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[Cancer Research 65, 3894-3902, May 1, 2005]
© 2005 American Association for Cancer Research

Experimental Therapeutics, Molecular Targets, and Chemical Biology

Poly(ADP-Ribose) Polymerase-1 Could Facilitate the Religation of Topoisomerase I-linked DNA Inhibited by Camptothecin

Shin-Young Park and Yung-Chi Cheng

Department of Pharmacology, Yale University School of Medicine, New Haven, Connecticut

Requests for reprints: Yung-Chi Cheng, Department of Pharmacology, Yale University School of Medicine, SHM B226, P.O. Box 208066, New Haven, CT 06520-8066. Phone: 203-785-7119; Fax: 203-785-7129; E-mail: yccheng{at}yale.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Poly(ADP-ribose) polymerase-1 (PARP-1) is known to have an important role in camptothecin sensitivity and interacts with topoisomerase I. In the present study, the impact of PARP-1 on the topoisomerase I-DNA complex stabilized by camptothecin was assessed. It was shown that NH2 terminus–truncated topoisomerase I (amino acids 201-765) showed at least 4-fold less sensitivity to camptothecin than full-length topoisomerase I in the oligonucleotide religation assay. PARP-1 could prevent the action of camptothecin on the religation activity of full-length topoisomerase I, which is linked to DNA in a stoichiometrical manner. However, the religation activity of NH2 terminus–truncated topoisomerase I, which is linked to DNA, could not be enhanced by PARP-1 in the presence of camptothecin. Both full-length and NH2 terminus–truncated topoisomerase I interact with PARP-1. This data suggests that PARP-1 destabilizes the topoisomerase I-camptothecin-DNA complex with the participation of the NH2-terminal domain of topoisomerase I. Poly(ADP-ribosyl)ation of topoisomerase I by PARP-1 in the presence its substrate, NAD, could also promote the religation activity of full-length topoisomerase I as well as NH2 terminus–truncated topoisomerase I. PARP-1 inhibitors (3-aminobenzamide, PJ34) could inhibit this process. Therefore, PARP-1 could facilitate the religation activity of topoisomerase I by itself through topoisomerase I-PARP-1 interaction (PARP-1 action) or by the formation of poly(ADP-ribosyl)ation of topoisomerase I (PARP-1/NAD action). This study also implies that PARP-1 and PARP-1/NAD actions need to be highly regulated by cellular factors for camptothecin to exert its cytotoxicity inside the cells. We propose ATP to be one of the important regulatory factors.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Human topoisomerase I plays a critical role in replication, transcription, and recombination by controlling the topological state of DNA (1–3). Transient breakage of DNA by topoisomerase I is one of its catalytic steps, and under normal conditions, these breaks can be religated immediately by topoisomerase I. This cleavage-religation coupling can be disrupted by drugs, such as camptothecin and its analogues, resulting in topoisomerase I-linked DNA (TLD), which will lead to double-stranded breaks during replication and transcription (4, 5). The stabilization of TLD by camptothecin is not sufficient to cause cell death. However, the religation process for TLD is very important because TLD has the potential to convert to double-stranded breaks, which can cause cell death.

Topoisomerase I is composed of 765 amino acid residues and has four distinct domains; the NH2-terminal domain (1-214), the core domain (215-635), the linker domain (636-712), and the COOH-terminal domain (713-765; ref. 3). The crystal structure of topoisomerase I was reported (6) but the NH2-terminal domain was not included and the function of this domain is unclear. The NH2-terminal domain is known to be dispensable for enzyme activity but plays an important role in the behavior of topoisomerase I including its sensitivity to camptothecin in vitro (7–9) It has four nuclear localization signals and binding sites for several proteins such as: nucleolin (10), SV40 large T antigen (11, 12), TATA-binding protein (13), and RING/SR proteins (14). Therefore, the NH2-terminal domain seems to influence the localization of topoisomerase I inside the cell (15, 16) and has regulatory functions through interaction with other proteins. The role of the NH2-terminal domain in the DNA religation process of topoisomerase I was unclear.

PARP-1 is a highly conserved nuclear enzyme which binds tightly to DNA and plays a role in DNA repair, recombination, proliferation, and genomic stability (17–19). DNA damage signals PARP-1 to catalyze the transfer of the ADP-ribose moiety from its substrate, NAD, to a limited number of protein acceptors such as histone, topoisomerase I, and PARP-1 itself. PARP-1, shown to be related to camptothecin resistance (20), is one of the components of the base excision repair protein complex, which consists of XRCC1, DNA polymerase ß, DNA ligase III{alpha}, AP endonuclease, and polynucleotide kinase (21–23). It has been reported that inhibitors of PARP-1 could potentiate the cytotoxicity of camptothecin and topotecan, another topoisomerase I inhibitor, suggesting that PARP-1 is somehow involved with topoisomerase I in modulating camptothecin action (24). It was also reported that PARP-1 poly(ADP-ribosyl)ated topoisomerase I, in the presence of NAD, results in the inhibition of topoisomerase I relaxation activity (25, 26). The underlying mechanism responsible for inhibiting activity was suggested to be due to the large increase in net negative charge upon poly(ADP-ribosyl)ation of topoisomerase I, which eventually causes repulsion between topoisomerase I and DNA.

The impact of PARP-1 on topoisomerase I activity was not only manifested through its catalytic action with NAD, but also by PARP-1 itself, which stimulates the relaxation activity of topoisomerase I in the absence of NAD (27). This stimulation was observed over a 1,000:1 PARP-1/topoisomerase I molar ratio range. It was postulated that the increased superhelicity of DNA induced by PARP-1 (28) may facilitate the formation of a more "tight-fisted" topoisomerase I-DNA complex that increases the rate of topoisomerase I catalytic activity. Since the DNA relaxation assay contains all steps of topoisomerase I action: binding, cleavage, strand rotation, and religation, it is difficult to differentiate the impact of PARP-1 on the individual steps of topoisomerase I action using the relaxation assay. Direct association of topoisomerase I and PARP-1 purified from calf thymus was shown by fluorescence resonance energy transfer analysis and by a binding assay using antibody (27, 29). The domains of topoisomerase I that can interact with PARP-1 were identified with the aid of glutathione S-transferase fusion peptides using the pull-down technique (30). Topoisomerase I has different binding regions to PARP-1, which uses core and COOH-terminal domains, and poly(ADP-ribosyl)ated PARP-1, which binds to the NH2-terminal and core domains.

It is still unclear what impact PARP-1 has on the formation of TLD and the religation process of TLD in the absence or presence of camptothecin. Based on the literature, it is possible that PARP-1 may alter the action of camptothecin on topoisomerase I either through protein-protein interaction or poly(ADP-ribosyl)ation reaction. Since TLD stabilized by camptothecin could lead to double-stranded breaks, we focused our attention on the impact of PARP-1 on the DNA cleavage and religation steps of topoisomerase I's action in the presence of camptothecin. Although the NH2-terminal domain of topoisomerase I has not been reported to interact with PARP-1 directly (30), it may still regulate the behavior of the topoisomerase I and PARP-1 complex. Therefore, we also compared the behaviors of full-length topoisomerase I (f-topoisomerase I) and NH2 terminus–truncated topoisomerase I (nt-topoisomerase I; amino acids, 201-765) toward PARP-1. Recently, it was published that the poly(ADP-ribose) polymer could enhance the religation step of topoisomerase I in the presence of camptothecin, whereas PARP-1 itself could not (31). Our study indicates that not only PARP-1/NAD, which produces poly(ADP-ribose) polymer but also PARP-1 itself could facilitate the religation process of full-length-TLD (f-TLD) inhibited by camptothecin in stoichiometric concentrations. Given that ATP was found to inhibit topoisomerase I action (32, 33), the effects of ATP in PARP-1 or PARP-1/NAD action were examined and the physiologic implication of the findings were discussed.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Drugs. Camptothecin was provided by Dr. Zong-Chao Liu of the Cancer Institute at Sun Yat-Sen University of Medical Sciences (Guangzhou, China). SN-38 was provided by Pharmacia Upjohn (Bridgewater, NJ). Topotecan was purchased from GlaxoSmithKline (Pittsburgh, PA). Dilutions of all drugs were made with PBS from 10 mmol/L stock solutions in 100% DMSO stored at –70°C. ATP and 3-aminobenzamide were purchased from Sigma (St. Louis, MO), and PJ34 was purchased from Calbiochem (San Diego, CA).

Expression and purification of full-length human topoisomerase I. A topoisomerase I expression plasmid, pGEX-TOP1 (34), was kindly provided by Dr. E. Rubin (UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ). The plasmid was digested with BamHI and EcoRI and the fragment encoding the full-length human DNA topoisomerase I was subcloned into the baculovirus transfer vector pVL1393 (PharMingen, San Diego, CA). Recombinant baculovirus was generated by cotransfecting Sf-9 cells with BaculoGold DNA (PharMingen) and pVL1393/topoisomerase I using a calcium-phosphate coprecipitation method. The correct clones of recombinant viruses were chosen by checking the protein produced by Sf-9 cells infected with individual clones. Two liters of Sf-9 cells were infected with an optimal amount of recombinant baculovirus. Four days later, cells were harvested and washed twice with PBS. The washed cells were lysed by vigorous shaking in 25 mL of buffer A [10 mmol/L Tris-HCl (pH 7.5), 10 mmol/L NaCl, 3 mmol/L MgCl2, 30 mmol/L sucrose, 0.5% NP40, 0.1 mg/mL aprotinin, and protease inhibitor cocktail (Roche Applied Science, Indianapolis, IN)]. The nuclei were washed once in 25 mL of buffer A and once with buffer B (buffer A minus NP40). Then the nuclei were resuspended in buffer B and adjusted to 10 mmol/L EDTA and lysed by adding 25 mL of buffer C [2 mol/L NaCl, 80 mmol/L Tris-HCl (pH 7.5), 20% glycerol, 2 mmol/L EDTA]. The nuclear extract was stirred for 30 minutes, then 25 mL of polyethylene glycol (PEG) buffer (18% PEG 8000, 1 mol/L NaCl, 10% glycerol) was added in a drop-wise manner to precipitate the DNA. After stirring for an additional 40 minutes, the PEG precipitate was pelleted by centrifugation at 20,000 x g for 20 minutes. The resulting PEG supernatant was dialyzed against 4 L of buffer D [50 mmol/L potassium phosphate buffer (pH 8.0), 500 mmol/L KCl, 0.5 mmol/L DTT, 10% glycerol, and 1 mmol/L phenylmethylsulfonyl fluoride]. The dialysate was centrifuged at 20,000 x g for 20 minutes to remove precipitates and the supernatant was loaded onto a 2 mL Ni-NTA agarose column (Qiagen, Valencia, CA) which was washed with 40 mL of buffer D alone and 40 mL of buffer D containing 20 mmol/L of imidazole. Full-length topoisomerase I was eluted with buffer D containing 40 mmol/L imidazole. The eluents containing topoisomerase I were combined and dialyzed against 4 L of buffer E [200 mmol/L potassium phosphate buffer (pH 7.4), 1 mmol/L EDTA, 1 mmol/L DTT, 10% glycerol, and 1 mmol/L phenylmethylsulfonyl fluoride]. The second step of purification used a 5 mL Econo-Pac CHT-II column (Bio-Rad, Hercules, CA) equilibrated with buffer E. The column was eluted with a 50 mL linear gradient of 200 to 800 mmol/L potassium phosphate. Topoisomerase I, which eluted as a single protein peak, was dialyzed into storage buffer [50 mmol/L potassium phosphate (pH 7.5), 0.1 mmol/L EDTA, 1 mmol/L DTT, and 30% glycerol] and stored in –20°C.

Other recombinant proteins. NH2 terminus–truncated topoisomerase I, whose amino acid starts from 201, was kindly provided by Dr. Y. Pommier (National Cancer Institute, Bethesda, MD). Human recombinant PARP-1 was purchased from Trevigen (Gaithersburg, MD) and Alexis (San Diego, CA).

Oligonucleotide. Oligonucleotides were purchased from IDT DNA (Coralville, IA) and the sequences are as follows:

ON4: 5'-TAAAAATTTTTCCGGGTCTTTTTTCp-3'
ON5: 5'-GAAAAAAGACTTGG-3'
ON6: 5'-GGAAAAATTTTTA-3'

ON5 was 5'-labeled by T4 polynucleotide kinase (New England Biolabs, Beverly, MA) using [{gamma}32P]-ATP (Perkin-Elmer, Boston, MA) as the phosphate group donor. Labeling reactions were done at 37°C for 20 minutes and unincorporated free ATP was removed by G25 column (Roche Applied Science). The duplex oligonucleotide substrate for topoisomerase I, ON5-ON4 was generated by mixing the two oligonucleotides in 1x annealing buffer [10 mmol/L Tris-HCl (pH 7.5), 100 mmol/L NaCl, 1 mmol/L EDTA] and heating for 2 minutes and then cooling slowly to room temperature for annealing.

Oligonucleotide cleavage reaction. Radiolabeled ON5-ON4 (~50 fmol) was incubated with topoisomerase I (220 fmol) in the presence or absence of PARP-1 and camptothecin in 10 µL reaction containing 10 mmol/L Tris-HCl (pH 7.5), 50 mmol/L KCl, 5 mmol/L MgCl2, 0.1 mmol/L EDTA, 30 µg/mL bovine serum albumin. The reaction was done at 37°C for 15 minutes and terminated by adding SDS to 0.5%. Covalent-linked topoisomerase I was digested by proteinase K (1 mg/mL final concentration) at 37°C for 1 hour. Samples were then mixed with loading buffer (98% formamide, 10 mmol/L EDTA, 10 mmol/L NaOH, 0.1% bromophenol blue, and 0.1% xylene cyanol), boiled, and analyzed by 20% denaturing urea/PAGE. The amount of cleavage was quantified by phosphoimager (Molecular Dynamics-Amersham Biosciences, Piscataway, NJ) and the percentage of cleavage product was calculated from the fraction of cleavage bands versus total bands.

Oligonucleotide religation reaction. Suicide cleavage complex was generated by cleavage reaction as mentioned above and religation was started by adding 5 pmol of ON6. Samples were incubated at 37°C for various times and analyzed by 20% urea/PAGE as described above. All components which were included in the religation reaction, such as PARP-1, NAD, 3-aminobenzamide, and ATP were premixed with ON6 on ice. The amount of religation product was expressed as the percentage of religation product out of total bands as measured on a phosphoimager.

Western blotting. Proteins were electrophoresed on 7.5% acrylamide gel and transferred to a nitrocellulose membrane (Bio-Rad) using a minitransblot electrophoretic transfer cell (Bio-Rad). Membranes were blocked with 5% nonfat milk in PBST (0.15% Tween 20 in PBS) for 1 hour at room temperature or overnight at 4°C. Membranes were incubated with anti-PAR (Biomol, Plymouth Meeting, PA), anti-PARP-1 (clone c-2-10, Biomol), or anti-topoisomerase I (clone 21, generated by our lab) antibodies for appropriate times and washed with 5% nonfat milk in PBST. Secondary antibodies were added to the membranes and incubated for 1 hour at room temperature. After another wash with PBST, enhanced chemiluminescence (Perkin-Elmer) was used to detect the peroxidase conjugate by exposure to X-ray film.

Binding assay. Topoisomerase I (800 fmol) were slot-blotted onto nitrocellulose membranes. Membranes were blocked with 5% nonfat milk in PBST for 1 hour and incubated with various amounts of PARP-1 for 2 hours at room temperature. Unbound PARP-1 was washed out thrice with 5% nonfat milk in PBST. Bound PARP-1 was detected by Western blotting method using anti-PARP-1 antibody.

Poly(ADP-ribose) polymerase-1 automodification. The reaction containing 50 mmol/L Tris-HCl (pH 8.0), 10 mmol/L MgCl2, 500 ng of PARP-1, 500 µmol/L of NAD, and 200 fmol of oligo duplex as an activator was done at 37°C for 30 minutes. The oligo duplex consisting of 20-mer and 33-mer does not have a topoisomerase I cleavage site and the sequences are shown as follows:

20-mer: 5'-GTGGCGCGGAGACTTAGAGA-3'
33-mer: 5'-CCCGCCCCAAATGTCTCTAAGTCTCCGCGCCAC-3'

As a control reaction, a reaction containing the same components described above, except without NAD, was set up and incubated at 37°C for 30 minutes. The residual NAD was removed by G25 miniquick spin column (Roche Applied Science). Automodified PARP-1 was prepared freshly for the topoisomerase I religation assay.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Human recombinant f-topoisomerase I was purified from insect cells and its purity was >95% (data not shown). Western blotting results indicated that f-topoisomerase I was about the same size as human topoisomerase I from cell lysate (data not shown). F-topoisomerase I was quite stable under the storage conditions used, such that degradation to the NH2terminus–truncated form was hardly observed. The purities of nt-topoisomerase I and PARP-1 were >95% (data not shown).

Poly(ADP-ribose) polymerase-1 inhibits the formation of topoisomerase I-linked DNA. The substrate, ON5-ON4 duplex, has two nucleotides downstream of the topoisomerase I cutting site and religation does not happen due to the dissociation of 2-mer at the 5' end of the cleavage product. Thus, the formation of TLD without the subsequent religation reaction could be studied using this substrate. To compare the behavior of f-topoisomerase I and nt-topoisomerase I, the same amounts of topoisomerase I (220 fmol), which gave about 50% cleavage product at 37°C for 15 minutes, were used. PARP-1 exhibited the inhibitory effects on f-topoisomerase I and nt-topoisomerase I cleavage reactions in a dose-dependent manner (Fig. 1). The degree of inhibition seemed to be higher in nt-topoisomerase I than f-topoisomerase I. Camptothecin did not affect or change the inhibitory activity of PARP-1 toward the formation of TLD.



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  		Figure 1. The impact of PARP-1 on cleavage activity of topoisomerase I. Cleavage reaction was done as described under Materials and Methods using f-topoisomerase I and nt-topoisomerase I in the absence and presence of camptothecin. Right, cleavage products were quantified with the phosphoimager and the percentage of cleavage product out of the total was plotted; arrow, cleavage site for topoisomerase I; S, substrate only.

 
Full-length topoisomerase I and NH2 terminus–truncated topoisomerase I show different sensitivities to camptothecin in religation reactions. The action of camptothecin in the religation reaction with f-topoisomerase I and nt-topoisomerase I was studied. The reaction conditions were described under Materials and Methods. Camptothecin decreased the amount of religation product, but f-topoisomerase I and nt-topoisomerase I showed quite different sensitivities to camptothecin (Fig. 2). Camptothecin could inhibit almost 90% of religation mediated by f-topoisomerase I at 20 µmol/L, but could inhibit only about 60% to 70% with nt-topoisomerase I, even at 70 µmol/L. The difference of sensitivities to camptothecin between the two enzymes was at least 4-fold with respect to the 50% inhibition concentration. For f-topoisomerase I, the inhibition of religation reaction reached a maximum with 40 µmol/L of camptothecin; therefore, this concentration of camptothecin was used for the rest of the studies. For nt-topoisomerase I, 70 µmol/L of camptothecin, which was the maximum concentration examined, was chosen for the studies.



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  		Figure 2. Difference in sensitivity to camptothecin in religation reaction. After 15 minutes of cleavage reaction with 220 fmol of topoisomerase I in the presence of various concentrations of camptothecin, ON6 and 430 fmol of PARP-1 were added and the religation reaction was continued for 30 minutes. The religation product was presented as a percentage of control (% of religation product with camptothecin / % of religation product without camptothecin x 100). Data present average values from three independent experiments; bar, SD.

 
PARP-1 facilitates religation mediated by full-length topoisomerase I but not by NH2 terminus–truncated topoisomerase I in the presence of camptothecin. The effect of PARP-1 on the religation activity of f-TLD and nt-TLD in the absence or presence of camptothecin was studied. The amount of PARP-1 used for this experiment was 430 fmol/Rx which is approximately 2:1 PARP-1/topoisomerase I molar ratio. At various times (5 minutes to 1 hour) after adding ON6, the religation product (25-mer) was analyzed using a phosphoimager. PARP-1 showed no enhancement effect on the religation activity of either f-TLD or nt-TLD in the absence of camptothecin (Fig. 3A and C), which is consistent with a previous report by others (30). In the presence of camptothecin, the impact of PARP-1 on the religation activities of the two topoisomerase I enzymes was different. For f-topoisomerase I, PARP-1 showed the activation effect on religation (Fig. 3A). To assess if this PARP-1 action was dose-dependent, various amounts of PARP-1 were added to the reaction and incubated for 30 minutes. It was found that the action of PARP-1 reached a plateau when the molar ratio of PARP-1 to f-topoisomerase I was near 2.0 (Fig. 3B). On the other hand, the religation activity of nt-TLD was not enhanced by adding PARP-1 (Fig. 3C). Even though the data of nt-topoisomerase I was not as obvious as that of f-topoisomerase I because it was less sensitive to camptothecin, the quantification resulting from three independent experiments showed that there was no difference with and without PARP-1. To access whether there is any difference between f-topoisomerase I and nt-topoisomerase I in terms of binding to PARP-1, 800 fmol of topoisomerase I was immobilized on nitrocellulose paper and incubated with various amounts of PARP-1. The PARP-1 bound to topoisomerase I was detected by anti-PARP-1 antibody. The result indicates that both f-topoisomerase I and nt-topoisomerase I could bind to PARP-1 (Fig. 3D). However, it was not clear if there was a difference in the binding affinity to PARP-1 between two enzymes. The effect of PARP-1 was not limited to camptothecin but was also observed with SN38 and topotecan (data not shown).



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  		Figure 3. The impact of PARP-1 on religation activity of topoisomerase I. A, the effect of PARP-1 on religation reaction with f-topoisomerase I in the absence and presence of camptothecin. After 15 minutes of cleavage reaction with 220 fmol of topoisomerase I, ON6 was added and reaction was continued for the indicated times in the absence and presence of 430 fmol of PARP-1. Religation product (25-mer) was quantified with the phosphoimager and the percentage of religation product out of the total was plotted. B, the action of PARP-1 in various amounts. After cleavage reaction as described above, ON6 was added and reaction was continued for 30 minutes with PARP-1 in the absence and presence of camptothecin. C, the effect of PARP-1 on religation reaction with nt-topoisomerase I in the absence and presence of camptothecin. D, binding of PARP-1 to topoisomerase I. Experiment was done as described under Materials and Methods.

 
The catalytic action of poly(ADP-ribose) polymerase-1 could facilitate the religation activity of full-length-topoisomerase I-linked DNA and NH2 terminus–truncated topoisomerase I-linked DNA in the presence of camptothecin. PARP-1 was reported to increase the DNA relaxation activity of topoisomerase I, whereas PARP-1 could decrease topoisomerase I relaxation activity in the presence of NAD (27). The impact of NAD on PARP-1 action with respect to the inhibitory action of camptothecin on the religation activity of TLD was studied. When a fixed amount of PARP-1 (215 fmol/Rx, 1:1 PARP-1/topoisomerase I molar ratio) and various amounts of NAD up to 1.8 mmol/L were added to religation reactions in the presence of camptothecin, the amount of religation product was increased with increasing amounts of NAD and increased to approximately the same amount as the one without camptothecin (Fig. 4A, top). NAD itself had no impact on the action of camptothecin on the religation reaction without PARP-1. The amount of PARP-1 required to desensitize the action of camptothecin was different in reactions with and without NAD. In the absence of NAD, PARP-1 started to have an effect from 107.5 fmol/Rx (0.5:1 PARP-1/topoisomerase I ratio). Whereas, in the presence of NAD, 10.7 fmol of PARP-1 (0.05:1 PARP-1/topoisomerase I ratio) had an effect (Fig. 4A, bottom). PARP-1/NAD could also stimulate religation mediated by nt-topoisomerase I in the presence of camptothecin (Fig. 4B); however, there was a difference in the amount of NAD required for the prevention of camptothecin action between f-topoisomerase I and nt-topoisomerase I. The amounts of NAD required to exhibit maximum effect in reactions with f-topoisomerase I and nt-topoisomerase I were 50 and 500 µmol/L, respectively [Fig. 4A (top) and B (top)]. Nt-topoisomerase I could reach only 80% to 85% of control levels in the presence of NAD, whereas, f-topoisomerase I could prevent almost 100% of camptothecin action. This data suggested that the NH2-terminal domain of topoisomerase I is important to have full activation of religation activity of f-TLD by PARP-1/NAD in the presence of camptothecin. The action of PARP-1/NAD on TLD in the presence of camptothecin could be inhibited by its inhibitors, 3-aminobenzamide (Fig. 4C) and PJ34 (data not shown), in a dose-dependent manner. Those inhibitors did not significantly affect PARP-1 action (Fig. 4D), implying that the binding of PARP-1 inhibitors to PARP-1 does not have influence in the interaction between f-topoisomerase I and PARP-1. The next question was whether the increased religation activity of TLD in the presence of NAD is due to the poly(ADP-ribosyl)ation of PARP-1 or topoisomerase I. Before answering this question, the antibodies against topoisomerase I, PARP-1, and PAR were employed to see the status of topoisomerase I and PARP-1 using Western blotting. Poly(ADP-ribosyl)ated PARP-1 was detected clearly by anti-PAR antibody but not by anti-PARP-1 antibody (Fig. 5A). Therefore, the PARP-1 shown in lane 3 of the Western blotting data using anti-PARP-1 antibody (Fig. 5A, bottom) is unmodified PARP-1, and the difference of intensity between lanes 1 and 3 indicates that the amount of PARP-1 was modified in lane 3, which was detected only by anti-PAR antibody. The efficiency of poly(ADP-ribosyl)ation was higher in the sample with oligo duplex (lane 3) than the sample without oligo (lane 2). In order to see if the poly(ADP-ribosyl)ation of topoisomerase I occurred with PARP-1 in the presence of NAD, we analyzed the reaction product with respect to the formation of poly(ADP-ribosyl)ated topoisomerase I using anti-PAR and anti-topoisomerase I antibody with f-topoisomerase I. It was observed that only the reaction containing NAD showed the ladder of ADP-ribose polymer (Fig. 5B). PARP-1 can be poly(ADP-ribosyl)ated (Fig. 5B, lane 4); therefore, the ladder shown in lane 3 of Fig. 5B might be the sum of poly(ADP-ribosyl)ated topoisomerase I and poly(ADP-ribosyl)ated PARP-1. The difference of intensity in the ADP-ribose ladder between lanes 3 and 4, especially the 97 kDa region, strongly indicates that topoisomerase I was modified by PARP-1/NAD. The formation of poly(ADP-ribosyl)ated topoisomerase I was further confirmed using anti-topoisomerase I antibody. The lack of a modified topoisomerase I band in the high molecular weight region in Fig. 5B is likely due to the interference of the ADP-ribose polymer on the interaction between topoisomerase I antibody and topoisomerase I. To address the effect of poly(ADP-ribosyl)ated PARP-1 on the religation activity of TLD, poly(ADP-ribosyl)ated PARP-1 was prepared and added to topoisomerase I religation reaction. Western blotting data showed that >70% of PARP-1 was modified (Fig. 5C, top). There was no difference in the effects between unmodified PARP-1 and modified PARP-1 on the religation activity of f-TLD (Fig. 5C, bottom). Both enzymes showed no changes without NAD and enhancing effects with NAD on the religation activity of f-TLD, indicating that the mechanism of action of PARP-1/NAD is due to the poly(ADP-ribosyl)ation of topoisomerase I.



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  		Figure 4. The effect of PARP-1/NAD on religation reaction. The religation reaction was done for 30 minutes with f-topoisomerase I (A) and nt-topoisomerase I (B). Top, dose response of NAD in the religation reaction with 215 fmol of PARP-1; bottom, the dose response of PARP-1 in the religation reaction with fixed concentration of NAD; 182 µmol/L for f-topoisomerase I, 1.8 mmol/L for nt-topoisomerase I. To compare f-topoisomerase I and nt-topoisomerase I, the religation product (25-mer) was presented as the percentage of control (% of religation product / % of religation product in sample without camptothecin x 100). 3-Aminobenzamide inhibits PARP-1/NAD action (C) but not PARP-1 action (D). C, the religation reaction including 220 fmol of f-topoisomerase I, 215 fmol of PARP-1, 182 µmol/L of NAD, and various amounts of 3-aminobenzamide were done for 30 minutes in the presence of 40 µmol/L of camptothecin. D, the religation reaction was done with 220 fmol of f-topoisomerase I, 430 fmol of PARP-1, and 40 µmol/L of camptothecin in the absence or presence of 500 µmol/L of 3-aminobenzamide. The reaction was terminated at the times indicated; R, religation in the absence of camptothecin; C, religation in the presence of camptothecin.

 


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  		Figure 5. Poly(ADP-ribosyl)ation of PARP-1 and topoisomerase I. A, poly(ADP-ribosyl)ated PARP-1 were prepared as described under Materials and Methods and analyzed by 7.5% SDS-PAGE and Western blotting. Lane 1, PARP-1; lane 2, PARP-1 and NAD; lane 3, PARP-1, NAD, and oligo duplex. B, the status of topoisomerase I after religation reaction was examined by 7.5% SDS-PAGE and Western blotting using anti-PAR and anti-topoisomerase I antibody. The religation reaction was done using nonradiolabeled substrate for 30 minutes with f-topoisomerase I in the presence of 40 µmol/L of camptothecin. Lane 1, topoisomerase I only; lane 2, topoisomerase I and PARP-1; lane 3, topoisomerase I, PARP-1, and NAD; lane 4, PARP-1 and NAD. C, the effect of poly(ADP-ribosyl)ated PARP-1 on religation reaction. Various amounts of PARP-1 and modified PARP-1 were added to religation reactions containing 220 fmol of f-topoisomerase I and 40 µmol/L of camptothecin in the presence or absence of 182 µmol/L of NAD. The reactions were done at 37°C for 30 minutes. Data present average values from three independent experiments; bar, SD.

 
Interference of poly(ADP-ribosyl)ation of topoisomerase I by ATP. Since ATP has been shown to inhibit the DNA relaxation activity of topoisomerase I (32, 33), the impact of ATP on PARP-1 action in the religation activity of f-TLD in the presence of camptothecin was examined with f-topoisomerase I. ATP did not show any effect on PARP-1 action in the presence of camptothecin (Fig. 6B) but showed an inhibitory effect on PARP-1/NAD action in a dose-dependent manner (Fig. 6C) in which ATP did not make any change in the religation activity of f-TLD (Fig. 6A). Given that topoisomerase I was poly(ADP-ribosyl)ated in the reaction containing PARP-1 and NAD in the presence of camptothecin (Fig. 5), the effect of ATP on the formation of poly(ADP-ribosyl)ated topoisomerase I under this condition was examined. As we expected, the amount of modified topoisomerase I was decreased by adding ATP into the reaction (Fig. 6D). The modification of PARP-1 was also inhibited by ATP. Since, as mentioned previously, anti-PARP-1 antibody cannot recognize modified PARP-1, the degree of modification was evaluated based on the amount of PARP-1 disappearing from native PARP-1.



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  		Figure 6. The impact of ATP on PARP-1 and PARP-1/NAD actions. Figure shows religation product (25-mer) only. Various amounts of ATP were applied to religation reactions containing 220 fmol of f-topoisomerase I (A), 220 fmol of f-topoisomerase I, and 430 fmol of PARP-1 in the presence of camptothecin (B), 220 fmol of f-topoisomerase I, 21.5 fmol of PARP-1, and 18.2 µmol/L of NAD (C) in the presence of camptothecin; the reactions were done for 30 minutes. D, the status of topoisomerase I and PARP-1 was examined by SDS-PAGE and Western blotting using anti-topoisomerase I and anti-PARP-1 antibody after the religation reaction, which include various amounts of ATP; T, topoisomerase I; P, PARP-1.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Camptothecin analogues are an important class of cancer chemotherapeutic drugs. The cytotoxicity is correlated with the formation of double-stranded breaks mediated through the formation of TLD stabilized by camptothecin. This study, together with others, indicated that the formation of TLD is not affected by camptothecin, whereas PARP-1 could inhibit this reaction. The NH2-terminal domain of f-topoisomerase I did not play a role in the formation of TLD, however, it played a key role in the sensitivity to camptothecin on the religation process. The impact of PARP-1 on f-topoisomerase I and nt-topoisomerase I was different with religation activity in the presence of camptothecin. PARP-1 could prevent the action of camptothecin in the religation reaction of f-TLD, but not of nt-TLD. Given the stoichiometric amounts of PARP-1 and topoisomerase I, the action of PARP-1 on the religation of topoisomerase I is likely the result of direct PARP-1-topoisomerase I interaction. It was suggested that the domains of topoisomerase I responsible for interaction with PARP-1 are the core and COOH-terminal domains, not the NH2-terminal domain (30). In this study, it was also observed that both f-topoisomerase I and nt-topoisomerase I could interact with PARP-1. Therefore, the difference induced by the NH2-terminal domain of f-topoisomerase I in the religation reaction indicates that the NH2-terminal domain could affect the interaction between PARP-1 and f-topoisomerase I by causing conformational changes of f-topoisomerase I stacked by camptothecin, which would render easier religation. Recently, Malanga and Althaus (31) reported that there was no enhancement of religation activity of TLD by PARP-1 itself. This is highly likely because of the topoisomerase I preparation which they used [we also purchased topoisomerase I prepared by TopoGene (Columbus, Ohio), where they purchased their topoisomerase I]. Western blot analysis indicated that most of the preparation is similar to our truncated topoisomerase I, which could be the result of the enzyme storage conditions. If they did study the truncated form of topoisomerase I, then their results are consistent with ours. However, we cannot rule out that the discrepancy between our results and those from Malanga and Althaus are due to the differences in assay conditions.

In addition to the direct interaction between PARP-1 and topoisomerase I, the catalytic action of PARP-1 with NAD could further enhance the religation activity of TLD in the presence of camptothecin. With PARP-1 only, the effect was observed at a stoichiometric ratio of topoisomerase I to PARP-1, whereas in the presence of NAD, the enhancement of religation was shown at even a 0.05:1 PARP-1/topoisomerase I molar ratio. Interestingly, the NH2-terminal domain of topoisomerase I also seems to have an impact on PARP-1/NAD action; therefore, different efficiencies of NAD as the substrate of PARP-1 catalytic action between f-topoisomerase I and nt-topoisomerase I were observed in the religation reaction with camptothecin. This suggested that the interaction of PARP-1 with different protein substrates could alter its enzymatic behavior with respect to NAD.

It was proposed that the role of poly(ADP-ribosyl)ation in DNA-based excision repair and maintenance of genomic stability is a sign of DNA damage through noncovalent binding of poly(ADP-ribose) to specific proteins (18). The poly(ADP-ribose)-binding sequence motif of 20 amino acids was identified in several proteins including p53, MSH6, DNA ligase III, XRCC1, and Ku70 (35). Noncovalent interaction between ADP-ribose polymer and p53 altered the function of p53 (31). Topoisomerase I was reported to have three regions for the noncovalent binding of ADP-ribose polymer and this interaction reprogrammed TLD to promote religation in the presence of camptothecin (31). Based on this information, it may be possible that poly(ADP-ribosyl)ated PARP-1 in the PARP-1 preparation used in our experiment could cause enhanced religation activity of f-TLD. However, that possibility could be ruled out by the Western blotting data of PARP-1 using anti-PAR antibody showing no modified PARP-1 (Fig. 5A, top). Our studies show that poly(ADP-ribosyl)ation of topoisomerase I, which is a covalent modification, is critical for the enhancement of the religation activity of TLD in the presence of camptothecin. This result does not disagree with the observation by Malanga and Althaus that noncovalent interaction between ADP-ribose polymer and topoisomerase I could activate the religation of topoisomerase I (31). The amount of ADP-ribose polymer bound to PARP-1 in our studies may not be sufficient to enhance the religation of f-TLD in the absence of NAD. It is not clear whether the region of topoisomerase I for the covalent modification by poly(ADP-ribosyl)ation and the region for noncovalent interaction with poly(ADP-ribose) polymer is the same or not.

ATP was shown to inhibit topoisomerase I catalytic action at 1 to 5 mmol/L, which is within physiologic range of the intracellular ATP (32). We previously suggested that topoisomerase I possesses a nucleoside triphosphate binding site, through which conformational changes can affect topoisomerase I and DNA interaction and hence the catalytic activity. This idea was supported by recent work of others (33, 36). Chen and Hwang (33) have shown that the inhibition of the DNA relaxation activity of topoisomerase I by ATP was at the binding step rather than at the cleavage or religation step. Their studies indicated that the religation activity of TLD in the presence of camptothecin could not be affected by ATP. Kun et al. (37) reported that ATP inhibits PARP-1 auto-poly(ADP-ribosyl)ation, but less so the transfer of ADP-ribose to histones [trans-poly(ADP-ribosyl)ation]. However, reduced poly(ADP-ribosyl)ation of DNA-PK, but not of the Ku 70/80 complex by ATP was shown (38). In our study, ATP had an inhibitory effect on poly(ADP-ribosyl)ation of topoisomerase I by PARP-1. Whether the inhibitory effect of ATP is due to the direct inhibition of trans-poly(ADP-ribosyl)ation of PARP-1 or the conformational change of topoisomerase I induced by ATP is not clear and requires further investigation. It is possible that the active site for different protein substrates of PARP-1 could have different sensitivities to ATP. This is supported by our observation that the NAD requirements for f-topoisomerase I and nt-topoisomerase I are different.

Removal of topoisomerase I from DNA is a critical issue to overcome camptothecin cytotoxicity. Here we showed that PARP-1 could destabilize the topoisomerase I-camptothecin-DNA complex either through stoichiometric interaction with topoisomerase I or through catalytic action with NAD and cause topoisomerase I to leave without any gap in DNA. Both topoisomerase I and PARP-1 are abundant proteins in nuclei. If those two proteins are physically associated in nuclei, it should render topoisomerase I less active and more resistant to camptothecin. Poly(ADP-ribosyl)ation of topoisomerase I by PARP-1 could also render topoisomerase I less active to carry out its role in DNA replication, DNA repair, and RNA transcription. This will argue that topoisomerase I and PARP-1 could not be physically associated in nuclei and that poly(ADP-ribosyl)ation of topoisomerase I could not be efficient under normal conditions. The observation that ATP could inhibit poly(ADP-ribosyl)ation of topoisomerase I by PARP-1 suggested that ATP or other nucleotides could be important regulators of preventing poly(ADP-ribosyl)ation of topoisomerase I by PARP-1. The factors responsible for the possible regulation of the interaction between PARP-1 and topoisomerase I are worthwhile for future investigation.


   Acknowledgments
 
Grant support: Supported in part by NIH grant 1R21 CA097750 and Y-C. Cheng is a fellow of the National Foundation for Cancer Research.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Drs. Y. Pommier (National Cancer Institute, Bethesda, MD) for providing NH2 terminus–truncated topoisomerase I and E. Rubin (UMDNJ-Robert Wood Johnson Medical School, New Brunswick, NJ) for providing the topoisomerase I expression plasmid, pGEX-TOP1.

Received 11/ 8/04. Revised 2/16/05. Accepted 2/19/05.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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