactive nick containing the THF abasic site sugar phosphate at the 5'-margin and FAB-dCMP at the 3'-margin (DNA2, Table I). Note that the sugar in the THF abasic site was not subject to removal by a beta -elimination mechanism.


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Fig. 2.   Structure of the photoreactive dCTP analogue (FAB-dCTP) and oligonucleotide DNA (DNA1, Table I). A, dC*TP, (exo-N-[beta -(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine 5'-triphosphate), the photoreactive arylazido group with a spacer arm was attached to the N-4 of dCTP. B, 34-base pair oligodeoxynucleotide containing the tetrahydrofuran residue (X), 3-hydroxy-2-hydroxymethyltetrahydrofuran (THF), opposite G.

                              
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Table I
Sequence of initial DNA probes and competitor DNA oligonucleotides


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Fig. 3.   Synthesis of the photoreactive DNA probe. A, a scheme illustrating in situ conversion of radiolabeled DNA1 (Table I) to the nicked DNA containing FAB-dCMP and deoxyribose phosphate at the 3'- and 5'-margins, respectively. B, photograph of an autoradiogram of a DNA sequencing gel showing primer extension products with FAB-dCMP and dCMP by the MEF extract. DNA1 was incubated for 30 min at 25 °C with extract and products were analyzed as described under "Experimental Procedures." Lane 1, DNA1 was preincubated with purified APE to produce labeled 15-mer primer; lane 2, DNA1 with extract, but without FAB-dCTP; lane 3, DNA1, with extract and FAB-dCTP. C, incorporation of dCMP and FABdCMP in vitro using purified APE and beta -pol, lanes 2 and 3, respectively. Lane 1 shows the 32P-labeled initial 15-mer primer, produced from DNA1 using purified APE.

Primer extension DNA synthesis leading to the formation of photoreactive probe in the cellular extract is illustrated in Fig. 3B, and for reference, formation of the probe by a system reconstituted with purified APE and beta -pol is shown in Fig. 3C. The electrophoretic mobility of the primer extended with FAB-dCMP is slower than that of the primer extended with dCMP (Fig. 3, B and C). The resulting DNA probe is regarded as a long patch BER intermediate because the 5'-margin sugar phosphate of the nick is refractory to beta -pol-mediated removal, and the persistence of the sugar phosphate prevents ligation of the unrepaired BER intermediate by DNA ligases. Limited strand displacement DNA synthesis by beta -pol followed by FEN-1 cleavage of the resulting flap structure are key subsequent steps in the long patch BER sub-pathway. Yet DNA2 is frozen at a stage prior to strand displacement, because the 3' FAB-dCMP is a poor substrate for further primer extension DNA synthesis.

Photoaffinity Labeling of BER Proteins in the MEF Extract-- Incubation of the 32P-labeled DNA photoaffinity labeling probe in a MEF crude extract, followed by near-UV light exposure, resulted in labeling of several proteins. A typical labeling pattern, when DNA2 is used as probe, is shown in Fig. 4A, lane 2. Only six proteins in the crude extract were strongly labeled by this probe, suggesting significant selectivity for this labeling procedure. Three of the labeled proteins were in a molecular mass range consistent with the well known BER enzymes APE, beta -pol, and FEN-1, and the abundantly labeled higher molecular mass product was consistent with PARP-1, DNA ligases, or DNA polymerases, among other proteins. Four of the six proteins were identified in the studies described below and are labeled accordingly in Fig. 4A. For comparison, a completely different cross-linking pattern was obtained by an alternative method involving UV light exposure at 254 nm and with DNA of similar structure as DNA2, but without the FAB photoaffinity labeling group (Fig. 4B). This result illustrates the selectivity of the labeling procedure used here and of the DNA2 probe.


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Fig. 4.   Photoaffinity labeling and identification of photoaffinity labeled proteins in MEF cellular extract. A shows a photograph depicting the pattern of cross-linking of cellular proteins to photoreactive nicked DNA. Experiments were as described under "Experimental Procedures." The 5'-32P-labeled DNA1 (0.4 µM) was preincubated with the usual beta -pol system MEF cellular extract expressing a FLAG epitope-tagged beta -pol for 30 min at 25 °C with 20 µM FAB-dCTP (lane 2) or without FAB-dCTP (lane 1) to introduce the photoreactive FAB-dCMP moiety into DNA1 (i.e. to form DNA2). Then the mixtures were irradiated with UV light (lambda max = 312 nm). The UV cross-linked products were separated by SDS-PAGE and visualized by autoradiography. The positions of the identified BER proteins and the protein markers are indicated. B shows the cross-linking pattern of cellular proteins to DNA5 without the photoreactive FAB-dCMP. 5'-32P-Labeled DNA5 (0.4 µM) was preincubated with MEF extract (as in A) and irradiated with UV light (lambda max = 254 nm). The UV cross-linked products were separated by SDS-PAGE and visualized by autoradiography. C shows the identification of photoaffinity labeled products by supplementing known DNA repair proteins to the extract. The reaction mixture containing DNA1 and FAB-dCTP was incubated for 30 min at 25 °C and then UV-irradiated either with no additional proteins (lane 1), 2 µM APE (lane 2), 1 µM beta -pol (lane 3), or 3 µM FEN-1 (lane 4). D shows the identification of cross-linked proteins in the MEF cellular extract by immunoprecipitation. After UV cross-linking, as in A, the photoaffinity labeled proteins were immunoprecipitated either with preimmune IgG (lane 2), anti-FEN-1 antibody (lane 3), or anti-FLAG antibody (beta -pol) (lane 4). Lane 1 represents 10% of the cross-linked reaction mixture used for immunoprecipitation. The positions of identified proteins and protein markers are indicated in the right and left margins, respectively. E shows a comparison of cross-linking patterns of cellular proteins to photoreactive nicked DNA (DNA2) obtained with beta -pol null cell extract (lane 3 and 4) and the usual MEF cellular extract expressing a FLAG epitope-tagged beta -pol (lanes 1 and 2). The 5'-32P-labeled DNA1 (0.4 µM) was preincubated for 40 min at 25 °C with 20 µM FAB-dCTP (lanes 2 and 4) or without FAB-dCTP (lanes 1 and 3) to introduce the photoreactive FAB-dCMP moiety into DNA. The level of DNA synthesis for incorporation of FAB-dCMP was found to be similar for the usual extract containing beta -pol and beta -pol null extract. The mixtures were then UV light (lambda max = 312 nm)-irradiated, and the UV-cross-linked products in the FEN-1 to APE molecular mass range were visualized as in A.

Several of the proteins strongly labeled in the experiment shown in Fig. 4A, lane 2, were identified. We first supplemented the MEF extract individually with well known BER enzymes in purified form to determine if these proteins could be labeled and if the products had gel mobility similar to those seen in Fig. 4A. With extract supplemented with APE, beta -pol, and FEN-1 (Fig. 4C, compare lane 1 with lanes 2-4), an increase in labeling was observed for products migrating at 42, 45, and 52 kDa, respectively. The mobility of each labeled species was consistent with addition of one molecule of the labeled DNA strand to APE, beta -pol, and FEN-1, respectively. Also, with addition of an excess of one enzyme, labeling of the other two enzymes was diminished. The doublet of labeled beta -pol seen in Fig. 4C, lane 3, was consistent with the inherent mass difference between the endogenous FLAG-tagged beta -pol in the MEF extract and the purified native beta -pol added to the extract. Overall, these results indicate that FEN-1, beta -pol, and APE can be labeled by the probe when added to the extract individually and that these purified proteins can bind the probe and compete with other proteins in the extract.

We next examined the question of whether these three labeled proteins in the MEF extract could be immunoprecipitated with antibody specific to each protein. Antibody to the FLAG epitope on beta -pol precipitated labeled protein corresponding to the 45-kDa product (i.e. FLAG-beta -pol) and a portion of the higher molecular mass product labeled as PARP-1 in Fig. 4D, lane 4. Antibody to FEN-1 precipitated labeled protein corresponding to the 52-kDa product (Fig. 4D, lane 3). Our antibody to APE failed to precipitate any labeled protein (data not shown), and similarly, non-immune IgG did not precipitate any labeled proteins (lane 2). Since APE was not identified by immunoprecipitation in these experiments, assignment of this protein as the 42-kDa labeled product is considered only preliminary. Finally, the proteins responsible for the two lower molecular mass labeled products seen in Fig. 4A, lane 2 (designated I and II), were not further evaluated, but they were not precipitated by any of the antibodies noted above. The identity of beta -pol was further confirmed using beta -pol null cell extract instead of the usual MEF extract (Fig. 4E). The results of cross-linking experiments indicated that the cross-linking product with molecular mass consistent with beta -pol was not detected with extract from beta -pol null cells (Fig. 4E, compare lane 2 to lane 4).

Next, we conducted experiments to identify the protein responsible for the higher molecular mass product, which was the major labeling product. The protein was eventually identified as PARP-1. In the first experiment, purified PARP-1 was added to the extract (Fig. 5A). The amount of the higher molecular mass product was observed to increase in proportion to the amount of PARP-1 added to the extract, suggesting that this product was due to PARP-1. This notion was further examined by adding NAD+ to the reaction mixture. The premise of this experiment is that in the presence of NAD+, PARP-1 will become auto-poly(ADP-ribosyl)ated, and as a result, the gel mobility of PARP-1 will change such that it will not enter the gel utilized in this experiment. When our incubation mixture was supplemented with NAD+ before the UV light cross-linking, the usual higher molecular mass labeled product was not observed, and a significant amount of labeled protein failed to enter the gel (Fig. 5B, compare lanes 1 and 2). Labeling of FEN-1, beta -pol, and APE was not altered by the addition of NAD+ (Fig. 5B, compare lanes 1 and 2). Thus, the protein responsible for the higher molecular mass product appeared to be poly(ADP-ribosyl)ated, but FEN-1, beta -pol, and APE were not. In the next experiment, we compared the amount of the higher molecular mass product formed by cellular extracts prepared from either wild-type or PARP-1 null MEF cell lines. This higher molecular mass product was detected with the extract from the wild-type cell line but not with extract from the PARP-1 null cell line (Fig. 5C).


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Fig. 5.   Identification of PARP-1. Several approaches were used to identify the high molecular mass product. A is a photograph of an autoradiogram showing only the high molecular mass product (PARP-1). The reaction mixture containing DNA1, FAB-dCTP, and cellular extract was preincubated for 30 min at 25 °C and then UV-irradiated (lambda max = 312 nm) either with (lanes 2-4) or without (lane 1) purified PARP-1. B shows the photocross-linking products obtained in cellular extract in the absence (lane 1) or in the presence (lane 2) of 1 mM NAD+ (lane 2). The positions of identified proteins and protein markers are indicated. C shows the cross-linking of the high molecular mass product in cellular extract derived either from PARP-1(-/-) (lane 1) or PARP-1(+/+) MEF (lane 2). D depicts a photograph of an autoradiogram of protein fractions precipitated with preimmune IgG (lane 2), anti-PARP-1 IgG (lane 3), or 10% of the reaction mixture by SDS-PAGE. The positions of the identified proteins and protein markers are indicated to the right and left of the panel.

Finally, we examined the question of whether the higher molecular mass labeled product observed with the usual MEF extract could be immunoprecipitated with antibody specific to PARP-1 (Fig. 5D). We found that this product was precipitated with the alpha -PARP-1 antibody but not with non-immune IgG, confirming that the labeled protein was indeed PARP-1. FEN-1, beta -pol, and APE were not co-immunoprecipitated along with PARP-1 by the alpha -PARP-1 antibody. We also examined whether purified PARP-1, FEN-1, beta -pol, and APE could be labeled using a mixture of these purified proteins. For an incubation mixture containing all four proteins and DNA2 as probe, we found four labeled products of masses identical to those seen with the crude cell extracts (data not shown) corresponding to PARP-1, FEN-1, beta -pol, and APE; similar labeling for beta -pol and APE was found either with or without the other two enzymes (data not shown).

Taken together, these results demonstrate that the higher molecular mass labeled product was due to PARP-1. Other DNA repair proteins, such as DNA ligase I, DNA ligase III, RPA, and DNA polymerase alpha  were also tested individually for their ability to cross-link to the probe. Although these purified proteins could be individually cross-linked, the mobility of the labeled product in each case (data not shown) was different than that of the major higher molecular mass labeled products observed here, i.e. in Fig. 4A, lane 2, the signal designated as PARP-1. Furthermore, the high molecular mass product did not immunoprecipitate with antibodies specific for DNA ligase I, DNA ligase III, or RPA.

DNA Structural Requirements for PARP-1 Labeling-- To examine further the specificity of PARP-1 labeling, we conducted competition experiments using a 32P-labeled probe designated DNA4 that was similar to the usual DNA probe described above (DNA2) and three unlabeled competitor DNA (Table I and Fig. 6). This DNA probe, DNA4, lacked a phosphate group on the 5'-tetrahydrofuran sugar in contrast to DNA2, yet we found that the amount of PARP-1 labeling by these two probes, DNA4 and DNA2, was similar. DNA4 was used as a labeled probe in competition experiments because preparation of closely related competitor DNA was more straightforward. Cell extract containing the probe was supplemented with an equimolar ratio of competitor DNA, and the mixture then was irradiated with near-UV light. As shown in Fig. 6, the amount of PARP-1 labeling was reduced by competitors DNA5 and DNA6 (see Table I) but not by the normal double-stranded DNA tested, DNA7. The results of the competition experiments using a reconstituted system that included purified PARP-1 were virtually identical (Fig. 6B). The results indicate that the double-stranded DNA competitor, DNA7, had much less affinity for PARP-1 than the probe DNA, DNA4. In contrast, DNA5 with dCMP in the position of the FAB-dCMP group in the probe, competed about 50% of the labeling, suggesting that DNA5 and the probe had similar affinity for PARP-1. The competitor DNA with an A:C base pair in the nick, DNA6, competed more strongly suggesting greater PARP-1 affinity than that of DNA5 or the probe DNA.


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Fig. 6.   Competition of photoaffinity labeling of PARP-1 with different DNAs. 32P-Labeled DNA3 (0.4 µM) was preincubated in MEF cellular extract (A) or in a reconstituted system (B) with FAB-dCTP (20 µM) for 30 min at 25 °C and then 0.4 µM unlabeled DNA5 (A and B, lane 2), DNA6 (A and B, lane 3), or DNA7 (A and B, lane 4) was added before UV irradiation. Lane 1 (A and B) shows PARP-1 cross-linking in cellular extract (A) or in a reconstituted system (B) without competitor DNA. Reaction mixtures were irradiated with UV light (lambda max = 312 nm). The UV cross-linked products were separated by SDS-PAGE and visualized by autoradiography. The positions of identified proteins and protein markers are indicated.

Different Labeling Patterns with Single Nucleotide and Long Patch BER Substrates-- Next, we made use of a modification in the structure of the labeled probe to examine further the specificity of PARP-1 labeling. We found that PARP-1 labeling was influenced by the DNA structure at the 5'-margin of the nick. An alternate DNA structure lacking a 5'-sugar moiety on the downstream polynucleotide (DNA9, Table I and Fig. 7) gave much less labeling of PARP-1 than DNA2 (Fig. 7A). In the case of DNA9, the probe contained FAB-dCMP and phosphate at the 3'- and 5'-margins of a nick (DNA9), respectively. Note that DNA9 was processed from DNA8 in situ by the extract (i.e. DNA9, Table I) but was not ligated into a double-stranded DNA product (data not shown). APE was not labeled by the DNA9 probe, yet beta -pol was equally labeled by DNA9 and DNA2 (Fig. 7A, lane 4). The photoreactive FAB-dCMP probe was inserted into DNA1 and DNA8 with similar efficiency to give rise to the extended products, DNA2 and DNA9, respectively (data not shown). As a control for these experiments, we found that similar results were obtained when the two probes were prepared by an alternate procedure involving annealing three preformed oligomers (Fig. 7B), instead of two. In this experiment, initial DNA substrates were created by annealing the oligonucleotides such that DNA3 contains 3'-OH and 5'-THF (without 5'-phosphate), and DNA10 contains 3'-OH and 5'-phosphate in the margin of the nick, respectively (see Table I). These experiments indicate that labeling of PARP-1 was much less when the DNA probe lacked the 5'-abasic site sugar characteristic of the early BER intermediate.


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Fig. 7.   Specificity of PARP-1 photocross-linking with single nucleotide (S-N) or long patch (LP) BER substrates. A, 32P-labeled DNA1 (lanes 1 and 2) or DNA8 (lanes 3 and 4) were preincubated with MEF cellular extract for 30 min at 25 °C with (lanes 2 and 4) and without (lanes 1 and 3) FAB-dCTP. Then the reaction mixtures were UV-irradiated at 312 nm. Photocross-linked proteins were separated by SDS-PAGE and visualized by autoradiography. B, lanes 1 and 2 and lanes 3 and 4 contain DNA3 and DNA10, as initial DNA, respectively. DNA3 contains 3'-OH and 5'-THF, whereas DNA10 bears 3'-OH and 5'-phosphate groups in the margin of the gap. Photocross-linking and the product analysis are as in A. Positions of identified proteins and protein markers are indicated.

Co-immunoprecipitation of BER Proteins with Anti-FLAG Antibody-- We used the approach of immunoprecipitation with an antibody to the FLAG epitope attached to beta -pol to probe for protein complexes in the MEF extract. The DNA2 probe/extract mixture was irradiated, and the mixture was then subjected to immunoprecipitation. The results revealed that a portion of the PARP-1 and FEN-1 in the extract were co-immunoprecipitated as labeled proteins (Fig. 8, lane 3) but that this was not the case for APE or other proteins. Negative controls for this experiment failed to reveal any protein immunoprecipitation (Fig. 8, lane 2), indicating that the result shown in Fig. 8, lane 3, was specific for the PARP-1, FEN-1, and beta -pol complex. It should be noted that extensive washing of the protein/DNA/antibody-agarose mixture eventually releases FEN-1 from the complex, as depicted in Fig. 4. This suggests that the interaction between FEN-1 and the beta -pol/PARP complex is weaker than the interaction between beta -pol and PARP.


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Fig. 8.   Immunoprecipitation of PARP-1, FEN-1, and beta -pol cross-linked to 32P-labeled DNA2. Experiments were conducted as described under "Experimental Procedures;" and as in Fig. 4, A, an autoradiogram of the cross-linking pattern of cellular proteins is shown. Lane 1, 10% of reaction mixture used for immunoprecipitation; lane 2, protein fraction precipitated with the preimmune IgG; lane 3, protein fraction precipitated with anti-FLAG epitope antibody (i.e. to beta -pol).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The identity of the various enzymes participating in mammalian BER is a subject of active investigation. In this study, we used a sensitive, novel photoaffinity labeling reagent to identify BER enzymes in the crude extract of MEF cells. For the two BER sub-pathways, a nicked DNA structure is considered a branch point in BER sub-pathway choice, and one can consider a nicked DNA structure with a reduced abasic site sugar, as used here, as representing an early intermediate frozen at the stage just prior to dRP lyase action in single nucleotide BER or at the stage just prior to strand displacement DNA synthesis in the long patch BER sub-pathway (6, 18). Thus, we evaluated cross-linking of MEF proteins to such a DNA structure as a novel approach toward probing for cellular enzymes involved in BER. The use of a 32P-labeled DNA with a sensitive photoreactive group at the 3'-margin of the nick permitted cross-linking with near-UV light instead of 254 nm UV light. This photoreactive BER intermediate was synthesized in situ (most likely by beta -pol) using a base-substituted dCTP analog carrying a photoreactive arylazido group at the fourth position of the base. The dNTP analog is known to be a good substrate for DNA polymerases (50-52) including beta -pol (52). This property permits introduction of the photoreactive dCMP moiety into the 3'-margin of a gap in DNA, thus creating the photoreactive BER intermediate within the cell extract (Fig. 3). The DNA probe does not participate in downstream processes of BER in the cell extract, namely strand displacement DNA synthesis or DNA ligation, because a polynucleotide ending in the FAB-dCMP moiety is a very poor substrate for either process. We provide evidence here that the main target for BER protein interaction with this DNA is the BER intermediate structure, rather than the two ends of the DNA oligomer or a nick with a 5'-phosphate group.

When irradiated with near-UV light, the DNA probe synthesized in situ could be cross-linked to the DNA polymerase or, after its dissociation, to other proteins capable of interacting with a nicked DNA structure. We regard this latter possibility as likely, because an excess of DNA probe over DNA polymerase or other individual cellular proteins was used in these experiments. Therefore, cellular proteins interacting preferentially with a photoreactive BER intermediate can be selected from the proteins in the crude cellular extract. This approach may have advantages in the study of specific transactions of DNA and DNA enzymes in cellular extract systems.

We found that protein labeling with the cellular extract was highly specific, as only six proteins were strongly labeled. Four of these turned out to be well known members of the BER machinery, PARP-1, FEN-1, beta -pol, and APE. These proteins, except for APE, were identified as the labeled products using various approaches including immunoprecipitation with specific antibodies (Figs. 4 and 5). Two additional proteins of molecular mass of ~30 kDa were observed to be affinity labeled also, but their identity was not pursued further in this study.

PARP-1 is an abundant cellular protein and is well known to be a nicked DNA sensor (2, 30, 35). Therefore, it is not surprising that PARP-1 was a target of labeling by the photoreactive BER intermediate used as probe in these experiments. We found that PARP-1 was by far the most heavily labeled protein and that several other well known BER proteins such as DNA ligases I and III and XRCC1 were not labeled. In view of the strong labeling of PARP-1 and the fact that PARP-1 has an important, but as yet unknown role in BER, we chose to examine requirements for PARP-1 labeling in detail. PARP-1 labeling in the MEF extract used in these experiments satisfied several criteria of affinity labeling using photoreactive nicked DNA; labeling varied as a function of the structure of the DNA probe; labeling was highest for a nick carrying an abasic site sugar phosphate at the 5'-margin, indicating that PARP-1 recognition of this BER intermediate was sensitive and specific; labeling could be competed by oligonucleotides representing BER intermediates but not by double-stranded DNA.

Immunoprecipitation experiments with an anti-FLAG-beta -pol antibody system revealed some co-precipitation of PARP-1, FEN-1, and beta -pol, each cross-linked individually to a molecule of labeled DNA probe. This indicated protein-protein interaction between PARP-1 and beta -pol and is in accord with the results reported by Dantzer et al. (29). These workers found that PARP-1 interacts with the C-terminal portion of beta -pol in the presence of DNA (29). Furthermore, they used extracts from cells made genetically deficient in beta -pol and/or PARP-1 and demonstrated that both proteins are involved in the BER of uracil-derived abasic sites. In the absence of both PARP-1 and beta -pol, both BER sub-pathways were reduced (29). The deletion of PARP-1 had a dramatic effect on long patch BER capacity measured in vitro using the cell extract, yet the mechanism of the role of PARP-1 in BER is still not clear (2, 30).

Our results on the requirements for PARP-1 recognition of BER intermediates show that the enzyme interacts most avidly with DNA nicks containing a sugar phosphate at the 5'-margin. This specificity of PARP-1 toward binding a sugar moiety at the 5'-margin of a nick in a single-stranded break suggests that PARP-1 recognizes a BER intermediate with this structure. This intermediate is formed in BER after introduction of a dNMP moiety by beta -pol but before sub-pathway choice leading to either single nucleotide BER or long patch BER. Therefore, our data point to a potential role of PARP-1 in both BER sub-pathways. The conclusion is consistent with the effect of PARP inhibitors on MMS-induced cytotoxicity in the cell lines used in these experiments. It appears that PARP plays a role in repair of MMS-induced lesions, and we inferred that this reflects the role of PARP in both BER pathways. We also found co-precipitation of PARP-1, FEN-1, and beta -pol cross-linked to the DNA probe using antibodies to the FLAG epitope attached to beta -pol (Fig. 8). These data, again, show interaction for these key BER proteins in a complex at a branch point intermediate of BER. It is interesting that preferential PARP-1 binding to this BER intermediate could stimulate the known rate-limiting steps of BER, such as the beta -pol-dependent strand displacement required in long patch BER and the dRP excision step required in single nucleotide BER. Thus, PARP-1 association in this way could be an important event in the overall efficiency of BER and in sub-pathway selection. Work is currently in progress to explore these possibilities.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Igor Safronov for providing the photoreactive dCTP analog. We thank Donna Joyce-Gray and Jana Naron for their excellent technical assistance. We thank Dr. William Beard for fruitful discussions and critical readings of this manuscript. We also thank Drs. Bennett Van Houten, William Copeland, Dmitry Gordenin, and Thomas Kunkel for their critical readings of this manuscript.

    FOOTNOTES

* This work was supported in part by Russian Funds for Basic Research Grants 99-04-49277 and 01-04-48895 (to O. I. L.).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.

|| To whom correspondence should be addressed: Laboratory of Structural Biology, NIEHS, National Institutes of Health, 111 T.W. Alexander Dr., Research Triangle Park, NC 27709. Tel.: 919-541-3267; Fax: 919-541-3592; E-mail: wilson5@niehs.nih.gov.

Published, JBC Papers in Press, May 4, 2001, DOI 10.1074/jbc.M102125200

    ABBREVIATIONS

The abbreviations used are: BER, base excision repair; APE, apurinic/apyrimidinic endonuclease; beta -pol, DNA polymerase beta ; FEN-1, flap endonuclease-1; PARP, poly(ADP-ribose) polymerase; XRCC1, x-ray cross complementing factor 1; RPA, replication protein A; dRP, 5'- deoxyribose phosphate; THF or tetrahydrofuran, 3-hydroxy-2-hydroxymethyltetrahydrofuran; PAGE, polyacrylamide gel electrophoresis; FAB-dCTP, exo-N-[beta -(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine 5'-triphosphate; FAB-dCMP, exo-N-[beta -(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine 5'-monophosphate; MEF(s), mouse embryonic fibroblast(s); FBS, fetal bovine serum; DMEM, Dulbecco's modified Eagle's medium; 3-AB, 3-aminobenzamide; 4-AN, 4-amino-1,8-naphthalimide; MMS, methyl methanesulfonate.

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