A rapid assay for measuring nucleotide excision repair by oligonucleotide retrieval.

ABSTRACT

Nucleotide excision repair (NER) excises bulky DNA lesions induced by mutagens and carcinogens. The repair process includes recognition of DNA damage, excision of a short patch of nucleotides containing the damaged base, re-synthesis of a new DNA strand and ligation of the nicks to restore the sequence integrity. Mutation or aberrant transcription of NER genes reduces repair efficiency and results in the accumulation of mutations that is associated with the development of cancer. Here we present a rapid, sensitive and quantitative assay to measure NER activity in human cells, which we term the Oligonucleotide Retrieval Assay (ORA). We used oligonucleotide constructs containing the UV-damaged adduct, cyclobutane pyrimidine dimer (CPD), to transfect human cells, and retrieved the oligonucleotides for quantification of the repaired, CPD-free DNA by real-time quantitative PCR. We demonstrate that ORA can quantify the extent of NER in diverse cell types, including immortalized, primary and stem-like cells.