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Recognition of DNA damage by the Rad4 nucleotide excision repair protein

Nature volume 449, pages 570–575 (2007)Cite this article

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Abstract

Mutations in the nucleotide excision repair (NER) pathway can cause the xeroderma pigmentosum skin cancer predisposition syndrome. NER lesions are limited to one DNA strand, but otherwise they are chemically and structurally diverse, being caused by a wide variety of genotoxic chemicals and ultraviolet radiation. The xeroderma pigmentosum C (XPC) protein has a central role in initiating global-genome NER by recognizing the lesion and recruiting downstream factors. Here we present the crystal structure of the yeast XPC orthologue Rad4 bound to DNA containing a cyclobutane pyrimidine dimer (CPD) lesion. The structure shows that Rad4 inserts a β-hairpin through the DNA duplex, causing the two damaged base pairs to flip out of the double helix. The expelled nucleotides of the undamaged strand are recognized by Rad4, whereas the two CPD-linked nucleotides become disordered. These findings indicate that the lesions recognized by Rad4/XPC thermodynamically destabilize the Watson–Crick double helix in a manner that facilitates the flipping-out of two base pairs.

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Figure 1: Overall structures of the Rad4–Rad23–DNA complex.
Figure 2: Rad4 contains three structurally homologous β-hairpin domains.
Figure 3: Rad4 binds to damaged DNA in two parts.
Figure 4: Rad4 undergoes conformational changes on DNA-binding.

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References

  1. Cleaver, J. E. Cancer in xeroderma pigmentosum and related disorders of DNA repair. Nature Rev. Cancer 5, 564–573 (2005)

    Article  CAS  Google Scholar 

  2. Gillet, L. C. & Scharer, O. D. Molecular mechanisms of mammalian global genome nucleotide excision repair. Chem. Rev. 106, 253–276 (2006)

    Article  CAS  PubMed  Google Scholar 

  3. Legerski, R. & Peterson, C. Expression cloning of a human DNA repair gene involved in xeroderma pigmentosum group C. Nature 359, 70–73 (1992)

    Article  ADS  CAS  PubMed  Google Scholar 

  4. Masutani, C. et al. Purification and cloning of a nucleotide excision repair complex involving the xeroderma pigmentosum group C protein and a human homologue of yeast RAD23. EMBO J. 13, 1831–1843 (1994)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Riedl, T., Hanaoka, F. & Egly, J.-M. The comings and goings of nucleotide excision repair factors on damaged DNA. EMBO J. 22, 5293–5303 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Sugasawa, K. et al. Xeroderma pigmentosum group C protein complex is the initiator of global genome nucleotide excision repair. Mol. Cell 2, 223–232 (1998)

    Article  CAS  PubMed  Google Scholar 

  7. Batty, D., Rapic'-Otrin, V., Levine, A. S. & Wood, R. D. Stable binding of human XPC complex to irradiated DNA confers strong discrimination for damaged sites. J. Mol. Biol. 300, 275–290 (2000)

    Article  CAS  PubMed  Google Scholar 

  8. Kusumoto, R. et al. Diversity of the damage recognition step in the global genomic nucleotide excision repair in vitro . Mutat. Res. 485, 219–227 (2001)

    Article  CAS  PubMed  Google Scholar 

  9. Hey, T. et al. The XPC–HR23B complex displays high affinity and specificity for damaged DNA in a true-equilibrium fluorescence assay. Biochemistry 41, 6583–6587 (2002)

    Article  CAS  PubMed  Google Scholar 

  10. Volker, M. et al. Sequential assembly of the nucleotide excision repair factors in vivo . Mol. Cell 8, 213–224 (2001)

    Article  CAS  PubMed  Google Scholar 

  11. Dip, R., Camenisch, U. & Naegeli, H. Mechanisms of DNA damage recognition and strand discrimination in human nucleotide excision repair. DNA Repair 3, 1409–1423 (2004)

    Article  CAS  PubMed  Google Scholar 

  12. Fitch, M. E., Nakajima, S., Yasui, A. & Ford, J. M. In vivo recruitment of XPC to UV-induced cyclobutane pyrimidine dimers by the DDB2 gene product. J. Biol. Chem. 278, 46906–46910 (2003)

    Article  CAS  PubMed  Google Scholar 

  13. Madura, K. Rad23 and Rpn10: perennial wallflowers join the melee. Trends Biochem. Sci. 29, 637–640 (2004)

    Article  CAS  PubMed  Google Scholar 

  14. Masutani, C. et al. Identification and characterization of XPC-binding domain of hHR23B. Mol. Cell. Biol. 17, 6915–6923 (1997)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Ng, J. M. Y. et al. A novel regulation mechanism of DNA repair by damage-induced and RAD23-dependent stabilization of xeroderma pigmentosum group C protein. Genes Dev. 17, 1630–1645 (2003)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Ortolan, T. G., Chen, L., Tongaonkar, P. & Madura, K. Rad23 stabilizes Rad4 from degradation by the Ub/proteasome pathway. Nucleic Acids Res. 32, 6490–6500 (2004)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Cosman, M. et al. Solution conformation of the (-)-cis-anti-benzo[a]pyrenyl-dG adduct opposite dC in a DNA duplex: intercalation of the covalently attached BP ring into the helix with base displacement of the modified deoxyguanosine into the major groove. Biochemistry 35, 9850–9863 (1996)

    Article  CAS  PubMed  Google Scholar 

  18. Mao, B. et al. Solution structure of the (+)-cis-anti-benzo[a]pyrene-dA ([BP]dA) adduct opposite dT in a DNA duplex. Biochemistry 38, 10831–10842 (1999)

    Article  CAS  PubMed  Google Scholar 

  19. de los Santos, C. et al. Influence of benzo[a]pyrene diol epoxide chirality on solution conformations of DNA covalent adducts: the (-)-trans-anti-[BP]ĠC adduct structure and comparison with the (+)-trans-anti-[BP]ĠC enantiomer. Biochemistry 31, 5245–5252 (1992)

    Article  CAS  PubMed  Google Scholar 

  20. Geacintov, N. E. et al. Thermodynamic and structural factors in the removal of bulky DNA adducts by the nucleotide excision repair machinery. Biopolymers 65, 202–210 (2002)

    Article  CAS  PubMed  Google Scholar 

  21. O'Handley, S. F. et al. Structural characterization of an N-acetyl-2-aminofluorene (AAF) modified DNA oligomer by NMR, energy minimization, and molecular dynamics. Biochemistry 32, 2481–2497 (1993)

    Article  CAS  PubMed  Google Scholar 

  22. Kim, J. K. & Choi, B. S. The solution structure of DNA duplex-decamer containing the (6-4) photoproduct of thymidylyl(3'→5')thymidine by NMR and relaxation matrix refinement. Eur. J. Biochem. 228, 849–854 (1995)

    Article  CAS  PubMed  Google Scholar 

  23. McAteer, K., Jing, Y., Kao, J., Taylor, J. S. & Kennedy, M. A. Solution-state structure of a DNA dodecamer duplex containing a cis-syn thymine cyclobutane dimer, the major UV photoproduct of DNA. J. Mol. Biol. 282, 1013–1032 (1998)

    Article  CAS  PubMed  Google Scholar 

  24. Jing, Y., Kao, J. F. & Taylor, J. S. Thermodynamic and base-pairing studies of matched and mismatched DNA dodecamer duplexes containing cis-syn, (6-4) and Dewar photoproducts of TT. Nucleic Acids Res. 26, 3845–3853 (1998)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Gunz, D., Hess, M. T. & Naegeli, H. Recognition of DNA adducts by human nucleotide excision repair. Evidence for a thermodynamic probing mechanism. J. Biol. Chem. 271, 25089–25098 (1996)

    Article  CAS  PubMed  Google Scholar 

  26. Buterin, T. et al. Unrepaired fjord region polycyclic aromatic hydrocarbon–DNA adducts in ras codon 61 mutational hot spots. Cancer Res. 60, 1849–1856 (2000)

    CAS  PubMed  Google Scholar 

  27. Gao, Y. G., Robinson, H., Sanishvili, R., Joachimiak, A. & Wang, A. H. Structure and recognition of sheared tandem ĠA base pairs associated with human centromere DNA sequence at atomic resolution. Biochemistry 38, 16452–16460 (1999)

    Article  CAS  PubMed  Google Scholar 

  28. Chou, S. H. & Chin, K. H. Solution structure of a DNA double helix incorporating four consecutive non-Watson–Crick base-pairs. J. Mol. Biol. 312, 769–781 (2001)

    Article  CAS  PubMed  Google Scholar 

  29. Sugasawa, K. et al. A multistep damage recognition mechanism for global genomic nucleotide excision repair. Genes Dev. 15, 507–521 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Buterin, T., Meyer, C., Giese, B. & Naegeli, H. DNA quality control by conformational readout on the undamaged strand of the double helix. Chem. Biol. 12, 913–922 (2005)

    Article  CAS  PubMed  Google Scholar 

  31. Anantharaman, V., Koonin, E. V. & Aravind, L. Peptide-N-glycanases and DNA repair proteins, Xp-C/Rad4, are, respectively, active and inactivated enzymes sharing a common transglutaminase fold. Hum. Mol. Genet. 10, 1627–1630 (2001)

    Article  CAS  PubMed  Google Scholar 

  32. Ikegami, T. et al. Solution structure of the DNA- and RPA-binding domain of the human repair factor XPA. Nature Struct. Biol. 5, 701–706 (1998)

    Article  CAS  PubMed  Google Scholar 

  33. Buschta-Hedayat, N., Buterin, T., Hess, M. T., Missura, M. & Naegeli, H. Recognition of nonhybridizing base pairs during nucleotide excision repair of DNA. Proc. Natl Acad. Sci. USA 96, 6090–6095 (1999)

    Article  ADS  CAS  PubMed  PubMed Central  Google Scholar 

  34. Yang, Z. et al. Specific and efficient binding of xeroderma pigmentosum complementation group A to double-strand/single-strand DNA junctions with 3′- and/or 5′-ssDNA Branches. Biochemistry 45, 15921–15930 (2006)

    Article  CAS  PubMed  Google Scholar 

  35. Wang, M., Mahrenholz, A. & Lee, S. H. RPA stabilizes the XPA-damaged DNA complex through protein–protein interaction. Biochemistry 39, 6433–6439 (2000)

    Article  CAS  PubMed  Google Scholar 

  36. Buchko, G. W. et al. DNA–XPA interactions: a 31P NMR and molecular modeling study of dCCAATAACC association with the minimal DNA-binding domain (M98–F219) of the nucleotide excision repair protein XPA. Nucleic Acids Res. 29, 2635–2643 (2001)

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  37. Camenisch, U., Dip, R., Schumacher, S. B., Schuler, B. & Naegeli, H. Recognition of helical kinks by xeroderma pigmentosum group A protein triggers DNA excision repair. Nature Struct. Mol. Biol. 13, 278–284 (2006)

    Article  CAS  Google Scholar 

  38. Sugasawa, K., Shimizu, Y., Iwai, S. & Hanaoka, F. A molecular mechanism for DNA damage recognition by the xeroderma pigmentosum group C protein complex. DNA Repair 1, 95–107 (2002)

    Article  CAS  PubMed  Google Scholar 

  39. Cosman, M. et al. Solution conformation of the (+)-trans-anti-[BPh]dA adduct opposite dT in a DNA duplex: intercalation of the covalently attached benzo[c]phenanthrene to the 5′-side of the adduct site without disruption of the modified base pair. Biochemistry 32, 12488–12497 (1993)

    Article  CAS  PubMed  Google Scholar 

  40. Cosman, M. et al. Solution conformation of the (-)-trans-anti-benzo[c]phenanthrene-dA ([BPh]dA) adduct opposite dT in a DNA duplex: intercalation of the covalently attached benzo[c]phenanthrenyl ring to the 3′-side of the adduct site and comparison with the (+)-trans-anti-[BPh]dA opposite dT stereoisomer. Biochemistry 34, 1295–1307 (1995)

    Article  CAS  PubMed  Google Scholar 

  41. Huffman, J. L., Sundheim, O. & Tainer, J. A. DNA base damage recognition and removal: new twists and grooves. Mutat. Res. 577, 55–76 (2005)

    Article  CAS  PubMed  Google Scholar 

  42. Cheng, X. & Blumenthal, R. M. Finding a basis for flipping bases. Structure 4, 639–645 (1996)

    Article  CAS  PubMed  Google Scholar 

  43. DeLano, W. L. The PyMOL Molecular Graphics System 〈http://www.pymol.org〉 (2002)

  44. Yang, A. et al. Flexibility and plasticity of human centrin 2 binding to the xeroderma pigmentosum group C protein (XPC) from nuclear excision repair. Biochemistry 45, 3653–3663 (2006)

    Article  CAS  PubMed  Google Scholar 

  45. Thompson, J. R., Ryan, Z. C., Salisbury, J. L. & Kumar, R. The structure of the human centrin 2-xeroderma pigmentosum group C protein complex. J. Biol. Chem. 281, 18746–18752 (2006)

    Article  CAS  PubMed  Google Scholar 

  46. Otwinowski, Z. & Minor, W. Processing of x-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326 (1997)

    Article  CAS  PubMed  Google Scholar 

  47. de La Fortelle, E. & Bricogne, G. Maximum-likelihood heavy-atom parameter refinement for multiple isomorphous replacement and multiwavelength anomalous diffraction methods. Methods Enzymol. 276, 472–494 (1997)

    Article  CAS  PubMed  Google Scholar 

  48. Jones, T. A., Zou, J. Y., Cowan, S. W. & Kjeldgaard, M. Improved methods for binding protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47, 110–119 (1991)

    Article  PubMed  Google Scholar 

  49. Brunger, A. T. et al. Crystallography and NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    Article  CAS  PubMed  Google Scholar 

  50. The CCP4 suite: programs for protein crystallography. Acta Crystallogr. D 50, 760–763 (1994)

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Acknowledgements

We thank D. King for mass spectroscopic analysis; H. Erdjument-Bromage for N-terminal sequencing; the staff of the Advanced Photon Source ID-24 and 8-BM beamlines for help with data collection; M. Minto for administrative assistance; and Y. Goldgur, A. Wong, A. Smalls-Mantey, A. Rozenbaum and the members of the Pavletich laboratory for help and discussions. This work was supported by the NIH and the Howard Hughes Medical Institute. J.-H.M. was supported by the Leukemia & Lymphoma Society as a Special Fellow.

Coordinates and structure factors of the Rad4–Rad23 and Rad4–Rad23–DNA complexes have been deposited in the Protein Data Bank under accession code 2QSF (Rad4–Rad23), 2QSG (Rad4–Rad23 bound to CPD-mismatch DNA) and 2QSH (Rad4–Rad23 bound to mismatch-only DNA).

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    Jung-Hyun Min & Nikola P. Pavletich

  2. Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021, USA ,

    Nikola P. Pavletich

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Correspondence to Nikola P. Pavletich.

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Supplementary Information

This file contains Supplementary Tables 1–2, Supplementary Figures 1–7 with Legends, and Supplementary Discussion. Supplementary Table 1 contains crystallographic statistics. Supplementary Table 2 contains DNA sequences used for crystallization and EMSA. Supplementary Figures and Discussion contain detailed characterization/description of the Rad4–Rad23 complex and its apo- and DNA-bound crystal structures presented in the paper. (PDF 2140 kb)

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Min, JH., Pavletich, N. Recognition of DNA damage by the Rad4 nucleotide excision repair protein. Nature 449, 570–575 (2007). https://doi.org/10.1038/nature06155

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