MEASURING DNA REPAIR
- Measure induction and removal of lesions; see figure
- Measure DNA breaks resulting from incision (first step of repair)
- Measure repair DNA synthesis
- Measure capacity of cell extract to perform repair (either incision or synthesis) on DNA substrate with defined lesions
Figure This is a biochemical rather than a cellular assay. An extract is prepared from the cells of interest, and incubated with a substrate consisting of nucleoids from cells treated with a specific DNA damaging agent. In this experiment, the substrate cells were treated with a chemical photosensitiser plus light to induce oxidative damage. As the extract finds damage and makes breaks in the substrate DNA, the breaks accumulate with time and are measured with the comet assay. There is a strong indication here of significant differences between different individuals in their capacity to repair this kind of damage.
Measuring the incision stage of nucleotide excision repair
Inhibitors of DNA polymerase allow incision to continue while blocking ligation, so breaks accumulate
Figure Cells are incubated for 30 min after UV irradiation, in the presence of inhibitors of the repair synthesis step of NER, such as hydroxyurea, aphidicolin, and cytosine arabinoside. These block the repair synthesis stage, but incision continues, so breaks accumulate in the DNA and can readily be detected. This figure shows the UV dose-dependent accumulation of breaks in wild-type mouse cells (solid squares), and in two mutant cell lines, one of which (US31, solid circles) is defective in incision, the other (US46) being defective at a later stage.
Detecting repair DNA synthesis: unscheduled DNA synthesis (UDS)
Figure Unscheduled DNA synthesis refers to the low level of DNA synthesis (seen here as incorporation of radioactive tritium-labelled thymidine into the DNA of non-S phase cells) accompanying nucleotide excision repair. In S phase cells repair also occurs, but is swampled by the much larger amount of replicative synthesis.
In vitro repair assay: measure repair activity of cell extract on substrate containing specific damage
There is little evidence for nutritional effects on DNA repair.