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.
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
- 1.Rupert, C. and Harm, W. (1966) Reactivation after photobiological damage. Adv. Radiat. Biol. 2, 1–81.Google Scholar
- 7.Invitrogen Life Technologies Lipofectamine 2000 CD Reagent, pp. 1–2; available at http://www.invitrogen.com.
- 8.Promega Luciferase Assay System Instructions, Technical Bulletin No. 281, pp. 1–13; available at http://www.promega.com.
- 9.BCA Protein Assay Reagent Kit 23227 Instructions, pp. 1–8; available at http://www.piercenet.com.
- 10.Miller, J. H. (1972) Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, pp. 352–355.Google Scholar
- 24.Maynard, K., Parsons, P. G., Cerny, T., and Margison, G. P. (1989) Relationships among cell survival, O6-alkylguanine-DNA alkyltransferase activity, and reactivation of methylated adenovirus 5 and herpes simplex virus type 1 in human melanoma cell lines. Cancer Res. 49, 4813–4817.PubMedGoogle Scholar
- 36.Fututa, T., Ueda, T., Aune, G., Sarasin, A., Kraemer, K. H., and Pommier, Y. (2002) Transcription-coupled nucleotide excision repair as a determinant of cisplatin sensitivity of human cells. Cancer Res. 65, 4899–4902.Google Scholar
- 37.Steier, H. and Cleaver, J. E. (1969) Exposure chamber for quantitative ultraviolet photo-biology. Lab Prac. 18, 1295.Google Scholar
- 38.Promega Transfection Guide, pp. 1–56; available at http://www.promega.com.