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Host Cell Reactivation: Assay for Actively Transcribed DNA (Nucleotide Excision) Repair Using Luciferase Family Expression Vectors

  • Jowaher S. Alanazi
  • Jean J. LatimerEmail author
Protocol
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Part of the Methods in Molecular Biology book series (MIMB, volume 2102)

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 (TCR), a subset of the overall nucleotide excision repair pathway that specifically targets transcribed gene sequences. This method features two ways to perform the assay using expression vectors with luciferase and beta galactosidase, as well as with firefly luciferase and Renilla luciferase using the same luminometer.

Key words

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

References

  1. 1.
    Rupert CS, Harm W (1966) Reactivation after photobiological damage. In: Advances in radiation biology, vol 2. Elsevier, Amsterdam, pp 1–81Google Scholar
  2. 2.
    Protić-Sabljić M, Kraemer KH (1985) One pyrimidine dimer inactivates expression of a transfected gene in xeroderma pigmentosum cells. Proc Natl Acad Sci 82(19):6622–6626PubMedCrossRefGoogle 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(21):5786–5793PubMedGoogle Scholar
  4. 4.
    Burger K, Kieser N, Gallinat S, Mielke H, Knott S, Bergemann J (2007) The influence of folic acid depletion on the nucleotide excision repair capacity of human dermal fibroblasts measured by a modified host cell reactivation assay. Biofactors 31(3, 4):181–190PubMedCrossRefGoogle Scholar
  5. 5.
    Qiao Y, Spitz MR, Guo Z, Hadeyati M, Grossman L, Kraemer KH, Wei Q (2002) Rapid assessment of repair of ultraviolet DNA damage with a modified host-cell reactivation assay using a luciferase reporter gene and correlation with polymorphisms of DNA repair genes in normal human lymphocytes. Mutat Res 509(1):165–174.  https://doi.org/10.1016/S0027-5107(02)00219-1PubMedCrossRefGoogle Scholar
  6. 6.
    Roguev A, Russev G (2000) Two-wavelength fluorescence assay for DNA repair. Anal Biochem 287(2):313–318PubMedCrossRefGoogle Scholar
  7. 7.
    Steier H, Cleaver JE (1969) Exposure chamber for quantitative ultraviolet photobiology. Lab Pract 18(12):1295PubMedGoogle Scholar
  8. 8.
    Wang L, Wei Q, Shi Q, Guo Z, Qiao Y, Spitz MR (2007) A modified host-cell reactivation assay to measure repair of alkylating DNA damage for assessing risk of lung adenocarcinoma. Carcinogenesis 28(7):1430–1436PubMedCrossRefGoogle Scholar
  9. 9.
    Cleaver JE, Lam ET, Revet I (2009) Disorders of nucleotide excision repair: the genetic and molecular basis of heterogeneity. Nat Rev Genet 10(11):756PubMedCrossRefGoogle Scholar
  10. 10.
    O’Driscoll M (2012) Diseases associated with defective responses to DNA damage. Cold Spring Harb Perspect Biol 4(12):a012773PubMedPubMedCentralGoogle Scholar
  11. 11.
    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(4):661–664PubMedCrossRefGoogle Scholar
  12. 12.
    Day RS III, Ziolkowski CHJ (1979) Human brain tumour cell strains with deficient host-cell reactivation of N-methyl-N′-nitro-N-nitrosoguanidine-damaged adenovirus 5. Nature 279(5716):797PubMedCrossRefGoogle Scholar
  13. 13.
    Berwick M, Vineis P (2000) Markers of DNA repair and susceptibility to cancer in humans: an epidemiologic review. J Natl Cancer Inst 92(11):874–897PubMedCrossRefGoogle Scholar
  14. 14.
    Invitrogen Life Technologies Lipofectamine 2000 CD Reagent, pp. 1–2. http://www.invitrogen.com. (n.d.)
  15. 15.
    Promega Corporation. (2015). Technical manual Dual-Luciferase® Reporter Assay System. Retrieved September 4, 2019. https://www.promega.com/resources/protocols/
  16. 16.
    BCA Protein Assay Reagent Kit 23227 Instructions, pp. 1–8. http://www.piercenet.com. (n.d.)
  17. 17.
    Rainbow A (1975). Host-cell reactivation of irradiated human adenovirus. In Molecular mechanisms for repair of DNA. Part BCrossRefGoogle Scholar
  18. 18.
    Slebos RJC, Taylor JA (2001) A novel host cell reactivation assay to assess homologous recombination capacity in human cancer cell lines. Biochem Biophys Res Commun 281(1):212–219PubMedCrossRefGoogle Scholar
  19. 19.
    Thoms K, Baesecke J, Emmert B, Hermann J, Roedling T, Laspe P et al (2007) Functional DNA repair system analysis in haematopoietic progenitor cells using host cell reactivation. Scand J Clin Lab Invest 67(6):580–588PubMedCrossRefGoogle Scholar
  20. 20.
    Yen L, Woo A, Christopoulopoulos G, Batist G, Panasci L, Roy R 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(3):179–189PubMedCrossRefGoogle Scholar
  21. 21.
    Hansson J, Wood RD (1989) Repair synthesis by human cell extracts in DNA damaged by cis-and trans-diamminedichloroplatinum (II). Nucleic Acids Res 17(20):8073–8091PubMedPubMedCentralCrossRefGoogle Scholar
  22. 22.
    Ratanaphan A, Canyuk B (2014) Host cell reactivation and transcriptional activation of carboplatin-modified BRCA1. Breast Cancer 8:BCBCR-S14224CrossRefGoogle Scholar
  23. 23.
    Selvakumaran M, Pisarcik DA, Bao R, Yeung AT, Hamilton TC (2003) Enhanced cisplatin cytotoxicity by disturbing the nucleotide excision repair pathway in ovarian cancer cell lines. Cancer Res 63(6):1311–1316PubMedGoogle Scholar
  24. 24.
    Costa RMA, Chiganças V, da Silva Galhardo R, Carvalho H, Menck CFM (2003) The eukaryotic nucleotide excision repair pathway. Biochimie 85(11):1083–1099.  https://doi.org/10.1016/j.biochi.2003.10.017PubMedCrossRefGoogle Scholar
  25. 25.
    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(11):2855–2858PubMedCrossRefGoogle Scholar
  26. 26.
    Hiroshi T, Mitsuhiko T, Mariko T (1975) Reparable lethal DNA damage produced by enzyme-activated 4-hydroxyaminoquinoline 1-oxide. Chem Biol Interact 10(1):11–18CrossRefGoogle Scholar
  27. 27.
    Wang L-E, Hu Z, Sturgis EM, Spitz MR, Strom SS, Amos CI et al (2010) Reduced DNA repair capacity for removing tobacco carcinogen–induced DNA adducts contributes to risk of head and neck cancer but not tumor characteristics. Clin Cancer Res 16(2):764–774PubMedPubMedCentralCrossRefGoogle Scholar
  28. 28.
    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(6):465–468PubMedGoogle Scholar
  29. 29.
    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 97(8):3832–3837PubMedCrossRefGoogle Scholar
  30. 30.
    Iakoucheva LM, Walker RK, van Houten B, Ackerman EJ (2002) Equilibrium and stop-flow kinetic studies of fluorescently labeled DNA substrates with DNA repair proteins XPA and replication protein a. Biochemistry 41(1):131–143PubMedCrossRefGoogle Scholar
  31. 31.
    Nagel ZD, Margulies CM, Chaim IA, McRee SK, Mazzucato P, Ahmad A et al (2014) Multiplexed DNA repair assays for multiple lesions and multiple doses via transcription inhibition and transcriptional mutagenesis. Proc Natl Acad Sci 111(18):E1823–E1832PubMedCrossRefGoogle Scholar
  32. 32.
    Kassam SN, Rainbow AJ (2007) Deficient base excision repair of oxidative DNA damage induced by methylene blue plus visible light in xeroderma pigmentosum group C fibroblasts. Biochem Biophys Res Commun 359(4):1004–1009PubMedCrossRefGoogle Scholar
  33. 33.
    Kassam SN, Rainbow AJ (2008) UV-inducible base excision repair of oxidative damaged DNA in human cells. Mutagenesis 24(1):75–83PubMedCrossRefGoogle Scholar
  34. 34.
    Parrish MC, Chaim IA, Nagel ZD, Tannenbaum SR, Samson LD, Engelward BP (2018) Nitric oxide induced S-nitrosation causes base excision repair imbalance. DNA Repair 68:25–33PubMedPubMedCentralCrossRefGoogle Scholar
  35. 35.
    Sattler U, Frit P, Salles B, Calsou P (2003) Long-patch DNA repair synthesis during base excision repair in mammalian cells. EMBO Rep 4(4):363–367PubMedPubMedCentralCrossRefGoogle Scholar
  36. 36.
    Kim Y-J, Wilson M III (2012) Overview of base excision repair biochemistry. Curr Mol Pharmacol 5(1):3–13PubMedPubMedCentralCrossRefGoogle Scholar
  37. 37.
    ProticĆ-SabyiĆ 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(10):1765–1770CrossRefGoogle Scholar
  38. 38.
    Matsumoto Y (1999) Base excision repair assay using Xenopus laevis oocyte extracts. In: DNA repair protocols. Springer, Berlin, pp 289–300Google Scholar
  39. 39.
    Rünger 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 Investig Dermatol 114(1):34–39PubMedCrossRefGoogle Scholar
  40. 40.
    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(7):e40PubMedPubMedCentralCrossRefGoogle Scholar
  41. 41.
    Latimer JJ, Johnson JM, Miles TD, Dimsdale JM, Edwards RP, Kelley JL, Grant SG (2008) Cell-type-specific level of DNA nucleotide excision repair in primary human mammary and ovarian epithelial cell cultures. Cell Tissue Res 333(3):461–467PubMedPubMedCentralCrossRefGoogle Scholar
  42. 42.
    Latimer JJ, Hultner ML, Cleaver JE, Pedersen RA (1996) Elevated DNA excision repair capacity in the extraembryonic mesoderm of the midgestation mouse embryo. Exp Cell Res 228(1):19–28PubMedPubMedCentralCrossRefGoogle Scholar
  43. 43.
    Latimer JJ, Majekwana VJ, Pab퐲n-PadÚn YR, Pimpley MR, Grant SG (2015) Regulation and disregulation of mammalian nucleotide excision repair: a pathway to nongermline breast carcinogenesis. Photochem Photobiol 91:493–500 (published online 11-13-14).  https://doi.org/10.1111/php.123.
  44. 44.
    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(1):17–24PubMedCrossRefGoogle Scholar
  45. 45.
    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(4):599–603Google Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2020

Authors and Affiliations

  1. 1.Department of Pharmaceutical SciencesNova Southeastern University and AutoNation Breast Cancer InstituteFort LauderdaleUSA

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