Measuring DNA repair with the comet assay

There are two main approaches to assessing the ability of cells to repair DNA damage; the cellular repair assay and the in vitro assay. In the first of these, cells are treated with an appropriate genotoxic agent to induce damage and incubated; the comet assay is used to measure the damage remaining at each time-point.

In the simplest case, rejoining of strand breaks can be followed after treating cells with ionising radiation or with H₂O₂. Typically, such repair is rapid, and samples need to be taken at intervals of a few minutes in order to estimate accurately either the initial rate of removal or the t_1/2 of the lesions (both valid measures of repair activity). Monitoring the repair of other lesions, such as oxidised bases (by base excision repair, BER), or UV-induced pyrimidine dimers (by nucleotide excision repair, NER), requires the use of enzymes that recognise the lesions and convert them to strand breaks.

Formamidopyrimidine DNA glycosylase (FPG) recognises 8-oxoguanine and other purine oxidation products; endonuclease III deals with oxidised pyrimidines; and T4 endonuclease V is able to incise at sites of pyrimidine dimers. Digestion with these enzymes is carried out after the initial lysis step. The excision repair pathways act more slowly than strand break rejoining (Collins & Horvathova, 2001), and samples should be taken over a period of a few hours.

The substrate consists of cells embedded in agarose and lysed - as in the standard comet assay - to leave the DNA in the form of nucleoids. These cells were previously treated with a specific DNA-damaging agent, and this is what defines the kind of repair activity that is assessed. Thus, the substrate cells can be irradiated with UV(C) in order to measure NER; or treated with the photosensitiser Ro 19-8022 and visible light to measure the activity of 8-oxoguanine DNA glycosylase (OGG). The repair reaction is carried out by adding extract to the substrate DNA in the gel, covering with a cover slip, and incubating for a fixed time. T4 endonuclease V or FPG is included as a positive control alongside the extracts. DNA breaks produced by glycosylase, endonuclease and/or lyase activities in the extract are measured by completing the comet assay as usual. Figure 2 illustrates the importance of cell concentration in the in vitro assay, and the dependence of the reaction on incubation time.

The two approaches examine different aspects of repair. The cellular assay looks at the overall process, removal of lesions, while the in vitro assay focuses on the intial, rate-limiting steps. In both, the end-result is comets, and the preferred parameter for representing DNA damage is % tail DNA, or relative tail intensity, conveniently measured with Perceptive Instruments Comet Assay IV. After scoring 50 or 100 comets per sample and time-point, the median % tail DNA is calculated.

Applications

The cellular repair assay is probably most useful for testing variant cell types for altered repair functions, and examining factors that inhibit or stimulate DNA repair. For example, we demonstrated an acceleration of strand break rejoining and BER of 8-oxoguanine on incubating cells with the carotenoid β-cryptoxanthin (Lorenzo et al., 2009).