Photoaffinity Labeling of Mouse Fibroblast Enzymes by a Base Excision Repair Intermediate
2001; Elsevier BV; Volume: 276; Issue: 27 Linguagem: Inglês
10.1074/jbc.m102125200
ISSN1083-351X
AutoresOlga I. Lavrik, Rajendra Prasad, Robert W. Sobol, Julie K. Horton, Eric J. Ackerman, Samuel H. Wilson,
Tópico(s)Cancer-related Molecular Pathways
ResumoTo 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 β, 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. 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 β, 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. base excision repair apurinic/apyrimidinic endonuclease DNA polymerase β flap endonuclease-1 poly(ADP-ribose) polymerase x-ray cross complementing factor 1 replication protein A 5′- deoxyribose phosphate 3-hydroxy-2-hydroxymethyltetrahydrofuran polyacrylamide gel electrophoresis exo-N-[β-(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine 5′-triphosphate exo-N-[β-(p-azidotetrafluorobenzamido)-ethyl]deoxycytidine 5′-monophosphate mouse embryonic fibroblast(s) fetal bovine serum Dulbecco's modified Eagle's medium 3-aminobenzamide 4-amino-1,8-naphthalimide methyl methanesulfonate 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 (1Lindahl T. Annu. Rev. Biochem. 1982; 51: 61-87Crossref PubMed Scopus (693) Google Scholar, 2Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1272) Google Scholar). 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 (3Ochs K. Sobol R.W. Wilson S.H. Kaina B. Cancer Res. 1999; 59: 1544-1551PubMed Google Scholar, 4Sobol 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 (783) Google Scholar, 5Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (264) Google Scholar). 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 (6Frosina G. Fortini P. Rossi O. Carrozzino F. Raspaglio G. Cox L.S. Lane D.P. Abbondandolo A. Dogliotti E. J. Biol. Chem. 1996; 271: 9573-9578Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 7Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 8Nicholl I.D. Nealon K. Kenny M.K. Biochemistry. 1997; 36: 7557-7566Crossref PubMed Scopus (77) Google Scholar, 9Srivastava D.K. Berg B.J. Prasad R. Molina J.T. Beard W.A. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1998; 273: 21203-21209Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). 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 (1Lindahl T. Annu. Rev. Biochem. 1982; 51: 61-87Crossref PubMed Scopus (693) Google Scholar, 10Lindahl T. Nyberg B. Biochemistry. 1974; 13: 3405-3410Crossref PubMed Scopus (576) Google Scholar, 11Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (246) Google Scholar). 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 β (β-pol) (12Doetsch P.W. Cunningham R.P. Mutat. Res. 1990; 236: 173-201Crossref PubMed Scopus (322) Google Scholar, 13Matsumoto Y. Kim K. Science. 1995; 269: 699-702Crossref PubMed Scopus (646) Google Scholar). The single nucleotide gap is filled by β-pol, and then the nick is eventually sealed by a DNA ligase, thus completing the %short patch" or %single nucleotide" BER sub-pathway (7Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 14Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 15Singhal R.K. Prasad R. Wilson S.H. J. Biol. Chem. 1995; 270: 949-957Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). In mammalian cells, repair of methylated bases, oxidized bases, and abasic sites appears to occur predominantly by this sub-pathway (16Dianov G. Bischoff C. Piotrowski J. Bohr V.A. J. Biol. Chem. 1998; 273: 33811-33816Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 17Klungland A. Hoss M. Gunz D. Constantinou A. Clarkson S.G. Doetsch P.W. Bolton P.H. Wood R.D. Lindahl T. Mol. Cell. 1999; 3: 33-42Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In other cases, for example where the sugar of the abasic site is not removed efficiently, the %long patch" BER sub-pathway mediates repair (18Prasad R. Dianov G.L. Bohr V.A. Wilson S.H. J. Biol. Chem. 2000; 275: 4460-4466Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). This sub-pathway involves limited strand displacement and DNA synthesis to replace between 2 and as many as 15 nucleotides in the damaged strand (5Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (264) Google Scholar, 19Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (371) Google Scholar, 20Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 21Lieber M.R. BioEssays. 1997; 19: 233-240Crossref PubMed Scopus (395) Google Scholar). 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 (14Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 17Klungland A. Hoss M. Gunz D. Constantinou A. Clarkson S.G. Doetsch P.W. Bolton P.H. Wood R.D. Lindahl T. Mol. Cell. 1999; 3: 33-42Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar, 20Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). The DNA synthesis step of long patch BER can be conducted by β-pol or other DNA polymerases (6Frosina G. Fortini P. Rossi O. Carrozzino F. Raspaglio G. Cox L.S. Lane D.P. Abbondandolo A. Dogliotti E. J. Biol. Chem. 1996; 271: 9573-9578Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 18Prasad R. Dianov G.L. Bohr V.A. Wilson S.H. J. Biol. Chem. 2000; 275: 4460-4466Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 20Kim K. Biade S. Matsumoto Y. J. Biol. Chem. 1998; 273: 8842-8848Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 22Biade S. Sobol R.W. Wilson S.H. Matsumoto Y. J. Biol. Chem. 1998; 273: 898-902Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 23Dianov G.L. Prasad R. Wilson S.H. Bohr V.A. J. Biol. Chem. 1999; 274: 13741-13743Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar, 24Gary R. Kim K. Cornelius H.L. Park M.S. Matsumoto Y. J. Biol. Chem. 1999; 274: 4354-4363Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 25Horton J.K. Prasad R. Hou E. Wilson S.H. J. Biol. Chem. 2000; 275: 2211-2218Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar, 26Matsumoto Y. Kim K. Bogenhagen D.F. Mol. Cell. Biol. 1994; 14: 6187-6197Crossref PubMed Scopus (260) Google Scholar). 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 (23Dianov G.L. Prasad R. Wilson S.H. Bohr V.A. J. Biol. Chem. 1999; 274: 13741-13743Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar,25Horton J.K. Prasad R. Hou E. Wilson S.H. J. Biol. Chem. 2000; 275: 2211-2218Abstract Full Text Full Text PDF PubMed Scopus (138) Google Scholar).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 (7Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 27Thompson L.H. West M.G. Mutat. Res. 2000; 459: 1-18Crossref PubMed Scopus (402) Google Scholar) and for β-pol and the following: APE (28Bennett R.A. Wilson III, D.M. Wong D. Demple B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7166-7169Crossref PubMed Scopus (325) Google Scholar), PARP-1 (29Dantzer F. de La Rubia G. Menissier-De Murcia J. Hostomsky Z. de Murcia G. Schreiber V. Biochemistry. 2000; 39: 7559-7569Crossref PubMed Scopus (405) Google Scholar), and DNA ligase I (14Prasad R. Singhal R.K. Srivastava D.K. Molina J.T. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1996; 271: 16000-16007Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar). 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. 30D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar). PARP-1 in mammalian cells exists in several isoforms (31Ame J.C. Rolli V. Schreiber V. Niedergang C. Apiou F. Decker P. Muller S. Hoger T. Menissier-de Murcia J. de Murcia G. J. Biol. Chem. 1999; 274: 17860-17868Abstract Full Text Full Text PDF PubMed Scopus (601) Google Scholar, 32Berghammer H. Ebner M. Marksteiner R. Auer B. FEBS Lett. 1999; 449: 259-263Crossref PubMed Scopus (58) Google Scholar, 33Sallmann F.R. Vodenicharov M.D. Wang Z.Q. Poirier G.G. J. Biol. Chem. 2000; 275: 15504-15511Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 34Smith S. Giriat I. Schmitt A. de Lange T. Science. 1998; 282: 1484-1487Crossref PubMed Scopus (895) Google Scholar). Recently, Dantzeret al. (29Dantzer F. de La Rubia G. Menissier-De Murcia J. Hostomsky Z. de Murcia G. Schreiber V. Biochemistry. 2000; 39: 7559-7569Crossref PubMed Scopus (405) Google Scholar) examined the role of PARP-1 in single nucleotide and long patch BER using biochemical studies of %knock out" MEF cell lines for the β-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 (29Dantzer F. de La Rubia G. Menissier-De Murcia J. Hostomsky Z. de Murcia G. Schreiber V. Biochemistry. 2000; 39: 7559-7569Crossref PubMed Scopus (405) Google Scholar). 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 (2Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1272) Google Scholar, 35de Murcia G. Menissier de Murcia J. Trends Biochem. Sci. 1994; 19: 172-176Abstract Full Text PDF PubMed Scopus (756) Google Scholar), and the physical interactions of PARP-1 with the known BER proteins XRCC1 and β-pol (27Thompson L.H. West M.G. Mutat. Res. 2000; 459: 1-18Crossref PubMed Scopus (402) Google Scholar, 29Dantzer F. de La Rubia G. Menissier-De Murcia J. Hostomsky Z. de Murcia G. Schreiber V. Biochemistry. 2000; 39: 7559-7569Crossref PubMed Scopus (405) Google Scholar, 36Caldecott K.W. Aoufouchi S. Johnson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar). In addition, PARP-1 null MEF cells are known to be hypersensitive to DNA-damaging agents that produce lesions repaired by BER (37Dantzer 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 (Paris). 1999; 81: 69-75Crossref PubMed Scopus (303) Google Scholar, 38de Murcia J.M. Niedergang C. Trucco C. Ricoul M. Dutrillaux B. Mark M. Oliver F.J. Masson M. Dierich A. LeMeur M. Walztinger C. Chambon P. de Murcia G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7303-7307Crossref PubMed Scopus (956) Google Scholar). It was also found that PARP-1 can activate DNA ligase during repair in vitro (39Creissen D. Shall S. Nature. 1982; 296: 271-272Crossref PubMed Scopus (300) Google Scholar), and recently it was shown that PARP-1, β-pol, and DNA ligase III/XRCC1 form a BER complex that can support the ATP requirement for DNA ligation of the nick (40Oei S.L. Ziegler M. J. Biol. Chem. 2000; 275: 23234-23239Abstract Full Text Full Text PDF PubMed Google Scholar). 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.DISCUSSIONThe 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 areduced 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 (6Frosina G. Fortini P. Rossi O. Carrozzino F. Raspaglio G. Cox L.S. Lane D.P. Abbondandolo A. Dogliotti E. J. Biol. Chem. 1996; 271: 9573-9578Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 18Prasad R. Dianov G.L. Bohr V.A. Wilson S.H. J. Biol. Chem. 2000; 275: 4460-4466Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). 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 β-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 (50Doronin S.V. Dobrikov M.I. Buckle M. Roux P. Buc H. Lavrik O.I. FEBS Lett. 1994; 354: 200-202Crossref PubMed Scopus (37) Google Scholar, 51Doronin S.V. Dobrikov M.I. Lavrik O.I. FEBS Lett. 1992; 313: 31-33Crossref PubMed Scopus (44) Google Scholar, 52Lavrik O.I. Prasad R. Beard W.A. Safronov I.V. Dobrikov M.I. Srivastava D.K. Shishkin G.V. Wood T.G. Wilson S.H. J. Biol. Chem. 1996; 271: 21891-21897Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar) including β-pol (52Lavrik O.I. Prasad R. Beard W.A. Safronov I.V. Dobrikov M.I. Srivastava D.K. Shishkin G.V. Wood T.G. Wilson S.H. J. Biol. Chem. 1996; 271: 21891-21897Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar). 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, β-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 (2Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1272) Google Scholar, 30D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar, 35de Murcia G. Menissier de Murcia J. Trends Biochem. Sci. 1994; 19: 172-176Abstract Full Text PDF PubMed Scopus (756) Google Scholar). 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-β-pol antibody system revealed some co-precipitation of PARP-1, FEN-1, and β-pol, each cross-linked individually to a molecule of labeled DNA probe. This indicated protein-protein interaction between PARP-1 and β-pol and is in accord with the results reported by Dantzer et al. (29Dantzer F. de La Rubia G. Menissier-De Murcia J. Hostomsky Z. de Murcia G. Schreiber V. Biochemistry. 2000; 39: 7559-7569Crossref PubMed Scopus (405) Google Scholar). These workers found that PARP-1 interacts with the C-terminal portion of β-pol in the presence of DNA (29Dantzer F. de La Rubia G. Menissier-De Murcia J. Hostomsky Z. de Murcia G. Schreiber V. Biochemistry. 2000; 39: 7559-7569Crossref PubMed Scopus (405) Google Scholar). Furthermore, they used extracts from cells made genetically deficient in β-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 β-pol, both BER sub-pathways were reduced (29Dantzer F. de La Rubia G. Menissier-De Murcia J. Hostomsky Z. de Murcia G. Schreiber V. Biochemistry. 2000; 39: 7559-7569Crossref PubMed Scopus (405) Google Scholar). The deletion of PARP-1 had a dramatic effect on long patch BER capacity measured in vitrousing the cell extract, yet the mechanism of the role of PARP-1 in BER is still not clear (2Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1272) Google Scholar, 30D'Amours D. Desnoyers S. D'Silva I. Poirier G.G. Biochem. J. 1999; 342: 249-268Crossref PubMed Scopus (0) Google Scholar).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 β-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 β-pol cross-linked to the DNA probe using antibodies to the FLAG epitope attached to β-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 β-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. 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 (1Lindahl T. Annu. Rev. Biochem. 1982; 51: 61-87Crossref PubMed Scopus (693) Google Scholar, 2Lindahl T. Wood R.D. Science. 1999; 286: 1897-1905Crossref PubMed Scopus (1272) Google Scholar). 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 (3Ochs K. Sobol R.W. Wilson S.H. Kaina B. Cancer Res. 1999; 59: 1544-1551PubMed Google Scholar, 4Sobol 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 (783) Google Scholar, 5Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (264) Google Scholar). 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 (6Frosina G. Fortini P. Rossi O. Carrozzino F. Raspaglio G. Cox L.S. Lane D.P. Abbondandolo A. Dogliotti E. J. Biol. Chem. 1996; 271: 9573-9578Abstract Full Text Full Text PDF PubMed Scopus (443) Google Scholar, 7Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 8Nicholl I.D. Nealon K. Kenny M.K. Biochemistry. 1997; 36: 7557-7566Crossref PubMed Scopus (77) Google Scholar, 9Srivastava D.K. Berg B.J. Prasad R. Molina J.T. Beard W.A. Tomkinson A.E. Wilson S.H. J. Biol. Chem. 1998; 273: 21203-21209Abstract Full Text Full Text PDF PubMed Scopus (342) Google Scholar). 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 (1Lindahl T. Annu. Rev. Biochem. 1982; 51: 61-87Crossref PubMed Scopus (693) Google Scholar, 10Lindahl T. Nyberg B. Biochemistry. 1974; 13: 3405-3410Crossref PubMed Scopus (576) Google Scholar, 11Slupphaug G. Eftedal I. Kavli B. Bharati S. Helle N.M. Haug T. Levine D.W. Krokan H.E. Biochemistry. 1995; 34: 128-138Crossref PubMed Scopus (246) Google Scholar). 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 β (β-pol) (12Doetsch P.W. Cunningham R.P. Mutat. Res. 1990; 236: 173-201Crossref PubMed Scopus (322) Google Scholar, 13Matsumoto Y. Kim K. 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Chem. 1998; 273: 33811-33816Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 17Klungland A. Hoss M. Gunz D. Constantinou A. Clarkson S.G. Doetsch P.W. Bolton P.H. Wood R.D. Lindahl T. Mol. Cell. 1999; 3: 33-42Abstract Full Text Full Text PDF PubMed Scopus (254) Google Scholar). In other cases, for example where the sugar of the abasic site is not removed efficiently, the %long patch" BER sub-pathway mediates repair (18Prasad R. Dianov G.L. Bohr V.A. Wilson S.H. J. Biol. Chem. 2000; 275: 4460-4466Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar). This sub-pathway involves limited strand displacement and DNA synthesis to replace between 2 and as many as 15 nucleotides in the damaged strand (5Wilson S.H. Mutat. Res. 1998; 407: 203-215Crossref PubMed Scopus (264) Google Scholar, 19Harrington J.J. Lieber M.R. EMBO J. 1994; 13: 1235-1246Crossref PubMed Scopus (371) Google Scholar, 20Kim K. Biade S. Matsumoto Y. J. Biol. 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