Title

Analysis of actively transcribed DNA repair using a transfection-based system

ISBN or ISSN

1064-3745

Publication Title

Methods in molecular biology

Volume

1105

Publication Date / Copyright Date

1-1-2014

First Page

533

Last Page

550

Publisher

Humana Press

DOI Number

10.1007/978-1-62703-739-6_37

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.

Disciplines

Medicine and Health Sciences | Pharmacy and Pharmaceutical Sciences

Keywords

Animals, Bacteriophages, Cell Line, DNA Repair, Genes, Reporter, Humans, Luciferases, Firefly, Plasmids, Transfection, Ultraviolet Rays