Molecular Toxicology Protocols

Volume 291 of the series Methods in Molecular Biology™ pp 321-335

Analysis of DNA Repair Using Transfection-Based Host Cell Reactivation

  • Jennifer M. JohnsonAffiliated withDepartment of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine
  • , Jean J. LatimerAffiliated withDepartment of Obstetrics, Gynecology and Reproductive Sciences, University of Pittsburgh School of Medicine

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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 UV irradiation as a source of damage to a luciferase reporter plasmid. Through measurement of the activity of a 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. Because 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.

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