Measurement of oxidative DNA damage and repair in specific DNA sequences

We describe two methods that were developed in our laboratory to measure th e production and repair of oxidative DNA damage in specific DNA sequences. Both of these methods rely on the use of monoclonal antibodies against modi fied nucleotides to separate DNA sequences that contain damage from those i n which repair has occurred. In one case, the modified base is bromodeoxyur idine, which is inserted into the DNA during the repair synthesis step of e xcision repair. An antibody against this modified base is used to detect th e production of the bromodeoxyuridine in the repair patch. This approach al lows for the measurement of repair of any DNA lesion whose removal is accom panied by the production of a DNA repair patch. In the other case, the modi fied base is thymine glycol, an oxidized base that is produced by hydrogen peroxide and ionizing radiation. Using a monoclonal antibody that recognize s this altered base, detection of the production and repair of a specific b ase 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. (C) 2000 Academic Press.