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Involvement of XRCC1 and DNA Ligase III Gene Products in DNA Base Excision Repair

1997; Elsevier BV; Volume: 272; Issue: 38 Linguagem: Inglês

10.1074/jbc.272.38.23970

1083-351X

Enrico Cappelli, Richard Taylor, Michela Cevasco, Angelo Abbondandolo, Keith W. Caldecott, Guido Fròsina,

Acute Lymphoblastic Leukemia research

DNA ligase III and the essential protein XRCC1 are present at greatly reduced levels in the xrcc1 mutant CHO cell line EM-C11. Cell-free extracts prepared from these cells were used to examine the role of the XRCC1 gene product in DNA base excision repairin vitro. EM-C11 cell extract was partially defective in ligation of base excision repair patches, in comparison to wild type CHO-9 extracts. Of the two branches of the base excision repair pathway, only the single nucleotide insertion pathway was affected; no ligation defect was observed in the proliferating cell nuclear antigen-dependent pathway. Full complementation of the ligation defect in EM-C11 extracts was achieved by addition to the repair reaction of recombinant human DNA ligase III but not by XRCC1. This is consistent with the notion that XRCC1 acts as an important stabilizing factor of DNA ligase III. These data demonstrate for the first time that xrcc1 mutant cells are partially defective in ligation of base excision repair patches and that the defect is specific to the polymerase β-dependent single nucleotide insertion pathway. DNA ligase III and the essential protein XRCC1 are present at greatly reduced levels in the xrcc1 mutant CHO cell line EM-C11. Cell-free extracts prepared from these cells were used to examine the role of the XRCC1 gene product in DNA base excision repairin vitro. EM-C11 cell extract was partially defective in ligation of base excision repair patches, in comparison to wild type CHO-9 extracts. Of the two branches of the base excision repair pathway, only the single nucleotide insertion pathway was affected; no ligation defect was observed in the proliferating cell nuclear antigen-dependent pathway. Full complementation of the ligation defect in EM-C11 extracts was achieved by addition to the repair reaction of recombinant human DNA ligase III but not by XRCC1. This is consistent with the notion that XRCC1 acts as an important stabilizing factor of DNA ligase III. These data demonstrate for the first time that xrcc1 mutant cells are partially defective in ligation of base excision repair patches and that the defect is specific to the polymerase β-dependent single nucleotide insertion pathway. DNA base excision repair (BER) 1The abbreviations used are: BER, base excision repair; AP sites, abasic sites; PCNA, proliferating cell nuclear antigen; bp, base pair(s); CHO, Chinese hamster ovary. 1The abbreviations used are: BER, base excision repair; AP sites, abasic sites; PCNA, proliferating cell nuclear antigen; bp, base pair(s); CHO, Chinese hamster ovary. counteracts the mutagenic and cytotoxic effects of various kinds of base alterations that do not significantly distort the secondary structure of the double helix. A common intermediate of this pathway is the abasic (AP) site, that arises as a consequence of removal of altered bases by DNA-N-glycosylases or as spontaneous detachment of normal bases from the deoxyribose-phosphate backbone. It has been calculated that 2000–10000 AP sites arise each day in a mammalian cell under physiological conditions (1Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4271) Google Scholar). Therefore, the task of BER is engaging and important, and data obtained in Escherichia coli and transgenic mice show that this process is essential for survival (2Saporito S.M. Gedenk M. Cunningham R.P. J. Bacteriol. 1989; 171: 2542-2546Crossref PubMed Google Scholar, 3Sobol 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, 4Xanthoudakis S. Smeyne R.J. Wallace J.D. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8919-8923Crossref PubMed Scopus (431) Google Scholar). We have recently shown that, in addition to the polymerase β-dependent single nucleotide insertion pathway previously investigated in mammalian cells (5Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (260) Google Scholar), a distinct proliferating cell nuclear antigen (PCNA)-dependent pathway is also present that incorporates a repair patch size of 7–14 nucleotides extending 3′ to the site of the lesion (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). Our knowledge of the enzymology of the two pathways has several gaps. In particular, the enzymology of the ligation step is poorly defined. A role for the XRCC1 protein has been suggested on the basis of the sensitivity of xrcc1 mutant cell lines (the CHO derivatives EM9 and EM-C11) to agents that introduce DNA base damage (7Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, 8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar) and because of their reduced rate of single-strand break rejoining following exposure to ionizing radiation or alkylating agents (9Thompson L.H Brookman K.W. Dillehay L.H. Carrano A.V. Mazrimas J.A. Mooney C.L. Minkler J.L. Mutat. Res. 1982; 95: 247-440Crossref Scopus (284) Google Scholar, 10Zdzienicka M.Z. van der Schans G.P. Natarajan A.T. Thompson L.H. Neuteboom I. Simons J.W.I.M. Mutagenesis. 1992; 7: 265-269Crossref PubMed Scopus (91) Google Scholar). Consistent with a role for XRCC1 in DNA ligation and BER is its observed interaction with DNA ligase III and DNA polymerase β (7Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, 11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar, 12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). Here, we have examined directly the role of XRCC1 and DNA ligase III in mammalian BER using a cell-free system. We report for the first time that (i) xrcc1 mutant cells are partially defective in ligation of BER patches and (ii) the defect involves only the polymerase β-dependent single nucleotide insertion pathway and not the PCNA-dependent pathway.DISCUSSIONWe report here that EM-C11 cell extracts, which possess greatly reduced levels of XRCC1 and DNA ligase III polypeptides, are defective in the DNA ligation step of BER. Of the two branches of the BER 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), only the single nucleotide insertion pathway was affected by the XRCC1 deficiency. No ligation defect was detectable in the PCNA-dependent pathway, thus indicating that ligase activities other than XRCC1-DNA ligase III are involved in this pathway. The ligation defect in EM-C11 extracts within the single nucleotide insertion pathway was partial, with approximately one-third of repair events remaining unligated after 1 h in comparison to CHO-9 wild type extracts. This indicates that the residual DNA ligase III is sufficient to complete some of the BER ligation events, or that other DNA ligases can partially compensate, at least in vitro, for the DNA ligase III defect. That the latter possibility is significant is supported by the finding that efficient complementation was achieved by addition of bacteriophage T4 DNA ligase. In vivo, one possible backup activity is DNA ligase I, since a partial defect in cell-free BER was observed for the DNA ligase I mutant, 46BR (16Prigent C. Satoh M.S. Daly G. Barnes D.E. Lindahl T Mol. Cell. Biol. 1994; 14: 310-317Crossref PubMed Scopus (128) Google Scholar) and a specific interaction between DNA polymerase β and DNA ligase I in a multiprotein BER complex has been recently found in bovine testis extracts (17Prasad 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). DNA ligase I could also be the major sealing activity in the PCNA-dependent pathway. The latter pathway is probably less efficient than the one nucleotide insertion pathway and can only partially compensate the defect of EM-C11.Levels of DNA ligase III protein and activity are severely reduced in EM-C11 cell extract (8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar) (Fig. 6). The cell-free BER defect in the EM-C11 extract was fully corrected by addition of recombinant DNA ligase III but not by recombinant XRCC1, indicating that it can be accounted for by the DNA ligase III deficiency. The lack of significant effect of adding recombinant XRCC1 to the BER reaction suggests that this protein is not required enzymatically for BER, at least in vitro. Rather, taken together, these results support the notion that XRCC1 is required to maintain normal cellular levels of DNA ligase III, a role presumably reflecting a dependence of the latter polypeptide on interaction with XRCC1 for physical stability (8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar). A very small reduction in DNA ligase I was observed in EM-C11 extracts as compared with parental CHO-9 extracts. This is unlikely to reflect any direct influence of XRCC1 protein on DNA ligase I, since these proteins do not appear to associate (11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar), but rather may reflect the decreased proliferation rate of EM-C11.In contrast to EM-C11, a DNA ligation defect was not detected in BER supported by EM9 cell extracts (12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). This discrepancy may similarly result from differences in levels of residual DNA ligase III, since levels of DNA ligase III were lower in cell extracts from EM-C11 cells than from EM9 cells (Fig. 6) (8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar). It is possible that the less severe DNA ligase III deficiency in EM9 is more readily complemented in the cell-free BER assay by the promiscuity of other DNA ligases. Such nonspecific complementation may not be possible in vivo due to the sequestration of different DNA ligases into different protein complexes separated spatially, and possibly temporally, within the nucleus.Although levels of DNA ligase III are the only cause of the BER defect observed in this cell-free system, it remains to be determined whether this is also the case in vivo, or whether XRCC1 has additional roles other than maintaining the level of DNA ligase III. Consistent with the latter possibility, we and others have recently reported that XRCC1 directly interacts with DNA polymerase β, and also possibly with poly(ADP)-ribose polymerase in addition to DNA ligase III (7Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, 11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar, 12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 18De Murcia G. Masson M. Trucco C. Dantzer F. Rolli V. Niedergang C. Ruf A. Schultz G. Menissier-De Murcia J. Workshop on Processing of DNA Damage: Molecular Mechanisms and Biological Effects. Noordwijkerhout, The Netherlands1996Google Scholar). On this basis, it has been proposed that XRCC1 might function as a scaffold protein physically linking together components of the BER machinery (12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar), or that XRCC1 may act as a molecular chaperone to actively target polymerase β and/or DNA ligase III to DNA repair events in vivo (11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar). Alternatively, XRCC1 may possess a novel catalytic activity. One such role suggested for XRCC1 is to promote single nucleotide incorporation and so prevent excessive repair synthesis by polymerase β. It was reported that XRCC1 mutant EM9 extracts display elevated repair patch size during polymerase β-dependent BER (12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). Such a defect was not apparent in our studies, since this would have manifested as elevated levels of incorporation in experiments measuring repair replication downstream of the AP site, which we did not see (Fig. 5, lanes 5 and 6). Indeed, a decrease of 52% in repair replication located 3′ to the the lesion was observed in our experiments. The latter finding suggests the possible involvement of XRCC1 protein in the PCNA-dependent pathway but clearly, whether and how XRCC1 participates in repair replication downstream of the AP site requires further study.In summary, we report here for the first time that XRCC1 mutant cell extracts are defective in the ligation step of BER and that this defect is specific to the single nucleotide insertion pathway catalyzed by DNA polymerase β. DNA base excision repair (BER) 1The abbreviations used are: BER, base excision repair; AP sites, abasic sites; PCNA, proliferating cell nuclear antigen; bp, base pair(s); CHO, Chinese hamster ovary. 1The abbreviations used are: BER, base excision repair; AP sites, abasic sites; PCNA, proliferating cell nuclear antigen; bp, base pair(s); CHO, Chinese hamster ovary. counteracts the mutagenic and cytotoxic effects of various kinds of base alterations that do not significantly distort the secondary structure of the double helix. A common intermediate of this pathway is the abasic (AP) site, that arises as a consequence of removal of altered bases by DNA-N-glycosylases or as spontaneous detachment of normal bases from the deoxyribose-phosphate backbone. It has been calculated that 2000–10000 AP sites arise each day in a mammalian cell under physiological conditions (1Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4271) Google Scholar). Therefore, the task of BER is engaging and important, and data obtained in Escherichia coli and transgenic mice show that this process is essential for survival (2Saporito S.M. Gedenk M. Cunningham R.P. J. Bacteriol. 1989; 171: 2542-2546Crossref PubMed Google Scholar, 3Sobol 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, 4Xanthoudakis S. Smeyne R.J. Wallace J.D. Curran T. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8919-8923Crossref PubMed Scopus (431) Google Scholar). We have recently shown that, in addition to the polymerase β-dependent single nucleotide insertion pathway previously investigated in mammalian cells (5Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (260) Google Scholar), a distinct proliferating cell nuclear antigen (PCNA)-dependent pathway is also present that incorporates a repair patch size of 7–14 nucleotides extending 3′ to the site of the lesion (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). Our knowledge of the enzymology of the two pathways has several gaps. In particular, the enzymology of the ligation step is poorly defined. A role for the XRCC1 protein has been suggested on the basis of the sensitivity of xrcc1 mutant cell lines (the CHO derivatives EM9 and EM-C11) to agents that introduce DNA base damage (7Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, 8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar) and because of their reduced rate of single-strand break rejoining following exposure to ionizing radiation or alkylating agents (9Thompson L.H Brookman K.W. Dillehay L.H. Carrano A.V. Mazrimas J.A. Mooney C.L. Minkler J.L. Mutat. Res. 1982; 95: 247-440Crossref Scopus (284) Google Scholar, 10Zdzienicka M.Z. van der Schans G.P. Natarajan A.T. Thompson L.H. Neuteboom I. Simons J.W.I.M. Mutagenesis. 1992; 7: 265-269Crossref PubMed Scopus (91) Google Scholar). Consistent with a role for XRCC1 in DNA ligation and BER is its observed interaction with DNA ligase III and DNA polymerase β (7Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, 11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar, 12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). Here, we have examined directly the role of XRCC1 and DNA ligase III in mammalian BER using a cell-free system. We report for the first time that (i) xrcc1 mutant cells are partially defective in ligation of BER patches and (ii) the defect involves only the polymerase β-dependent single nucleotide insertion pathway and not the PCNA-dependent pathway. DISCUSSIONWe report here that EM-C11 cell extracts, which possess greatly reduced levels of XRCC1 and DNA ligase III polypeptides, are defective in the DNA ligation step of BER. Of the two branches of the BER 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), only the single nucleotide insertion pathway was affected by the XRCC1 deficiency. No ligation defect was detectable in the PCNA-dependent pathway, thus indicating that ligase activities other than XRCC1-DNA ligase III are involved in this pathway. The ligation defect in EM-C11 extracts within the single nucleotide insertion pathway was partial, with approximately one-third of repair events remaining unligated after 1 h in comparison to CHO-9 wild type extracts. This indicates that the residual DNA ligase III is sufficient to complete some of the BER ligation events, or that other DNA ligases can partially compensate, at least in vitro, for the DNA ligase III defect. That the latter possibility is significant is supported by the finding that efficient complementation was achieved by addition of bacteriophage T4 DNA ligase. In vivo, one possible backup activity is DNA ligase I, since a partial defect in cell-free BER was observed for the DNA ligase I mutant, 46BR (16Prigent C. Satoh M.S. Daly G. Barnes D.E. Lindahl T Mol. Cell. Biol. 1994; 14: 310-317Crossref PubMed Scopus (128) Google Scholar) and a specific interaction between DNA polymerase β and DNA ligase I in a multiprotein BER complex has been recently found in bovine testis extracts (17Prasad 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). DNA ligase I could also be the major sealing activity in the PCNA-dependent pathway. The latter pathway is probably less efficient than the one nucleotide insertion pathway and can only partially compensate the defect of EM-C11.Levels of DNA ligase III protein and activity are severely reduced in EM-C11 cell extract (8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar) (Fig. 6). The cell-free BER defect in the EM-C11 extract was fully corrected by addition of recombinant DNA ligase III but not by recombinant XRCC1, indicating that it can be accounted for by the DNA ligase III deficiency. The lack of significant effect of adding recombinant XRCC1 to the BER reaction suggests that this protein is not required enzymatically for BER, at least in vitro. Rather, taken together, these results support the notion that XRCC1 is required to maintain normal cellular levels of DNA ligase III, a role presumably reflecting a dependence of the latter polypeptide on interaction with XRCC1 for physical stability (8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar). A very small reduction in DNA ligase I was observed in EM-C11 extracts as compared with parental CHO-9 extracts. This is unlikely to reflect any direct influence of XRCC1 protein on DNA ligase I, since these proteins do not appear to associate (11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar), but rather may reflect the decreased proliferation rate of EM-C11.In contrast to EM-C11, a DNA ligation defect was not detected in BER supported by EM9 cell extracts (12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). This discrepancy may similarly result from differences in levels of residual DNA ligase III, since levels of DNA ligase III were lower in cell extracts from EM-C11 cells than from EM9 cells (Fig. 6) (8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar). It is possible that the less severe DNA ligase III deficiency in EM9 is more readily complemented in the cell-free BER assay by the promiscuity of other DNA ligases. Such nonspecific complementation may not be possible in vivo due to the sequestration of different DNA ligases into different protein complexes separated spatially, and possibly temporally, within the nucleus.Although levels of DNA ligase III are the only cause of the BER defect observed in this cell-free system, it remains to be determined whether this is also the case in vivo, or whether XRCC1 has additional roles other than maintaining the level of DNA ligase III. Consistent with the latter possibility, we and others have recently reported that XRCC1 directly interacts with DNA polymerase β, and also possibly with poly(ADP)-ribose polymerase in addition to DNA ligase III (7Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, 11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar, 12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 18De Murcia G. Masson M. Trucco C. Dantzer F. Rolli V. Niedergang C. Ruf A. Schultz G. Menissier-De Murcia J. Workshop on Processing of DNA Damage: Molecular Mechanisms and Biological Effects. Noordwijkerhout, The Netherlands1996Google Scholar). On this basis, it has been proposed that XRCC1 might function as a scaffold protein physically linking together components of the BER machinery (12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar), or that XRCC1 may act as a molecular chaperone to actively target polymerase β and/or DNA ligase III to DNA repair events in vivo (11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar). Alternatively, XRCC1 may possess a novel catalytic activity. One such role suggested for XRCC1 is to promote single nucleotide incorporation and so prevent excessive repair synthesis by polymerase β. It was reported that XRCC1 mutant EM9 extracts display elevated repair patch size during polymerase β-dependent BER (12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). Such a defect was not apparent in our studies, since this would have manifested as elevated levels of incorporation in experiments measuring repair replication downstream of the AP site, which we did not see (Fig. 5, lanes 5 and 6). Indeed, a decrease of 52% in repair replication located 3′ to the the lesion was observed in our experiments. The latter finding suggests the possible involvement of XRCC1 protein in the PCNA-dependent pathway but clearly, whether and how XRCC1 participates in repair replication downstream of the AP site requires further study.In summary, we report here for the first time that XRCC1 mutant cell extracts are defective in the ligation step of BER and that this defect is specific to the single nucleotide insertion pathway catalyzed by DNA polymerase β. We report here that EM-C11 cell extracts, which possess greatly reduced levels of XRCC1 and DNA ligase III polypeptides, are defective in the DNA ligation step of BER. Of the two branches of the BER 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), only the single nucleotide insertion pathway was affected by the XRCC1 deficiency. No ligation defect was detectable in the PCNA-dependent pathway, thus indicating that ligase activities other than XRCC1-DNA ligase III are involved in this pathway. The ligation defect in EM-C11 extracts within the single nucleotide insertion pathway was partial, with approximately one-third of repair events remaining unligated after 1 h in comparison to CHO-9 wild type extracts. This indicates that the residual DNA ligase III is sufficient to complete some of the BER ligation events, or that other DNA ligases can partially compensate, at least in vitro, for the DNA ligase III defect. That the latter possibility is significant is supported by the finding that efficient complementation was achieved by addition of bacteriophage T4 DNA ligase. In vivo, one possible backup activity is DNA ligase I, since a partial defect in cell-free BER was observed for the DNA ligase I mutant, 46BR (16Prigent C. Satoh M.S. Daly G. Barnes D.E. Lindahl T Mol. Cell. Biol. 1994; 14: 310-317Crossref PubMed Scopus (128) Google Scholar) and a specific interaction between DNA polymerase β and DNA ligase I in a multiprotein BER complex has been recently found in bovine testis extracts (17Prasad 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). DNA ligase I could also be the major sealing activity in the PCNA-dependent pathway. The latter pathway is probably less efficient than the one nucleotide insertion pathway and can only partially compensate the defect of EM-C11. Levels of DNA ligase III protein and activity are severely reduced in EM-C11 cell extract (8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar) (Fig. 6). The cell-free BER defect in the EM-C11 extract was fully corrected by addition of recombinant DNA ligase III but not by recombinant XRCC1, indicating that it can be accounted for by the DNA ligase III deficiency. The lack of significant effect of adding recombinant XRCC1 to the BER reaction suggests that this protein is not required enzymatically for BER, at least in vitro. Rather, taken together, these results support the notion that XRCC1 is required to maintain normal cellular levels of DNA ligase III, a role presumably reflecting a dependence of the latter polypeptide on interaction with XRCC1 for physical stability (8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar). A very small reduction in DNA ligase I was observed in EM-C11 extracts as compared with parental CHO-9 extracts. This is unlikely to reflect any direct influence of XRCC1 protein on DNA ligase I, since these proteins do not appear to associate (11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar), but rather may reflect the decreased proliferation rate of EM-C11. In contrast to EM-C11, a DNA ligation defect was not detected in BER supported by EM9 cell extracts (12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). This discrepancy may similarly result from differences in levels of residual DNA ligase III, since levels of DNA ligase III were lower in cell extracts from EM-C11 cells than from EM9 cells (Fig. 6) (8Caldecott K.W. Tucker J.D. Stanker L.H. Thompson L.H. Nucleic Acids Res. 1995; 23: 4836-4843Crossref PubMed Scopus (257) Google Scholar). It is possible that the less severe DNA ligase III deficiency in EM9 is more readily complemented in the cell-free BER assay by the promiscuity of other DNA ligases. Such nonspecific complementation may not be possible in vivo due to the sequestration of different DNA ligases into different protein complexes separated spatially, and possibly temporally, within the nucleus. Although levels of DNA ligase III are the only cause of the BER defect observed in this cell-free system, it remains to be determined whether this is also the case in vivo, or whether XRCC1 has additional roles other than maintaining the level of DNA ligase III. Consistent with the latter possibility, we and others have recently reported that XRCC1 directly interacts with DNA polymerase β, and also possibly with poly(ADP)-ribose polymerase in addition to DNA ligase III (7Caldecott K.W. McKeown C.K. Tucker J.D. Ljungquist S. Thompson L.H. Mol. Cell. Biol. 1994; 14: 68-76Crossref PubMed Google Scholar, 11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar, 12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar, 18De Murcia G. Masson M. Trucco C. Dantzer F. Rolli V. Niedergang C. Ruf A. Schultz G. Menissier-De Murcia J. Workshop on Processing of DNA Damage: Molecular Mechanisms and Biological Effects. Noordwijkerhout, The Netherlands1996Google Scholar). On this basis, it has been proposed that XRCC1 might function as a scaffold protein physically linking together components of the BER machinery (12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar), or that XRCC1 may act as a molecular chaperone to actively target polymerase β and/or DNA ligase III to DNA repair events in vivo (11Caldecott K.W. Aoufouchi S. Jonhson P. Shall S. Nucleic Acids Res. 1996; 24: 4387-4394Crossref PubMed Scopus (543) Google Scholar). Alternatively, XRCC1 may possess a novel catalytic activity. One such role suggested for XRCC1 is to promote single nucleotide incorporation and so prevent excessive repair synthesis by polymerase β. It was reported that XRCC1 mutant EM9 extracts display elevated repair patch size during polymerase β-dependent BER (12Kubota Y. Nash R.A. Klungland A. Schar P. Barnes D.E. Lindahl T. EMBO J. 1996; 15: 6662-6670Crossref PubMed Scopus (688) Google Scholar). Such a defect was not apparent in our studies, since this would have manifested as elevated levels of incorporation in experiments measuring repair replication downstream of the AP site, which we did not see (Fig. 5, lanes 5 and 6). Indeed, a decrease of 52% in repair replication located 3′ to the the lesion was observed in our experiments. The latter finding suggests the possible involvement of XRCC1 protein in the PCNA-dependent pathway but clearly, whether and how XRCC1 participates in repair replication downstream of the AP site requires further study. In summary, we report here for the first time that XRCC1 mutant cell extracts are defective in the ligation step of BER and that this defect is specific to the single nucleotide insertion pathway catalyzed by DNA polymerase β. We thank M. Zdzienicka for the EM-C11 cell line, S. Boiteux for E. coli uracil-DNA glycosylase, T. Lindahl for TL-6 anti-DNA ligase I antibody, E. Dogliotti for useful discussions, and M. Isola for photography.