Skip to main content
PMC home page
Molecular and Cellular Biology logoLink to Molecular and Cellular Biology
. 1995 Apr;15(4):1993–1998. doi: 10.1128/mcb.15.4.1993

Mutations in XPA that prevent association with ERCC1 are defective in nucleotide excision repair.

L Li 1, C A Peterson 1, X Lu 1, R J Legerski 1
PMCID: PMC230426  PMID: 7891694

Abstract

The human repair proteins XPA and ERCC1 have been shown to be absolutely required for the incision step of nucleotide excision repair, and recently we identified an interaction between these two proteins both in vivo and in vitro (L. Li, S. J. Elledge, C. A. Peterson, E. S. Bales, and R. J. Legerski, Proc. Natl. Acad. Sci. USA 91:5012-5016, 1994). In this report, we demonstrate the functional relevance of this interaction. The ERCC1-binding domain on XPA was previously mapped to a region containing two highly conserved XPA sequences, Gly-72 to Phe-75 and Glu-78 to Glu-84, which are termed the G and E motifs, respectively. Site-specific mutagenesis was used to independently delete these motifs and create two XPA mutants referred to as delta G and delta E. In vitro, the binding of ERCC1 to delta E was reduced by approximately 70%, and binding to delta G was undetectable; furthermore, both mutants failed to complement XPA cell extracts in an in vitro DNA repair synthesis assay. In vivo, the delta E mutant exhibited an intermediate level of complementation of XPA cells and the delta G mutant exhibited little or no complementation. In addition, the delta G mutant inhibited repair synthesis in wild-type cell extracts, indicating that it is a dominant negative mutant. The delta E and delta G mutations, however, did not affect preferential binding of XPA to damaged DNA. These results suggest that the association between XPA and ERCC1 is a required step in the nucleotide excision repair pathway and that the probable role of the interaction is to recruit the ERCC1 incision complex to the damage site. Finally, the affinity of the XPA-ERCC1 complex was found to increase as a function of salt concentration, indicating a hydrophobic interaction; the half-life of the complex was determined to be approximately 90 min.

Full Text

The Full Text of this article is available as a PDF (267.2 KB).

Selected References

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

  1. Alting-Mees M. A., Short J. M. pBluescript II: gene mapping vectors. Nucleic Acids Res. 1989 Nov 25;17(22):9494–9494. doi: 10.1093/nar/17.22.9494. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Arias J. A., Dynan W. S. Promoter-dependent transcription by RNA polymerase II using immobilized enzyme complexes. J Biol Chem. 1989 Feb 25;264(6):3223–3229. [PubMed] [Google Scholar]
  3. Bailly V., Sommers C. H., Sung P., Prakash L., Prakash S. Specific complex formation between proteins encoded by the yeast DNA repair and recombination genes RAD1 and RAD10. Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8273–8277. doi: 10.1073/pnas.89.17.8273. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bankmann M., Prakash L., Prakash S. Yeast RAD14 and human xeroderma pigmentosum group A DNA-repair genes encode homologous proteins. Nature. 1992 Feb 6;355(6360):555–558. doi: 10.1038/355555a0. [DOI] [PubMed] [Google Scholar]
  5. Bardwell A. J., Bardwell L., Tomkinson A. E., Friedberg E. C. Specific cleavage of model recombination and repair intermediates by the yeast Rad1-Rad10 DNA endonuclease. Science. 1994 Sep 30;265(5181):2082–2085. doi: 10.1126/science.8091230. [DOI] [PubMed] [Google Scholar]
  6. Biggerstaff M., Szymkowski D. E., Wood R. D. Co-correction of the ERCC1, ERCC4 and xeroderma pigmentosum group F DNA repair defects in vitro. EMBO J. 1993 Sep;12(9):3685–3692. doi: 10.1002/j.1460-2075.1993.tb06043.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Chen C., Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. doi: 10.1128/mcb.7.8.2745. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cleaver J. E. Defective repair replication of DNA in xeroderma pigmentosum. Nature. 1968 May 18;218(5142):652–656. doi: 10.1038/218652a0. [DOI] [PubMed] [Google Scholar]
  9. Collins A. R. Mutant rodent cell lines sensitive to ultraviolet light, ionizing radiation and cross-linking agents: a comprehensive survey of genetic and biochemical characteristics. Mutat Res. 1993 Jan;293(2):99–118. doi: 10.1016/0921-8777(93)90062-l. [DOI] [PubMed] [Google Scholar]
  10. Drapkin R., Reardon J. T., Ansari A., Huang J. C., Zawel L., Ahn K., Sancar A., Reinberg D. Dual role of TFIIH in DNA excision repair and in transcription by RNA polymerase II. Nature. 1994 Apr 21;368(6473):769–772. doi: 10.1038/368769a0. [DOI] [PubMed] [Google Scholar]
  11. Hoeijmakers J. H., Bootsma D. Molecular genetics of eukaryotic DNA excision repair. Cancer Cells. 1990 Oct;2(10):311–320. [PubMed] [Google Scholar]
  12. Hoeijmakers J. H. Nucleotide excision repair I: from E. coli to yeast. Trends Genet. 1993 May;9(5):173–177. doi: 10.1016/0168-9525(93)90164-d. [DOI] [PubMed] [Google Scholar]
  13. Hoeijmakers J. H. Nucleotide excision repair. II: From yeast to mammals. Trends Genet. 1993 Jun;9(6):211–217. doi: 10.1016/0168-9525(93)90121-w. [DOI] [PubMed] [Google Scholar]
  14. Jones C. J., Wood R. D. Preferential binding of the xeroderma pigmentosum group A complementing protein to damaged DNA. Biochemistry. 1993 Nov 16;32(45):12096–12104. doi: 10.1021/bi00096a021. [DOI] [PubMed] [Google Scholar]
  15. Lambert C., Couto L. B., Weiss W. A., Schultz R. A., Thompson L. H., Friedberg E. C. A yeast DNA repair gene partially complements defective excision repair in mammalian cells. EMBO J. 1988 Oct;7(10):3245–3253. doi: 10.1002/j.1460-2075.1988.tb03191.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Legerski R. J., Gray H. B., Jr, Robberson D. L. A sensitive endonuclease probe for lesions in deoxyribonucleic acid helix structure produced by carcinogenic or mutagenic agents. J Biol Chem. 1977 Dec 10;252(23):8740–8746. [PubMed] [Google Scholar]
  17. Legerski R., Peterson C. Expression cloning of a human DNA repair gene involved in xeroderma pigmentosum group C. Nature. 1992 Sep 3;359(6390):70–73. doi: 10.1038/359070a0. [DOI] [PubMed] [Google Scholar]
  18. Li L., Elledge S. J., Peterson C. A., Bales E. S., Legerski R. J. Specific association between the human DNA repair proteins XPA and ERCC1. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5012–5016. doi: 10.1073/pnas.91.11.5012. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Miyamoto I., Miura N., Niwa H., Miyazaki J., Tanaka K. Mutational analysis of the structure and function of the xeroderma pigmentosum group A complementing protein. Identification of essential domains for nuclear localization and DNA excision repair. J Biol Chem. 1992 Jun 15;267(17):12182–12187. [PubMed] [Google Scholar]
  20. O'Donovan A., Davies A. A., Moggs J. G., West S. C., Wood R. D. XPG endonuclease makes the 3' incision in human DNA nucleotide excision repair. Nature. 1994 Sep 29;371(6496):432–435. doi: 10.1038/371432a0. [DOI] [PubMed] [Google Scholar]
  21. Park C. H., Sancar A. Formation of a ternary complex by human XPA, ERCC1, and ERCC4(XPF) excision repair proteins. Proc Natl Acad Sci U S A. 1994 May 24;91(11):5017–5021. doi: 10.1073/pnas.91.11.5017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Peterson C., Legerski R. High-frequency transformation of human repair-deficient cell lines by an Epstein-Barr virus-based cDNA expression vector. Gene. 1991 Nov 15;107(2):279–284. doi: 10.1016/0378-1119(91)90328-9. [DOI] [PubMed] [Google Scholar]
  23. Reardon J. T., Thompson L. H., Sancar A. Excision repair in man and the molecular basis of xeroderma pigmentosum syndrome. Cold Spring Harb Symp Quant Biol. 1993;58:605–617. doi: 10.1101/sqb.1993.058.01.067. [DOI] [PubMed] [Google Scholar]
  24. Riboni R., Botta E., Stefanini M., Numata M., Yasui A. Identification of the eleventh complementation group of UV-sensitive excision repair-defective rodent mutants. Cancer Res. 1992 Dec 1;52(23):6690–6691. [PubMed] [Google Scholar]
  25. Robins P., Jones C. J., Biggerstaff M., Lindahl T., Wood R. D. Complementation of DNA repair in xeroderma pigmentosum group A cell extracts by a protein with affinity for damaged DNA. EMBO J. 1991 Dec;10(12):3913–3921. doi: 10.1002/j.1460-2075.1991.tb04961.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schaeffer L., Roy R., Humbert S., Moncollin V., Vermeulen W., Hoeijmakers J. H., Chambon P., Egly J. M. DNA repair helicase: a component of BTF2 (TFIIH) basic transcription factor. Science. 1993 Apr 2;260(5104):58–63. doi: 10.1126/science.8465201. [DOI] [PubMed] [Google Scholar]
  27. Scherly D., Nouspikel T., Corlet J., Ucla C., Bairoch A., Clarkson S. G. Complementation of the DNA repair defect in xeroderma pigmentosum group G cells by a human cDNA related to yeast RAD2. Nature. 1993 May 13;363(6425):182–185. doi: 10.1038/363182a0. [DOI] [PubMed] [Google Scholar]
  28. Shimamoto T., Kohno K., Tanaka K., Okada Y. Molecular cloning of human XPAC gene homologs from chicken, Xenopus laevis and Drosophila melanogaster. Biochem Biophys Res Commun. 1991 Dec 31;181(3):1231–1237. doi: 10.1016/0006-291x(91)92070-z. [DOI] [PubMed] [Google Scholar]
  29. Stefanini M., Giliani S., Nardo T., Marinoni S., Nazzaro V., Rizzo R., Trevisan G. DNA repair investigations in nine Italian patients affected by trichothiodystrophy. Mutat Res. 1992 Mar;273(2):119–125. doi: 10.1016/0921-8777(92)90073-c. [DOI] [PubMed] [Google Scholar]
  30. Stefanini M., Vermeulen W., Weeda G., Giliani S., Nardo T., Mezzina M., Sarasin A., Harper J. I., Arlett C. F., Hoeijmakers J. H. A new nucleotide-excision-repair gene associated with the disorder trichothiodystrophy. Am J Hum Genet. 1993 Oct;53(4):817–821. [PMC free article] [PubMed] [Google Scholar]
  31. Sung P., Reynolds P., Prakash L., Prakash S. Purification and characterization of the Saccharomyces cerevisiae RAD1/RAD10 endonuclease. J Biol Chem. 1993 Dec 15;268(35):26391–26399. [PubMed] [Google Scholar]
  32. Tanaka K., Miura N., Satokata I., Miyamoto I., Yoshida M. C., Satoh Y., Kondo S., Yasui A., Okayama H., Okada Y. Analysis of a human DNA excision repair gene involved in group A xeroderma pigmentosum and containing a zinc-finger domain. Nature. 1990 Nov 1;348(6296):73–76. doi: 10.1038/348073a0. [DOI] [PubMed] [Google Scholar]
  33. Thompson L. H., Brookman K. W., Weber C. A., Salazar E. P., Reardon J. T., Sancar A., Deng Z., Siciliano M. J. Molecular cloning of the human nucleotide-excision-repair gene ERCC4. Proc Natl Acad Sci U S A. 1994 Jul 19;91(15):6855–6859. doi: 10.1073/pnas.91.15.6855. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Tomkinson A. E., Bardwell A. J., Bardwell L., Tappe N. J., Friedberg E. C. Yeast DNA repair and recombination proteins Rad1 and Rad10 constitute a single-stranded-DNA endonuclease. Nature. 1993 Apr 29;362(6423):860–862. doi: 10.1038/362860a0. [DOI] [PubMed] [Google Scholar]
  35. Troelstra C., van Gool A., de Wit J., Vermeulen W., Bootsma D., Hoeijmakers J. H. ERCC6, a member of a subfamily of putative helicases, is involved in Cockayne's syndrome and preferential repair of active genes. Cell. 1992 Dec 11;71(6):939–953. doi: 10.1016/0092-8674(92)90390-x. [DOI] [PubMed] [Google Scholar]
  36. Weber C. A., Salazar E. P., Stewart S. A., Thompson L. H. ERCC2: cDNA cloning and molecular characterization of a human nucleotide excision repair gene with high homology to yeast RAD3. EMBO J. 1990 May;9(5):1437–1447. doi: 10.1002/j.1460-2075.1990.tb08260.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Weeda G., van Ham R. C., Vermeulen W., Bootsma D., van der Eb A. J., Hoeijmakers J. H. A presumed DNA helicase encoded by ERCC-3 is involved in the human repair disorders xeroderma pigmentosum and Cockayne's syndrome. Cell. 1990 Aug 24;62(4):777–791. doi: 10.1016/0092-8674(90)90122-u. [DOI] [PubMed] [Google Scholar]
  38. Wood R. D. DNA repair. Seven genes for three diseases. Nature. 1991 Mar 21;350(6315):190–190. doi: 10.1038/350190a0. [DOI] [PubMed] [Google Scholar]
  39. Wood R. D., Robins P., Lindahl T. Complementation of the xeroderma pigmentosum DNA repair defect in cell-free extracts. Cell. 1988 Apr 8;53(1):97–106. doi: 10.1016/0092-8674(88)90491-6. [DOI] [PubMed] [Google Scholar]
  40. van Duin M., de Wit J., Odijk H., Westerveld A., Yasui A., Koken M. H., Hoeijmakers J. H., Bootsma D. Molecular characterization of the human excision repair gene ERCC-1: cDNA cloning and amino acid homology with the yeast DNA repair gene RAD10. Cell. 1986 Mar 28;44(6):913–923. doi: 10.1016/0092-8674(86)90014-0. [DOI] [PubMed] [Google Scholar]
  41. van Vuuren A. J., Appeldoorn E., Odijk H., Yasui A., Jaspers N. G., Bootsma D., Hoeijmakers J. H. Evidence for a repair enzyme complex involving ERCC1 and complementing activities of ERCC4, ERCC11 and xeroderma pigmentosum group F. EMBO J. 1993 Sep;12(9):3693–3701. doi: 10.1002/j.1460-2075.1993.tb06044.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. van Vuuren A. J., Vermeulen W., Ma L., Weeda G., Appeldoorn E., Jaspers N. G., van der Eb A. J., Bootsma D., Hoeijmakers J. H., Humbert S. Correction of xeroderma pigmentosum repair defect by basal transcription factor BTF2 (TFIIH). EMBO J. 1994 Apr 1;13(7):1645–1653. doi: 10.1002/j.1460-2075.1994.tb06428.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis

RESOURCES