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