ATP for the DNA Ligation Step in Base Excision Repair Is Generated from Poly(ADP-ribose)

Abstract

In mammalian cells, the base excision repair (BER) pathway is the main route to counteract the mutagenic effects of DNA lesions. DNA nicks induce, among others, DNA polymerase activities and the synthesis of poly(ADP-ribose). It is shown here that poly(ADP-ribose) serves as an energy source for the final and rate-limiting step of BER, DNA ligation. This conclusion was drawn from experiments in which the fate of [32P]poly(ADP-ribose) or [32P]NAD added to HeLa nuclear extracts was systematically followed. ATP was synthesized from poly(ADP-ribose) in a pathway that strictly depended on nick-induced DNA synthesis. NAD was used for the synthesis of poly(ADP-ribose), which, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl moiety was then preferentially used to adenylate DNA ligase III, from which it was transferred to the 5′-phosphoryl end of the nicked DNA. Finally, ligation to the 3′-OH end resulted in the release of AMP. When using NAD, but not poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire process was blocked, confirming poly(ADP-ribosyl)ation to be the essential initial step. Thus, poly(ADP-ribose) polymerase-1, DNA polymerase β, and ligase III interact with x-ray repair cross-complementing protein-1 within the BER complex, which ensures that ATP is generated and specifically used for DNA ligation. In mammalian cells, the base excision repair (BER) pathway is the main route to counteract the mutagenic effects of DNA lesions. DNA nicks induce, among others, DNA polymerase activities and the synthesis of poly(ADP-ribose). It is shown here that poly(ADP-ribose) serves as an energy source for the final and rate-limiting step of BER, DNA ligation. This conclusion was drawn from experiments in which the fate of [32P]poly(ADP-ribose) or [32P]NAD added to HeLa nuclear extracts was systematically followed. ATP was synthesized from poly(ADP-ribose) in a pathway that strictly depended on nick-induced DNA synthesis. NAD was used for the synthesis of poly(ADP-ribose), which, in turn, was converted to ATP by pyrophosphorylytic cleavage utilizing the pyrophosphate generated from dNTPs during DNA synthesis. The adenylyl moiety was then preferentially used to adenylate DNA ligase III, from which it was transferred to the 5′-phosphoryl end of the nicked DNA. Finally, ligation to the 3′-OH end resulted in the release of AMP. When using NAD, but not poly(ADP-ribose), in the presence of 3-aminobenzamide, the entire process was blocked, confirming poly(ADP-ribosyl)ation to be the essential initial step. Thus, poly(ADP-ribose) polymerase-1, DNA polymerase β, and ligase III interact with x-ray repair cross-complementing protein-1 within the BER complex, which ensures that ATP is generated and specifically used for DNA ligation. base excision repair 3-amionobenzamide DNA ligase methyl-N′-nitro-N′-nitrosoguanidine polyacrylamide gel electrophoresis poly(ADP-ribose) poly(ADP-ribosyl) polymerase-1 DNA polymerase β x-ray repair cross-complementing protein-1 The maintenance of an intact genome is crucial to each individual. Therefore, DNA damages need to be efficiently removed, which is accomplished by complex DNA repair mechanisms (reviewed in Ref. 1Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1240) Google Scholar). The major pathway, BER,1 is initiated by DNA glycosylases that cleave the base-deoxyribose glycosyl bond of a damaged nucleotide residue. Then, endonucleases are recruited that cleave the chain on the 5′ side of the abasic site. As a result, nicked DNA intermediates occur. Nicked DNA, in turn, triggers the catalytic activities of DNA polymerase β (Pol β) and poly(ADP-ribose) polymerase-1 (PARP-1) (2Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (754) Google Scholar, 3Benjamin R.C. Gill D.M. J. Biol. Chem. 1980; 255: 10502-10508Abstract Full Text PDF PubMed Google Scholar, 4Durkacz B.W. Omidiji O. Gray D.A. Shall S. Nature. 1980; 283: 593-596Crossref PubMed Scopus (864) Google Scholar). Several different enzymes with poly(ADP-ribosyl)ation activity (EC2.4.2.30) have been described recently, but the major cellular pathway of NAD catabolism in response to the appearance of DNA lesions has been ascribed to the catalytic activity of the 116-kDa protein PARP-1 (Ref.4Durkacz B.W. Omidiji O. Gray D.A. Shall S. Nature. 1980; 283: 593-596Crossref PubMed Scopus (864) Google Scholar; reviewed in Ref. 5D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). Besides a potential participation of PARP-1 in transcription (6Oei S.L. Griesenbeck J. Ziegler M. Schweiger M. Biochemistry. 1998; 37: 1465-1469Crossref PubMed Scopus (57) Google Scholar, 7Oei S.L. Griesenbeck J. Schweiger M. Ziegler M. J. Biol. Chem. 1998; 273: 31644-31647Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar), recombination, apoptosis, and necrosis (5D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar), a large number of molecular and genetic studies have clearly implicated PARP-1 activity in positively regulating BER (8Molinete M. Vermeulen W. Burkle A. Menissier-de Murcia J. Kupper J.H. Hoeijmakers J.H. de Murcia G. EMBO J. 1993; 12: 2109-2117Crossref PubMed Scopus (221) Google Scholar, 9Dantzer F. Schreiber V. Niedergang C. Trucco C. Flatter E. De La Rubia G. Oliver J. Rolli V. Menissier-de Murcia J. de Murcia G. Biochimie. 1999; 81: 69-75Crossref PubMed Scopus (296) Google Scholar). Originally, it was suggested that poly(ADP-ribosyl)ation may activate a DNA ligase required for DNA repair in mammalian cells (10Creissen D. Shall S. Nature. 1982; 296: 271-272Crossref PubMed Scopus (297) Google Scholar). Several further investigations confirmed a positive influence of PARP-1 activity on DNA repair, especially on DNA ligation (Refs. 11Ding R. Pommier Y. Kang V.H. Smulson M. J. Biol. Chem. 1992; 267: 12804-12812Abstract Full Text PDF PubMed Google Scholar, 12Trucco C. Oliver F.J. de Murcia G. Menissier-de Murcia J. Nucleic Acids Res. 1998; 26: 2644-2649Crossref PubMed Scopus (299) Google Scholar, 13Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (666) Google Scholar; reviewed in Ref. 9Dantzer F. Schreiber V. Niedergang C. Trucco C. Flatter E. De La Rubia G. Oliver J. Rolli V. Menissier-de Murcia J. de Murcia G. Biochimie. 1999; 81: 69-75Crossref PubMed Scopus (296) Google Scholar). However, the actual function of PARP-1 in the BER process and the mechanism whereby poly(ADP-ribose) synthesis stimulates ligation (14Satoh M.S. Lindahl T. Nature. 1992; 356: 356-358Crossref PubMed Scopus (898) Google Scholar) have still remained obscure (reviewed in Ref. 9Dantzer F. Schreiber V. Niedergang C. Trucco C. Flatter E. De La Rubia G. Oliver J. Rolli V. Menissier-de Murcia J. de Murcia G. Biochimie. 1999; 81: 69-75Crossref PubMed Scopus (296) Google Scholar). X-ray repair cross-complementing protein-1 (XRCC1) was the first human gene product isolated that mediates the cellular response to ionizing radiation (15Thompson L.H. Brookman K.W. Jones N.J. Allen S.A. Carrano A.V. Mol. Cell. Biol. 1990; 10: 6160-6171Crossref PubMed Scopus (370) Google Scholar). This protein is apparently essential and required for the BER pathway (16Tebbs R.S. Flannery M.L. Meneses J.J. Hartmann A. Tucker J.D. Thompson L.H. Cleaver J.E. Pedersen R.A. Dev. Biol. 1999; 208: 513-529Crossref PubMed Scopus (286) Google Scholar). Recent investigations demonstrated specific interactions of XRCC1 with Pol β, DNA ligase III (Lig III), and PARP-1 (13Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (666) Google Scholar,17Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, 18Masson M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. Mol. Cell. Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (795) Google Scholar, 19Caldecott K.W. Aoufouchi S. Johnson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (526) Google Scholar, 20Cappelli E. Taylor R. Cevasco M. Abbondandolo A. Caldecott K. Frosina G. J. Biol. Chem. 1997; 272: 23970-23975Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). Therefore, the complex of these proteins is supposed to control BER (1Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1240) Google Scholar, 9Dantzer F. Schreiber V. Niedergang C. Trucco C. Flatter E. De La Rubia G. Oliver J. Rolli V. Menissier-de Murcia J. de Murcia G. Biochimie. 1999; 81: 69-75Crossref PubMed Scopus (296) Google Scholar, 19Caldecott K.W. Aoufouchi S. Johnson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (526) Google Scholar). Most situations requiring highly efficient DNA repair are accompanied by a dramatic decrease of the cellular ATP concentration. Considering that ligation represents the rate-limiting, ATP-dependent step in BER, it appears reasonable to expect a compensatory mechanism that would enable efficient DNA repair even in situations of energy deprivation. In the present report, it is demonstrated that poly(ADP-ribose) synthesized by PARP-1 may serve as a source of ATP that is specifically used for ligation. This finding provides a molecular mechanism for previous observations, in vivo and in vitro, demonstrating the specific stimulation of ligation by poly(ADP-ribosyl)ation during BER. All reactions were carried out with nuclear HeLa cell extracts purchased from Promega or prepared (21Schreiber E. Matthias P. Müller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3880) Google Scholar). In standard reactions (10 mm Hepes, pH 7.9, 10% glycerol, 7 mm MgCl2, 50 mm KCl, 100 mm NaCl, 0, 1 mm EDTA, 0.25 mmdithiothreitol, 0.25 mm phenylmethylsulfonyl fluoride, 10 μg/ml aphidicolin), 8 μg of nuclear protein were incubated with 200 ng of nicked plasmid DNA. Further additions are indicated in the legends to the figures. The reaction volume was 10 μl, and incubations were conducted at 30 °C for the time periods indicated. All data presented are representative of at least three independent experiments. Nicked plasmid was obtained by controlled incubation of an empty vector plasmid, pUC 9, with DNase I and subsequent purification of nicked plasmids by CsCl ethidium bromide centrifugation. Poly(ADP-ribose) was synthesized with 10 μg/ml purified recombinant PARP-1 from 100 μm NAD as described before (22Griesenbeck J. Oei S.L. Mayer-Kuckuk P. Ziegler M. Buchlow G. Schweiger M. Biochemistry. 1997; 36: 7297-7304Crossref PubMed Scopus (43) Google Scholar). Synthesized polymers were freed of residual amounts of DNA by DNase treatment followed by phenol/chloroform extraction and precipitation. Reactions were stopped by precipitation with 10 volumes of acetone. Precipitated nucleotides were redissolved in 10 mm Tris-HCl, pH 8.0, 0.1 mmEDTA. Samples containing equal amounts of radioactivity (as estimated by Cerenkov counting) were subjected to cellulose-coated plates (Machery-Nagel). Chromatography was performed as described before (23Hagen T. Ziegler M. Biochim. Biophys. Acta. 1997; 1340: 7-12Crossref PubMed Scopus (7) Google Scholar) using the solvent system isobutyric acid/25% NH4OH/H2O (96/4/19 (v/v/v)). After separation was completed, the cellulose plates were dried and subjected to autoradiography. Reactions were stopped, and proteins were separated by 6% polyacrylamide gel electrophoresis. For analysis of plasmids, reactions were stopped, and after phenol/chloroform extraction, DNA was precipitated with ethanol, resuspended, and applied to 1% agarose gels in 90 mm Tris, 90 mm boric acid, 2 mm EDTA, pH 8.3, and 50 μg/ml chloroquine. HeLa cells were grown in suspension in RPMI 1640 medium supplemented with 10% fetal calf serum in a humidified 5% CO2 atmosphere at 37 °C. A medium change of confluent cells was performed 2 h before treatment with methyl-N′-nitro-N′-nitrosoguanidine (MNNG). At a density of 106 cells/ml, a 10-ml culture was treated with 100 μm MNNG for 5 min. The cells were then centrifuged and washed twice, and the culture was continued in a culture flask at 37 °C. 1-ml aliquots of the cell culture were taken after the time intervals indicated, and nuclear extracts were prepared (21Schreiber E. Matthias P. Müller M.M. Schaffner W. Nucleic Acids Res. 1989; 17: 6419Crossref PubMed Scopus (3880) Google Scholar). and nick-induced DNA synthesis activities of nuclear extracts were the as described However, the reactions μg of nuclear of was by using 1 μm and for at were stopped by precipitation. washed was by Cerenkov DNA synthesis was by using 0.1 mm and and 1 were continued for 30 at 30 °C. The reactions were stopped, and after phenol/chloroform extraction, DNA was precipitated with ethanol, resuspended, and subjected to agarose gel electrophoresis. The gel was dried and subjected to autoradiography. of nucleotides was by Cerenkov of gel All data presented are representative of at least three independent experiments. the of poly(ADP-ribose) incubated in the presence of nicked DNA was in HeLa nuclear The major product of is 1 from the activity of poly(ADP-ribose) activity present in nuclear extracts R. Res. 1989; PubMed Scopus Google Scholar, W. J. Biol. Chem. 1997; 272: Full Text Full Text PDF PubMed Scopus Google Scholar). In the presence of 1 mm which nick-induced DNA in to and ATP was 1 of or dNTPs to the of A of at least three dNTPs the synthesis of ATP from the of 1 mm pyrophosphate of dNTPs not to the of amounts of ATP 1 Thus, synthesis of ATP strictly depended on DNA synthesis. the for dNTPs indicated that the pyrophosphate during DNA synthesis is specifically to the added pyrophosphate was It be out that the pathway for the of ATP from poly(ADP-ribose) the cleavage of the bond of by a ATP and H. S. Biochem. Biophys. Res. 1997; PubMed Scopus Google Scholar). was used in synthesis of ATP was at (10 of pyrophosphate The of ATP on the presence of polymers was further by incubations of nuclear extracts with dNTPs and NAD 1 The synthesis of ATP from NAD was not the reaction was performed in the presence of the of 1 However, the presence of not the synthesis of ATP from 1 of ATP an for added or synthesized by ATP from NAD or 1 a after of incubation in 1 and 8 and an of to that of NAD or in was used it was to in a was 1 In ATP synthesized from to DNA synthesis was apparently not to A and This that the ATP is to as or but is to enzymes ATP to AMP. Thus, the pathway of ATP synthesis using as product to ATP for a specific In the first step of DNA ATP is used to an ligase In subsequent the adenylyl is transferred to the 5′-phosphoryl and is the 5′-phosphoryl and the 3′-OH are to the that PARP-1 and III are of the BER complex K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, 18Masson M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. Mol. Cell. Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (795) Google Scholar, 19Caldecott K.W. Aoufouchi S. Johnson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (526) Google Scholar, 20Cappelli E. Taylor R. Cevasco M. Abbondandolo A. Caldecott K. Frosina G. J. Biol. Chem. 1997; 272: 23970-23975Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar), it to be a that III is specifically using ATP generated from as described nuclear extracts were incubated in the presence of nicked DNA, and NAD for time to reactions shown in 1 were then separated by and subjected to 1 As the major protein PARP-1 or 1 in with previous that the reaction of poly(ADP-ribosyl)ation represents of PARP-1 K. M. O. J. Biol. Chem. Full Text PDF PubMed Google Scholar). A further protein with an molecular of was on the of which was to treatment with 1 on with of proteins is especially at of nuclear extracts with and separation of proteins on followed by proteins by 1 As proteins molecular to of human DNA 1997; PubMed Scopus Google Scholar), I III and 1 to the reaction using NAD and dNTPs were incubated with the nuclear the of proteins was that the protein was not protein been by ATP during the in the presence of NAD and of III from dNTPs or NAD was by an of the with NAD and dNTPs was conducted in the presence of of the protein with was The subsequent step after of DNA is the of the DNA by the adenylyl to the of a DNA The complex then with the 3′-OH to the ligation of the with the 3′-OH release of AMP. The complex be the final step of the ligation is This was accomplished by using The specific synthesis of ATP from was still a of and was used of dNTPs Therefore, during nick-induced DNA the of or nicked plasmids would in to intermediates of nicked plasmids with to an This was in ligation reactions in nuclear extracts using nicked DNA, NAD, and and 1 The of was clearly but in the of 1 that was to occur. for the conclusion that the adenylyl of poly(ADP-ribose) may be used to activate the 5′-phosphoryl of nicked DNA. In pathway, the activities of DNA and ligation to the ligation of nicked DNA All the enzymes implicated in mechanism Pol β, and interact with the protein XRCC1 within the BER of XRCC1 and III to ligation activity E. Taylor R. Cevasco M. Abbondandolo A. Caldecott K. Frosina G. J. Biol. Chem. 1997; 272: 23970-23975Abstract Full Text Full Text PDF PubMed Scopus (268) Google Scholar). the of XRCC1 with PARP-1 or Pol β in a of the catalytic activity (13Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (666) Google Scholar, 18Masson M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. Mol. Cell. Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (795) Google Scholar). It was confirmed in that in HeLa cells during BER Pol β and PARP-1 activities are 2 of HeLa cells with 100 μm a of the BER pathway, to of PARP-1 and Pol β activities followed by a after 30 min. The of DNA synthesis used in was to Pol β DNA were by DNA W. H. Scholar). As for PARP-1 in the of activity was not by of protein This for the after h the MNNG vivo experiments the conclusion of previous studies that during DNA repair PARP-1 and Pol β activities are in by with The time of the of PARP-1 and Pol β the of poly(ADP-ribose) and of the cellular NAD, and in studies Biochem. Biophys. Res. PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar). The of the final step of BER, with these The ligation step is for DNA it is rate-limiting and The data presented suggested that synthesis of poly(ADP-ribose) may serve as an to DNA repair of cellular ATP It would be then that of ATP a This was by 5 mm ATP to incubations of HeLa nuclear extracts in the presence of nicked DNA, and these of nuclear proteins PARP-1 or was not but 2 as to the poly(ADP-ribosyl)ation in the of ATP 1 of III was not of nucleotides confirmed that in the presence of 5 mm of the added was 2 of or at the not A of ATP on PARP-1 was the catalytic activity of the isolated not a ATP of isolated recombinant of PARP-1 resulted in the of poly(ADP-ribosyl)ation in the presence of ATP 2 However, of III was still not The of PARP-1 is to specific with proteins (22Griesenbeck J. Oei S.L. Mayer-Kuckuk P. Ziegler M. Buchlow G. Schweiger M. Biochemistry. 1997; 36: 7297-7304Crossref PubMed Scopus (43) Google Scholar), in XRCC1 M. Niedergang C. Schreiber V. Muller S. Menissier-de Murcia J. de Murcia G. Mol. Cell. Biol. 1998; 18: 3563-3571Crossref PubMed Scopus (795) Google Scholar). Therefore, it appears that of the of the PARP-1 to and to the BER PARP-1 was but the BER complex was to The suggested of the added of PARP-1 was further by analysis of nick-induced DNA synthesis and ligation by the nuclear DNA but was in the of ATP 2 the presence of 5 mm ATP to ligation but nick-induced DNA synthesis 2 of the of PARP-1 nick-induced DNA synthesis in the presence of the plasmid not of the added dNTPs It is that DNA repair need a of Therefore, in are conducted in the presence of 2 mm ATP and a system R.D. P. Lindahl T. Cell. Full Text PDF PubMed Scopus Google Scholar). It was demonstrated in the shown in 2 that in the of added ATP or an ATP system nicked plasmids be by HeLa nuclear However, the for the ATP synthesis from are of nicked to was added or from NAD by and dNTPs were present 2 5 and As added not serve as a for The of the present the conclusion that poly(ADP-ribosyl)ation may to the process of BER by a source of ATP for the ligation step. It is to that the effects of NAD and on ligation have been Originally, the stimulation of was to be by poly(ADP-ribosyl)ation of a ligase (10Creissen D. Shall S. Nature. 1982; 296: 271-272Crossref PubMed Scopus (297) Google Scholar). However, has not been In initial in vivo was used to cells of It was that the of PARP-1 resulted in a of DNA R. Pommier Y. Kang V.H. Smulson M. J. Biol. Chem. 1992; 267: 12804-12812Abstract Full Text PDF PubMed Google Scholar). of cells from a of the DNA ligation activity C. Oliver F.J. de Murcia G. Menissier-de Murcia J. Nucleic Acids Res. 1998; 26: 2644-2649Crossref PubMed Scopus (299) Google Scholar). In the the BER complex has been and interactions of the proteins have been in and in It was clearly demonstrated that NAD, which provides the for the ligation step for Pol β activity (13Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (666) Google Scholar). Considering the dramatic decrease of the cellular ATP to Biochem. Biophys. Res. PubMed Scopus Google Scholar, Biochemistry. PubMed Scopus Google Scholar), a of poly(ADP-ribosyl)ation may be the extraction of energy from NAD and for DNA repair, specifically ligation. the of PARP-1 for DNA nicks would that NAD is preferentially used at in need of DNA previous the of a complex of DNA polymerase β, III, and PARP-1 F. Schreiber V. Niedergang C. Trucco C. Flatter E. De La Rubia G. Oliver J. Rolli V. Menissier-de Murcia J. de Murcia G. Biochimie. 1999; 81: 69-75Crossref PubMed Scopus (296) Google for the of these of the BER complex to ATP from poly(ADP-ribose) provides a for the of these proteins In the influence of NAD specifically on the ligation reaction be to the of which is apparently to the to the data the pathway of ATP synthesis not the cleavage of poly(ADP-ribose) to However, may be generated within the complex of enzymes and may not be for by added to the for pyrophosphate ATP using pyrophosphate generated from dNTPs during nick-induced DNA synthesis is to the of a added is in ATP to of in the nuclear the of DNA synthesis to ATP from the of (10 of added pyrophosphate were to ATP from in the of dNTPs Therefore, it would that the ATP synthesis cleavage of as activity of reaction has been in HeLa cell extracts H. S. Biochem. Biophys. Res. 1997; PubMed Scopus Google Scholar). It is of to the that the specific of ATP the synthesis of DNA, which is accompanied by the of It is ATP that is at least preferentially used by III for and subsequent of the DNA mediates ligation. the final of BER, nick-induced DNA of ATP from and appears to be accomplished by an ATP and pyrophosphate are apparently not used by a cellular energy ATP may serve as a that ligation of nicked DNA or of the Pol repair pathway (2Sobol R.W. Horton J.K. Kuhn R. Gu H. Singhal R.K. Prasad R. Rajewsky K. Wilson S.H. Nature. 1996; 379: 183-186Crossref PubMed Scopus (754) Google Scholar, Prasad R. Wilson S.H. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). 2 to the potential function of III and PARP-1 as molecular proteins highly that to Niedergang C. Menissier-de Murcia M. J. K. J. de Murcia G. J. Biol. Chem. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). to the data presented in and at ATP III is At the poly(ADP-ribosyl)ation and DNA synthesis are A would be that of III within the BER complex to DNA nicks or at least the BER complex to a that DNA of PARP-1 and a mechanism would the of NAD as an energy source to situations of ATP G. Buchlow for