Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK
10.1101/gad.14.3.349
Crossref journal-article
Cold Spring Harbor Laboratory
Genes & Development (246)
Abstract

During human nucleotide excision repair, damage is recognized, two incisions are made flanking a DNA lesion, and residues are replaced by repair synthesis. A set of proteins required for repair of most lesions is RPA, XPA, TFIIH, XPC–hHR23B, XPG, and ERCC1–XPF, but additional components have not been excluded. The most complex and difficult to analyze factor is TFIIH, which has a 6-subunit core (XPB, XPD, p44, p34, p52, p62) and a 3-subunit kinase (CAK). TFIIH has roles both in basal transcription initiation and in DNA repair, and several inherited human disorders are associated with mutations in TFIIH subunits. To identify the forms of TFIIH that can function in repair, recombinant XPA, RPA, XPC–hHR23B, XPG, and ERCC1–XPF were combined with TFIIH fractions purified from HeLa cells. Repair activity coeluted with the peak of TFIIH and with transcription activity. TFIIH from cells with XPB or XPD mutations was defective in supporting repair, whereas TFIIH from spinal muscular atrophy cells with a deletion of one p44 gene was active. Recombinant TFIIH also functioned in repair, both a 6- and a 9-subunit form containing CAK. The CAK kinase inhibitor H-8 improved repair efficiency, indicating that CAK can negatively regulate NER by phosphorylation. The 15 recombinant polypeptides define the minimal set of proteins required for dual incision of DNA containing a cisplatin adduct. Complete repair was achieved by including highly purified human DNA polymerase δ or ε, PCNA, RFC, and DNA ligase I in reaction mixtures, reconstituting adduct repair for the first time with recombinant incision factors and human replication proteins.

Bibliography

Araújo, S. J., Tirode, F., Coin, F., Pospiech, H., Syväoja, J. E., Stucki, M., Hübscher, U., Egly, J.-M., & Wood, R. D. (2000). Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK. Genes & Development, 14(3), 349–359.

Authors 9
  1. Sofia J. Araújo (first)
  2. Franck Tirode (additional)
  3. Frederic Coin (additional)
  4. Helmut Pospiech (additional)
  5. Juhani E. Syväoja (additional)
  6. Manuel Stucki (additional)
  7. Ulrich Hübscher (additional)
  8. Jean-Marc Egly (additional)
  9. Richard D. Wood (additional)
References 61 Referenced 252
  1. 10.1016/0092-8674(95)90289-9
  2. 10.1002/j.1460-2075.1996.tb00538.x / EMBO J. / MAT1, cdk7 and cyclin H form a kinase complex which is UV light-sensitive upon association with TFIIH. by Adamczewski (1996)
  3. 10.1016/S0921-8777(99)00042-7
  4. 10.1093/nar/24.3.433
  5. Biggerstaff M. Wood R.D. (1999) Assay for nucleotide excision repair proteins using mammalian cell extracts and UV damaged plasmid DNA. in DNA repair protocols: Eukaryotic systems, ed Henderson D.S. (Humana Press, Totowa, NJ), pp 357–372. (10.1007/978-1-4612-1608-7_29)
  6. 10.1086/302063
  7. {'key': '2021111419181290000_14.3.349.7', 'first-page': '72', 'article-title': 'The gene encoding p44, a subunit of the transcription factor TFIIH, is involved in large-scale deletions associated with Werdnig-Hoffmann disease.', 'volume': '60', 'author': 'Burglen', 'year': '1997', 'journal-title': 'Am. J. Hum. Genet.'} / Am. J. Hum. Genet. / The gene encoding p44, a subunit of the transcription factor TFIIH, is involved in large-scale deletions associated with Werdnig-Hoffmann disease. by Burglen (1997)
  8. 10.1074/jbc.270.14.7799
  9. 10.1038/2491
  10. 10.1093/emboj/18.5.1357
  11. 10.1074/jbc.274.9.5637
  12. 10.1101/gad.13.7.768
  13. 10.1073/pnas.93.13.6488
  14. 10.1016/S0021-9258(17)36837-0 / J. Biol. Chem. / Inhibitors of transcription such as 5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole and isoquinoline sulfonamide derivatives (H-8 and H-7) promote dephosphorylation of the carboxyl-terminal domain of RNA polymerase II largest subunit. by Dubois (1994)
  15. 10.1093/emboj/16.3.625
  16. 10.1093/emboj/16.21.6559
  17. 10.1016/0092-8674(94)90040-X
  18. 10.1016/S0021-9258(18)54801-8 / J. Biol. Chem. / Purification and interaction properties of the human RNA polymerase B(II) general transcription factor BTF2. by Gerard (1991)
  19. 10.1074/jbc.270.22.12973
  20. 10.1074/jbc.272.35.21665
  21. 10.1074/jbc.273.11.6292
  22. 10.1074/jbc.271.45.28243
  23. 10.1016/S0021-9258(19)78100-9 / J. Biol. Chem. / Recombinant replication protein-A—Expression, complex-formation, and functional-characterization. by Henricksen (1994)
  24. Hübscher U. Mossi R. Ferrari E. Stucki M. Jónsson Z.O. (1999) Functional analysis of DNA replication accessory proteins. in Eukaryotic DNA replication, ed Cotterill S. (Oxford University Press, Oxford, UK), pp 119–137. (10.1093/oso/9780199636815.003.0005)
  25. 10.1073/pnas.96.2.424
  26. 10.1074/jbc.271.27.15898
  27. 10.1242/jcs.76.1.115 / J. Cell Sci. / Xeroderma pigmentosum D-HeLa hybrids with low and high ultraviolet sensitivity associated with normal and diminished DNA repair ability, respectively. by Johnson (1985)
  28. 10.1021/bi00096a021
  29. 10.1128/MCB.16.12.6783 / Mol. Cell. Biol. / Dual requirement for the yeast mms19 gene in DNA repair and RNA polymerase II transcription. by Lauder (1996)
  30. 10.1093/nar/25.20.3974
  31. 10.1074/jbc.272.17.11550
  32. 10.1006/meth.1997.0476
  33. 10.1128/MCB.17.12.6915 / Mol. Cell. Biol. / Identification and characterization of XPC-binding domain of hHR23B. by Masutani (1997)
  34. 10.1074/jbc.271.12.7177
  35. 10.1074/jbc.270.6.2415
  36. 10.1074/jbc.271.14.8285
  37. 10.1074/jbc.272.46.28971
  38. 10.1093/nar/21.4.841
  39. 10.1073/pnas.93.13.6482
  40. 10.1074/jbc.273.45.29481
  41. 10.1016/S0021-9258(19)61935-6 / J. Biol. Chem. / Structural and functional homology between mammalian DNase IV and the 5′ nuclease domain of Escherichia coli DNA polymerase I. by Robins (1994)
  42. 10.1093/emboj/16.7.1628
  43. 10.1016/0092-8674(94)90039-6
  44. 10.1021/bi00015a012
  45. 10.1038/sj.onc.1202352
  46. Shivji M.K.K. Moggs J.G. Kuraoka I. Wood R.D. (1999) Dual incision assays for nucleotide excision repair using DNA with a lesion at a specific site. in DNA repair protocols: Eukaryotic systems, ed Henderson D.S. (Humana Press, Totowa, NJ), pp 373–392. (10.1007/978-1-4612-1608-7_30)
  47. 10.1016/S0092-8674(00)80155-5
  48. {'key': '2021111419181290000_14.3.349.48', 'first-page': '817', 'article-title': 'A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy.', 'volume': '53', 'author': 'Stefanini', 'year': '1993', 'journal-title': 'Am. J. Hum. Genet.'} / Am. J. Hum. Genet. / A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy. by Stefanini (1993)
  49. 10.1016/0092-8674(95)90447-6
  50. 10.1016/0968-0004(96)10046-3
  51. 10.1016/S0021-9258(19)81640-X / J. Biol. Chem. / Characterization of a large form of DNA polymerase δ from HeLa cells that is insensitive to proliferating cell nuclear antigen. by Syväoja (1989)
  52. {'key': '2021111419181290000_14.3.349.52', 'first-page': '5656', 'article-title': 'Defects in the DNA repair and transcription gene ERCC2 in the cancer-prone disorder xeroderma pigmentosum group D.', 'volume': '55', 'author': 'Takayama', 'year': '1995', 'journal-title': 'Cancer Res.'} / Cancer Res. / Defects in the DNA repair and transcription gene ERCC2 in the cancer-prone disorder xeroderma pigmentosum group D. by Takayama (1995)
  53. 10.1073/pnas.94.16.8658
  54. 10.1016/S1097-2765(00)80177-X
  55. 10.1128/MCB.14.9.6135
  56. 10.1101/SQB.1994.059.01.036
  57. 10.1128/MCB.17.2.635 / Mol. Cell. Biol. / The RAD7, RAD16, and RAD23 genes of Saccharomyces cerevisiae—Requirement for transcription-independent nucleotide excision repair in vitro and interactions between the gene products. by Wang (1997)
  58. 10.1016/0092-8674(90)90122-U
  59. 10.1016/S0021-9258(18)99242-2 / J. Biol. Chem. / Biochemical and functional comparison of DNA polymerases α, δ, and ε from calf thymus. by Weiser (1991)
  60. Winkler, G.S., S.J. Araújo, U. Fiedler, W. Vermeulen, F. Coin, J.-M. Egly, J.H.J. Hoeijmakers, R.D. Wood, H.T.M. Timmers, and G. Weeda. 2000. TFIIH with inactive XPD helicase functions in transcription initiation but is defective in DNA repair. J. Biol. Chem. 275: (in press).. (10.1074/jbc.275.6.4258)
  61. 10.1016/0092-8674(88)90491-6
Dates
Type When
Created 3 years, 9 months ago (Nov. 14, 2021, 10:27 p.m.)
Deposited 1 year, 9 months ago (Nov. 12, 2023, 11:29 a.m.)
Indexed 4 days, 19 hours ago (Aug. 19, 2025, 6:28 a.m.)
Issued 25 years, 6 months ago (Feb. 1, 2000)
Published 25 years, 6 months ago (Feb. 1, 2000)
Published Online 25 years, 6 months ago (Feb. 1, 2000)
Published Print 25 years, 6 months ago (Feb. 1, 2000)
Funders 0

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@article{Ara_jo_2000, title={Nucleotide excision repair of DNA with recombinant human proteins: definition of the minimal set of factors, active forms of TFIIH, and modulation by CAK}, volume={14}, ISSN={1549-5477}, url={http://dx.doi.org/10.1101/gad.14.3.349}, DOI={10.1101/gad.14.3.349}, number={3}, journal={Genes & Development}, publisher={Cold Spring Harbor Laboratory}, author={Araújo, Sofia J. and Tirode, Franck and Coin, Frederic and Pospiech, Helmut and Syväoja, Juhani E. and Stucki, Manuel and Hübscher, Ulrich and Egly, Jean-Marc and Wood, Richard D.}, year={2000}, month=feb, pages={349–359} }