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. 1997 Jun 2;16(11):3341–3348. doi: 10.1093/emboj/16.11.3341

Second pathway for completion of human DNA base excision-repair: reconstitution with purified proteins and requirement for DNase IV (FEN1).

A Klungland 1, T Lindahl 1
PMCID: PMC1169950  PMID: 9214649

Abstract

Two forms of DNA base excision-repair (BER) have been observed: a 'short-patch' BER pathway involving replacement of one nucleotide and a 'long-patch' BER pathway with gap-filling of several nucleotides. The latter mode of repair has been investigated using human cell-free extracts or purified proteins. Correction of a regular abasic site in DNA mainly involves incorporation of a single nucleotide, whereas repair patches of two to six nucleotides in length were found after repair of a reduced or oxidized abasic site. Human AP endonuclease, DNA polymerase beta and a DNA ligase (either III or I) were sufficient for the repair of a regular AP site. In contrast, the structure-specific nuclease DNase IV (FEN1) was essential for repair of a reduced AP site, which occurred through the long-patch BER pathway. DNase IV was required for cleavage of a reaction intermediate generated by template strand displacement during gap-filling. XPG, a related nuclease, could not substitute for DNase IV. The long-patch BER pathway was largely dependent on DNA polymerase beta in cell extracts, but the reaction could be reconstituted with either DNA polymerase beta or delta. Efficient repair of gamma-ray-induced oxidized AP sites in plasmid DNA also required DNase IV. PCNA could promote the Pol beta-dependent long-patch pathway by stimulation of DNase IV.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Abbotts J., SenGupta D. N., Zmudzka B., Widen S. G., Notario V., Wilson S. H. Expression of human DNA polymerase beta in Escherichia coli and characterization of the recombinant enzyme. Biochemistry. 1988 Feb 9;27(3):901–909. doi: 10.1021/bi00403a010. [DOI] [PubMed] [Google Scholar]
  2. Bjørås M., Klungland A., Johansen R. F., Seeberg E. Purification and properties of the alkylation repair DNA glycosylase encoded the MAG gene from Saccharomyces cerevisiae. Biochemistry. 1995 Apr 11;34(14):4577–4582. doi: 10.1021/bi00014a010. [DOI] [PubMed] [Google Scholar]
  3. Breen A. P., Murphy J. A. Reactions of oxyl radicals with DNA. Free Radic Biol Med. 1995 Jun;18(6):1033–1077. doi: 10.1016/0891-5849(94)00209-3. [DOI] [PubMed] [Google Scholar]
  4. Carr A. M., Sheldrick K. S., Murray J. M., al-Harithy R., Watts F. Z., Lehmann A. R. Evolutionary conservation of excision repair in Schizosaccharomyces pombe: evidence for a family of sequences related to the Saccharomyces cerevisiae RAD2 gene. Nucleic Acids Res. 1993 Mar 25;21(6):1345–1349. doi: 10.1093/nar/21.6.1345. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cloud K. G., Shen B., Strniste G. F., Park M. S. XPG protein has a structure-specific endonuclease activity. Mutat Res. 1995 Jul;347(2):55–60. doi: 10.1016/0165-7992(95)90070-5. [DOI] [PubMed] [Google Scholar]
  6. DeMott M. S., Shen B., Park M. S., Bambara R. A., Zigman S. Human RAD2 homolog 1 5'- to 3'-exo/endonuclease can efficiently excise a displaced DNA fragment containing a 5'-terminal abasic lesion by endonuclease activity. J Biol Chem. 1996 Nov 22;271(47):30068–30076. doi: 10.1074/jbc.271.47.30068. [DOI] [PubMed] [Google Scholar]
  7. Demple B., Harrison L. Repair of oxidative damage to DNA: enzymology and biology. Annu Rev Biochem. 1994;63:915–948. doi: 10.1146/annurev.bi.63.070194.004411. [DOI] [PubMed] [Google Scholar]
  8. Dianov G., Price A., Lindahl T. Generation of single-nucleotide repair patches following excision of uracil residues from DNA. Mol Cell Biol. 1992 Apr;12(4):1605–1612. doi: 10.1128/mcb.12.4.1605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Evans E., Fellows J., Coffer A., Wood R. D. Open complex formation around a lesion during nucleotide excision repair provides a structure for cleavage by human XPG protein. EMBO J. 1997 Feb 3;16(3):625–638. doi: 10.1093/emboj/16.3.625. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Frosina G., Fortini P., Rossi O., Carrozzino F., Raspaglio G., Cox L. S., Lane D. P., Abbondandolo A., Dogliotti E. Two pathways for base excision repair in mammalian cells. J Biol Chem. 1996 Apr 19;271(16):9573–9578. doi: 10.1074/jbc.271.16.9573. [DOI] [PubMed] [Google Scholar]
  11. Gottlieb T. M., Jackson S. P. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell. 1993 Jan 15;72(1):131–142. doi: 10.1016/0092-8674(93)90057-w. [DOI] [PubMed] [Google Scholar]
  12. Goulian M., Richards S. H., Heard C. J., Bigsby B. M. Discontinuous DNA synthesis by purified mammalian proteins. J Biol Chem. 1990 Oct 25;265(30):18461–18471. [PubMed] [Google Scholar]
  13. Habraken Y., Sung P., Prakash L., Prakash S. Structure-specific nuclease activity in yeast nucleotide excision repair protein Rad2. J Biol Chem. 1995 Dec 15;270(50):30194–30198. doi: 10.1074/jbc.270.50.30194. [DOI] [PubMed] [Google Scholar]
  14. Hadi S. M., Goldthwait D. A. Endonuclease II of Escherichia coli. Degradation of partially depurinated deoxyribonucleic acid. Biochemistry. 1971 Dec 21;10(26):4986–4993. doi: 10.1021/bi00802a024. [DOI] [PubMed] [Google Scholar]
  15. Harrington J. J., Lieber M. R. Functional domains within FEN-1 and RAD2 define a family of structure-specific endonucleases: implications for nucleotide excision repair. Genes Dev. 1994 Jun 1;8(11):1344–1355. doi: 10.1101/gad.8.11.1344. [DOI] [PubMed] [Google Scholar]
  16. Harrington J. J., Lieber M. R. The characterization of a mammalian DNA structure-specific endonuclease. EMBO J. 1994 Mar 1;13(5):1235–1246. doi: 10.1002/j.1460-2075.1994.tb06373.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Ishimi Y., Claude A., Bullock P., Hurwitz J. Complete enzymatic synthesis of DNA containing the SV40 origin of replication. J Biol Chem. 1988 Dec 25;263(36):19723–19733. [PubMed] [Google Scholar]
  18. Kubota Y., Nash R. A., Klungland A., Schär P., Barnes D. E., Lindahl T. Reconstitution of DNA base excision-repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein. EMBO J. 1996 Dec 2;15(23):6662–6670. [PMC free article] [PubMed] [Google Scholar]
  19. Li X., Li J., Harrington J., Lieber M. R., Burgers P. M. Lagging strand DNA synthesis at the eukaryotic replication fork involves binding and stimulation of FEN-1 by proliferating cell nuclear antigen. J Biol Chem. 1995 Sep 22;270(38):22109–22112. doi: 10.1074/jbc.270.38.22109. [DOI] [PubMed] [Google Scholar]
  20. Lindahl T., Gally J. A., Edelman G. M. Deoxyribonuclease IV: a new exonuclease from mammalian tissues. Proc Natl Acad Sci U S A. 1969 Feb;62(2):597–603. doi: 10.1073/pnas.62.2.597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Lindahl T., Nyberg B. Rate of depurination of native deoxyribonucleic acid. Biochemistry. 1972 Sep 12;11(19):3610–3618. doi: 10.1021/bi00769a018. [DOI] [PubMed] [Google Scholar]
  22. Lundquist R. C., Olivera B. M. Transient generation of displaced single-stranded DNA during nick translation. Cell. 1982 Nov;31(1):53–60. doi: 10.1016/0092-8674(82)90404-4. [DOI] [PubMed] [Google Scholar]
  23. Lyamichev V., Brow M. A., Dahlberg J. E. Structure-specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerases. Science. 1993 May 7;260(5109):778–783. doi: 10.1126/science.7683443. [DOI] [PubMed] [Google Scholar]
  24. Mackenney V. J., Barnes D. E., Lindahl T. Specific function of DNA ligase I in simian virus 40 DNA replication by human cell-free extracts is mediated by the amino-terminal non-catalytic domain. J Biol Chem. 1997 Apr 25;272(17):11550–11556. doi: 10.1074/jbc.272.17.11550. [DOI] [PubMed] [Google Scholar]
  25. Matsumoto Y., Kim K., Bogenhagen D. F. Proliferating cell nuclear antigen-dependent abasic site repair in Xenopus laevis oocytes: an alternative pathway of base excision DNA repair. Mol Cell Biol. 1994 Sep;14(9):6187–6197. doi: 10.1128/mcb.14.9.6187. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Matsumoto Y., Kim K. Excision of deoxyribose phosphate residues by DNA polymerase beta during DNA repair. Science. 1995 Aug 4;269(5224):699–702. doi: 10.1126/science.7624801. [DOI] [PubMed] [Google Scholar]
  27. Murante R. S., Rust L., Bambara R. A. Calf 5' to 3' exo/endonuclease must slide from a 5' end of the substrate to perform structure-specific cleavage. J Biol Chem. 1995 Dec 22;270(51):30377–30383. doi: 10.1074/jbc.270.51.30377. [DOI] [PubMed] [Google Scholar]
  28. Murray J. M., Tavassoli M., al-Harithy R., Sheldrick K. S., Lehmann A. R., Carr A. M., Watts F. Z. Structural and functional conservation of the human homolog of the Schizosaccharomyces pombe rad2 gene, which is required for chromosome segregation and recovery from DNA damage. Mol Cell Biol. 1994 Jul;14(7):4878–4888. doi: 10.1128/mcb.14.7.4878. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Nealon K., Nicholl I. D., Kenny M. K. Characterization of the DNA polymerase requirement of human base excision repair. Nucleic Acids Res. 1996 Oct 1;24(19):3763–3770. doi: 10.1093/nar/24.19.3763. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. O'Donovan A., Scherly D., Clarkson S. G., Wood R. D. Isolation of active recombinant XPG protein, a human DNA repair endonuclease. J Biol Chem. 1994 Jun 10;269(23):15965–15968. [PubMed] [Google Scholar]
  31. Podust L. M., Podust V. N., Sogo J. M., Hübscher U. Mammalian DNA polymerase auxiliary proteins: analysis of replication factor C-catalyzed proliferating cell nuclear antigen loading onto circular double-stranded DNA. Mol Cell Biol. 1995 Jun;15(6):3072–3081. doi: 10.1128/mcb.15.6.3072. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Povirk L. F., Steighner R. J. Oxidized apurinic/apyrimidinic sites formed in DNA by oxidative mutagens. Mutat Res. 1989 Sep;214(1):13–22. doi: 10.1016/0027-5107(89)90193-0. [DOI] [PubMed] [Google Scholar]
  33. Price A., Lindahl T. Enzymatic release of 5'-terminal deoxyribose phosphate residues from damaged DNA in human cells. Biochemistry. 1991 Sep 3;30(35):8631–8637. doi: 10.1021/bi00099a020. [DOI] [PubMed] [Google Scholar]
  34. Reagan M. S., Pittenger C., Siede W., Friedberg E. C. Characterization of a mutant strain of Saccharomyces cerevisiae with a deletion of the RAD27 gene, a structural homolog of the RAD2 nucleotide excision repair gene. J Bacteriol. 1995 Jan;177(2):364–371. doi: 10.1128/jb.177.2.364-371.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Robins P., Pappin D. J., Wood R. D., Lindahl T. Structural and functional homology between mammalian DNase IV and the 5'-nuclease domain of Escherichia coli DNA polymerase I. J Biol Chem. 1994 Nov 18;269(46):28535–28538. [PubMed] [Google Scholar]
  36. Satoh M. S., Poirier G. G., Lindahl T. Dual function for poly(ADP-ribose) synthesis in response to DNA strand breakage. Biochemistry. 1994 Jun 14;33(23):7099–7106. doi: 10.1021/bi00189a012. [DOI] [PubMed] [Google Scholar]
  37. Seeberg E., Eide L., Bjørås M. The base excision repair pathway. Trends Biochem Sci. 1995 Oct;20(10):391–397. doi: 10.1016/s0968-0004(00)89086-6. [DOI] [PubMed] [Google Scholar]
  38. Shivji K. K., Kenny M. K., Wood R. D. Proliferating cell nuclear antigen is required for DNA excision repair. Cell. 1992 Apr 17;69(2):367–374. doi: 10.1016/0092-8674(92)90416-a. [DOI] [PubMed] [Google Scholar]
  39. Singhal R. K., Prasad R., Wilson S. H. DNA polymerase beta conducts the gap-filling step in uracil-initiated base excision repair in a bovine testis nuclear extract. J Biol Chem. 1995 Jan 13;270(2):949–957. doi: 10.1074/jbc.270.2.949. [DOI] [PubMed] [Google Scholar]
  40. Singhal R. K., Wilson S. H. Short gap-filling synthesis by DNA polymerase beta is processive. J Biol Chem. 1993 Jul 25;268(21):15906–15911. [PubMed] [Google Scholar]
  41. Slupphaug G., Eftedal I., Kavli B., Bharati S., Helle N. M., Haug T., Levine D. W., Krokan H. E. Properties of a recombinant human uracil-DNA glycosylase from the UNG gene and evidence that UNG encodes the major uracil-DNA glycosylase. Biochemistry. 1995 Jan 10;34(1):128–138. doi: 10.1021/bi00001a016. [DOI] [PubMed] [Google Scholar]
  42. Sobol R. W., Horton J. K., Kühn R., Gu H., Singhal R. K., Prasad R., Rajewsky K., Wilson S. H. Requirement of mammalian DNA polymerase-beta in base-excision repair. Nature. 1996 Jan 11;379(6561):183–186. doi: 10.1038/379183a0. [DOI] [PubMed] [Google Scholar]
  43. Waga S., Bauer G., Stillman B. Reconstitution of complete SV40 DNA replication with purified replication factors. J Biol Chem. 1994 Apr 8;269(14):10923–10934. [PubMed] [Google Scholar]
  44. Walker L. J., Robson C. N., Black E., Gillespie D., Hickson I. D. Identification of residues in the human DNA repair enzyme HAP1 (Ref-1) that are essential for redox regulation of Jun DNA binding. Mol Cell Biol. 1993 Sep;13(9):5370–5376. doi: 10.1128/mcb.13.9.5370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Wang Z., Wu X., Friedberg E. C. DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae. Mol Cell Biol. 1993 Feb;13(2):1051–1058. doi: 10.1128/mcb.13.2.1051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Wu X., Li J., Li X., Hsieh C. L., Burgers P. M., Lieber M. R. Processing of branched DNA intermediates by a complex of human FEN-1 and PCNA. Nucleic Acids Res. 1996 Jun 1;24(11):2036–2043. doi: 10.1093/nar/24.11.2036. [DOI] [PMC free article] [PubMed] [Google Scholar]

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