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

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Molecular Toxicology Protocols

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

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Author information

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, Nova Southeastern University, 3301 College Avenue, Fort Lauderdale-Davie, FL, 33314-7796, USA

    Jean J. Latimer

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  1. Jean J. Latimer
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Correspondence to Jean J. Latimer .

Editor information

Editors and Affiliations

  1. Occupational Health, University of Pittsburgh Dept. Environmental &, Pittsburgh, Pennsylvania, USA

    Phouthone Keohavong

  2. Dept. of Pharmaceutical Sciences, Nova Southeastern University College of Pharmacy, Fort Lauderdale, Florida, USA

    Stephen G. Grant

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© 2014 Humana Press

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Latimer, J.J. (2014). Analysis of Actively Transcribed DNA Repair Using a Transfection-Based System. In: Keohavong, P., Grant, S. (eds) Molecular Toxicology Protocols. Methods in Molecular Biology, vol 1105. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-739-6_37

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  • DOI: https://doi.org/10.1007/978-1-62703-739-6_37

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-62703-738-9

  • Online ISBN: 978-1-62703-739-6

  • eBook Packages: Springer Protocols

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