Skip to main content
SpringerLink
Log in

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

  • Protocol
  • First Online:
Molecular Toxicology Protocols

Part of the book series: Methods in Molecular Biology ((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.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Log in via an institution
Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 129.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever
Softcover Book
USD 169.99
Price excludes VAT (USA)
  • Compact, lightweight edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info
Hardcover Book
USD 249.99
Price excludes VAT (USA)
  • Durable hardcover edition
  • Dispatched in 3 to 5 business days
  • Free shipping worldwide - see info

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Rupert CS, Harm W (1966) Reactivation after photobiological damage. In: Advances in radiation biology, vol 2. Elsevier, Amsterdam, pp 1–81

    Google Scholar 

  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):6622–6626

    Article  PubMed  PubMed Central  Google Scholar 

  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):5786–5793

    CAS  PubMed  Google Scholar 

  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):181–190

    Article  CAS  PubMed  Google Scholar 

  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)00219-1

    Article  CAS  PubMed  Google Scholar 

  6. Roguev A, Russev G (2000) Two-wavelength fluorescence assay for DNA repair. Anal Biochem 287(2):313–318

    Article  CAS  PubMed  Google Scholar 

  7. Steier H, Cleaver JE (1969) Exposure chamber for quantitative ultraviolet photobiology. Lab Pract 18(12):1295

    CAS  PubMed  Google Scholar 

  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):1430–1436

    Article  PubMed  Google Scholar 

  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):756

    Article  CAS  PubMed  Google Scholar 

  10. O’Driscoll M (2012) Diseases associated with defective responses to DNA damage. Cold Spring Harb Perspect Biol 4(12):a012773

    PubMed  PubMed Central  Google Scholar 

  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):661–664

    Article  CAS  PubMed  Google Scholar 

  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):797

    Article  CAS  PubMed  Google Scholar 

  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):874–897

    Article  CAS  PubMed  Google Scholar 

  14. Invitrogen Life Technologies Lipofectamine 2000 CD Reagent, pp. 1–2. http://www.invitrogen.com. (n.d.)

  15. Promega Corporation. (2015). Technical manual Dual-Luciferase® Reporter Assay System. Retrieved September 4, 2019. https://www.promega.com/resources/protocols/

  16. BCA Protein Assay Reagent Kit 23227 Instructions, pp. 1–8. http://www.piercenet.com. (n.d.)

  17. Rainbow A (1975). Host-cell reactivation of irradiated human adenovirus. In Molecular mechanisms for repair of DNA. Part B

    Chapter  Google Scholar 

  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):212–219

    Article  CAS  PubMed  Google Scholar 

  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):580–588

    Article  CAS  PubMed  Google Scholar 

  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):179–189

    Article  CAS  PubMed  Google Scholar 

  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):8073–8091

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Ratanaphan A, Canyuk B (2014) Host cell reactivation and transcriptional activation of carboplatin-modified BRCA1. Breast Cancer 8:BCBCR-S14224

    Article  Google Scholar 

  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):1311–1316

    CAS  PubMed  Google Scholar 

  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.2003.10.017

    Article  CAS  PubMed  Google Scholar 

  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):2855–2858

    Article  CAS  PubMed  Google Scholar 

  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):11–18

    Article  Google Scholar 

  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):764–774

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):465–468

    CAS  PubMed  Google Scholar 

  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):3832–3837

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):131–143

    Article  CAS  PubMed  Google Scholar 

  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–E1832

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):1004–1009

    Article  CAS  PubMed  Google Scholar 

  33. Kassam SN, Rainbow AJ (2008) UV-inducible base excision repair of oxidative damaged DNA in human cells. Mutagenesis 24(1):75–83

    Article  PubMed  Google Scholar 

  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 68:25–33

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):363–367

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Kim Y-J, Wilson M III (2012) Overview of base excision repair biochemistry. Curr Mol Pharmacol 5(1):3–13

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):1765–1770

    Article  Google Scholar 

  38. Matsumoto Y (1999) Base excision repair assay using Xenopus laevis oocyte extracts. In: DNA repair protocols. Springer, Berlin, pp 289–300

    Google Scholar 

  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):34–39

    Article  PubMed  Google Scholar 

  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):e40

    Article  PubMed  PubMed Central  Google Scholar 

  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):461–467

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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):19–28

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  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. 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):17–24

    Article  CAS  PubMed  Google Scholar 

  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):599–603

    CAS  PubMed  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Nova Southeastern University and AutoNation Breast Cancer Institute, Fort Lauderdale, FL, USA

    Jowaher S. Alanazi & Jean J. Latimer

Authors
  1. Jowaher S. Alanazi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jean J. Latimer
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Jean J. Latimer .

Editor information

Editors and Affiliations

  1. Department of Environmental and Occupational Health, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA

    Phouthone Keohavong

  2. Department of Environmental Toxicology, The Institute of Environmental and Human Health (TIEHH), Texas Tech University, Lubbock, TX, USA

    Kamaleshwar P. Singh

  3. Department of Occupational and Environmental Health Sciences, School of Public Health West Virginia University, Morgantown, WV, USA

    Weimin Gao

Rights and permissions

Reprints and permissions

Copyright information

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

About this protocol

Check for updates. Verify currency and authenticity via CrossMark

Cite this protocol

Alanazi, J.S., Latimer, J.J. (2020). Host Cell Reactivation: Assay for Actively Transcribed DNA (Nucleotide Excision) Repair Using Luciferase Family Expression Vectors. In: Keohavong, P., Singh, K., Gao, W. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 2102. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0223-2_28

Download citation

  • DOI: https://doi.org/10.1007/978-1-0716-0223-2_28

  • Published:

  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-0222-5

  • Online ISBN: 978-1-0716-0223-2

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics

Search

Navigation