Methods for Measuring DNA Repair: Introduction and Cellular Repair

Abstract

DNA repair pathways provide a critically important cellular defence system, effectively protecting us from mutations and cancer. Distinct pathways deal with various classes of damage: single- and double-strand breaks, oxidized and alkylated bases, bulky adducts, intra- and inter-strand cross-links. A simple approach to measuring the DNA repair capacity of cell lines, or samples of blood cells, for instance, is the cellular repair assay, or challenge assay, in which cells are treated with a specific DNA-damaging agent, and incubated; at intervals, samples are taken and the residual damage is measured. The comet assay is well suited for measuring strand break rejoining, excision repair of oxidized or alkylated bases (with a lesion-specific endonuclease to convert the altered bases into breaks), and nucleotide excision repair of UV-induced lesions (again, using an appropriate enzyme to detect the damage). An alternative way to assess nucleotide excision repair capability is the incision assay: after UV irradiation, cells are incubated with inhibitors of repair DNA synthesis, so that incomplete repair sites accumulate as DNA breaks.

We provide protocols for these DNA repair assays, and discuss their applications—and their limitations. We also raise some important, so far unanswered questions concerning the regulation of repair, and the factors that might account for the wide variations seen in individual repair capacities.