Analysis of Actively Transcribed DNA Repair Using a Transfection-Based System

Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1105)

Abstract

Host cell reactivation (HCR) is a transfection-based assay in which intact cells repair damage localized to exogenous DNA. This chapter provides instructions for the application of this technique, using as an exemplar UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a successfully transcribed and translated reporter enzyme, the amount of damaged plasmid that a cell can “reactivate” or repair and express can be quantitated. Different DNA repair pathways can be analyzed by this technique by damaging the reporter plasmid in different ways. Since it involves repair of a transcriptionally active gene, when applied to UV damage the HCR assay measures the capacity of the host cells to perform transcription-coupled repair, a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences.

Key words

DNA damage Host cell reactivation (HCR) Transcription-coupled repair (TCR) Global genomic repair (GGR) Nucleotide excision repair (NER) Transfection Luciferase UV irradiation Thymine dimers 6-4 photoproducts 

References

  1. 1.
    Rupert C, Harm W (1966) Reactivation after photobiological damage. Adv Radiat Biol 2:1–81CrossRefGoogle Scholar
  2. 2.
    Protic-Sabljic M, Kraemer KH (1985) One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. Proc Natl Acad Sci U S A 82:6622–6626PubMedCentralPubMedCrossRefGoogle Scholar
  3. 3.
    Athas WF, Hedayati MA, Matanoski GM, Farmer ER, Grossman L (1991) Development and field-test validation of an assay for DNA repair in circulating human lymphocytes. Cancer Res 51:5786–5793PubMedGoogle Scholar
  4. 4.
    Bohr VA, Smith CA, Okumoto DS, Hanawalt PC (1985) DNA repair in an active gene: removal of pyrimidine dimers from the DHFR gene of CHO cells is much more efficient than in the genome overall. Cell 40:359–369PubMedCrossRefGoogle Scholar
  5. 5.
    Cleaver JE, Lam ET, Revet I (2009) Disorders of nucleotide excision repair: the genetic and molecular basis of heterogeneity. Nat Rev Genet 10:756–768PubMedCrossRefGoogle Scholar
  6. 6.
    Matijasevic Z, Precopio ML, Snyder JE, Ludlum DB (2001) Repair of sulfur mustard-induced DNA damage in mammalian cells measured by a host cell reactivation assay. Carcinogenesis 22:661–664PubMedCrossRefGoogle Scholar
  7. 7.
    Berwick M, Veneis P (2000) Markers of DNA repair and susceptibility to cancer in humans: an epidemiologic review. J Natl Cancer Inst 92:847–897CrossRefGoogle Scholar
  8. 8.
    Invitrogen Life Technologies Lipofectamine 2000 CD Reagent, pp 1–2. Available at http://www.invitrogen.com
  9. 9.
    Promega Luciferase Assay System Instructions. Technical Bulletin No. 281, pp 1–13. Available at http://www.promega.com
  10. 10.
    BCA Protein Assay Reagent Kit 23227 Instructions, pp 1–8. Available at http://www.piercenet.com
  11. 11.
    Rainbow A (1975) Host-cell reactivation of irradiated human adenovirus. Basic Life Sci 5B:753–754PubMedGoogle Scholar
  12. 12.
    Slebos RJ, Taylor JA (2001) A novel host cell reactivation assay to assess homologous recombination capacity in human cancer cell lines. Biochem Biophys Res Commun 281:212–219PubMedCrossRefGoogle Scholar
  13. 13.
    Hansson J, Wood RD (1989) Repair synthesis by human cell extracts in DNA damaged by cis- and trans-diamminedichloroplatinum(II). Nucleic Acids Res 17:8073–8091PubMedCentralPubMedCrossRefGoogle Scholar
  14. 14.
    Yen L, Woo A, Christopoulopoulos G et al (1995) Enhanced host cell reactivation capacity and expression of DNA repair genes in human breast cancer cells resistant to bi-functional alkylating agents. Mutat Res 337:179–189PubMedCrossRefGoogle Scholar
  15. 15.
    Dean SW, Sykes HR, Lehmann AR (1988) Inactivation by nitrogen mustard of plasmids introduced into normal and Fanconi’s anaemia cells. Mutat Res 194:57–63PubMedGoogle Scholar
  16. 16.
    Sun Y, Moses RE (1991) Reactivation of psoralen-reacted plasmid in Fanconi anemia, xeroderma pigmentosum, and normal human fibroblast cells. Somat Cell Mol Genet 17:229–238PubMedCrossRefGoogle Scholar
  17. 17.
    Stevnsner T, Frandsen H, Autrup H (1995) Repair of DNA lesions induced by ultraviolet irradiation and aromatic amines in normal and repair-deficient human lymphoblastoid cell lines. Carcinogenesis 16:2855–2858PubMedCrossRefGoogle Scholar
  18. 18.
    Tanooka H, Tada M (1975) Reparable lethal DNA damage produced by enzyme-activated 4-hydroxyaminoquinoline 1-oxide. Chem Biol Interact 10:11–18PubMedCrossRefGoogle Scholar
  19. 19.
    Cheng L, Eicher SA, Guo Z, Hong WK, Spitz MR, Wei Q (1998) Reduced DNA repair capacity in head and neck cancer patients. Cancer Epidemiol Biomarkers Prev 7:465–468PubMedGoogle Scholar
  20. 20.
    Kuraoka I, Bender C, Romieu A, Cadet J, Wood RD, Lindahl T (2000) Removal of oxygen free-radical-induced 5′,8-purine cyclodeoxynucleosides from DNA by the nucleotide excision repair pathway in human cells. Proc Natl Acad Sci U S A 97:3832–3837PubMedCentralPubMedCrossRefGoogle Scholar
  21. 21.
    Iakoucheva LM, Walker RK, van Houten B, Ackerman EJ (2002) Equilibrium and stop-slow kinetic studies of fluorescently labeled DNA substrates with DNA repair proteins XPA and replication protein A. Biochemistry 41:131–143PubMedCrossRefGoogle Scholar
  22. 22.
    Day RS III, Ziolkowski CH (1979) Human brain tumour cell strains with deficient host-cell reactivation of N-methyl-N′-nitro-N-nitrosoguanidine-damaged adenovirus 5. Nature 279:797–799PubMedCrossRefGoogle Scholar
  23. 23.
    Maynard K, Parsons PG, Cerny T, Margison GP (1989) Relationships among cell survival, O6-alkylguanine-DNA alkyltransferase activity, and reactivation of methylated adenovirus 5 and herpes simplex virus type 1 in human melanoma cell lines. Cancer Res 49:4813–4817PubMedGoogle Scholar
  24. 24.
    L’Herault P, Chung YS (1982) Host cell reactivation of ozone-treated T3 bacteriophage by different strains of Escherichia coli. Experientia 38:1491–1492PubMedCrossRefGoogle Scholar
  25. 25.
    Diem C, Runger TM (1997) Processing of three different types of DNA damage in cell lines of a cutaneous squamous cell carcinoma progression model. Carcinogenesis 18:657–662PubMedCrossRefGoogle Scholar
  26. 26.
    Protic-Sabljic M, Kraemer KH (1986) Host cell reactivation by human cells of DNA expression vectors damaged by ultraviolet radiation or by acid/heat treatment. Carcinogenesis 7:1765–1770PubMedCrossRefGoogle Scholar
  27. 27.
    Matsumoto Y (1999) Base excision repair assay using Xenopus laevis oocyte extracts. In: Henderson DS (ed) DNA repair protocols: eukaryotic systems, vol 113, Methods in molecular biology. Humana, Totowa, NJ, pp 289–300CrossRefGoogle Scholar
  28. 28.
    Runger TM, Emmert S, Schadendorf D, Diem C, Epe B, Hellfritsch D (2000) Alterations of DNA repair in melanoma cell lines resistant to cisplatin, fotemustine, or etoposide. J Invest Dermatol 114:34–39PubMedCrossRefGoogle Scholar
  29. 29.
    Perlow RA, Schinecker TM, Kim SJ, Geacintov NE, Scicchitano DA (2003) Construction and purification of site-specifically modified DNA templates for transcription assays. Nucleic Acids Res 31:e40PubMedCentralPubMedCrossRefGoogle Scholar
  30. 30.
    Latimer JJ, Hultner ML, Cleaver JE, Pedersen RA (1996) Elevated DNA excision repair capacity in the extraembryonic mesoderm of the mid-gestation mouse embryo. Exp Cell Res 228:19–28PubMedCrossRefGoogle Scholar
  31. 31.
    Cheng L, Guan Y, Li L et al (1999) Expression in normal human tissues of five nucleotide excision repair genes measured simultaneously by multiplex reverse transcription-polymerase chain reaction. Cancer Epidemiol Biomarkers Prev 8:801–807PubMedGoogle Scholar
  32. 32.
    Latimer JJ, Johnson JM, Miles TD et al (2008) Cell-type-specific level of DNA nucleotide excision repair in primary human mammary and ovarian epithelial cell cultures. Cell Tissue Res 333:461–467PubMedCrossRefGoogle Scholar
  33. 33.
    Ford JM, Baron EL, Hanawalt PC (1998) Human fibroblasts expressing the human papillomavirus E6 gene are deficient in global genomic nucleotide excision repair and sensitive to ultraviolet irradiation. Cancer Res 58:599–603PubMedGoogle Scholar
  34. 34.
    Bowman KK, Sicard DM, Ford JM, Hanawalt PC (2000) Reduced global genomic repair of ultraviolet light-induced cyclobutane pyrimidine dimers in simian virus 40-transformed human cells. Mol Carcinog 29:17–24PubMedCrossRefGoogle Scholar
  35. 35.
    Fututa T, Ueda T, Aune G, Sarasin A, Kraemer KH, Pommier Y (2002) Transcription-coupled nucleotide excision repair as a determinant of cisplatin sensitivity of human cells. Cancer Res 65:4899–4902Google Scholar
  36. 36.
    Steier H, Cleaver JE (1969) Exposure chamber for quantitative ultraviolet photobiology. Lab Pract 18:1295PubMedGoogle Scholar
  37. 37.
    Promega Transfection Guide, pp 1–56. Available at http://www.promega.com

Copyright information

© Humana Press 2014

Authors and Affiliations

  1. 1.Department of Pharmaceutical SciencesNova Southeastern UniversityFort Lauderdale-DavieUSA

Personalised recommendations