Analysis of DNA repair using transfection-based host cell reactivation.
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.
Johnson
Jennifer M
JM
Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, PA, USA.
Latimer
Jean J
JJ
D000818:Animals; D020137:Base Pair Mismatch / Q000235:genetics; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry; D004249:DNA Damage / Q000235:genetics; D004260:DNA Repair; D017930:Genes, Reporter / Q000235:genetics*; D006801:Humans; D008156:Luciferases / Q000032:analysis / Q000235:genetics*; D010957:Plasmids / Q000235:genetics*; D011740:Pyrimidine Dimers / Q000032:analysis; D014162:Transfection
D011740:Pyrimidine Dimers; D004247:DNA; D008156:Luciferases