Measurement of Oxidative DNA Damage and Repair in Specific DNA Sequences

We describe two methods that were developed in our laboratory to measure the production and repair of oxidative DNA damage in specific DNA sequences. Both of these methods rely on the use of monoclonal antibodies against modified nucleotides to separate DNA sequences that contain damage from those in which repair has occurred. In one case, the modified base is bromodeoxyuridine, which is inserted into the DNA during the repair synthesis step of excision repair. An antibody against this modified base is used to detect the production of the bromodeoxyuridine in the repair patch. This approach allows for the measurement of repair of any DNA lesion whose removal is accompanied by the production of a DNA repair patch. In the other case, the modified base is thymine glycol, an oxidized base that is produced by hydrogen peroxide and ionizing radiation. Using a monoclonal antibody that recognizes this altered base, detection of the production and repair of a specific base damage in a DNA sequence can be accomplished. These approaches are used in our laboratory to examine the transcription-coupled repair of oxidative DNA damage in both yeast and mammalian cells.