Repair of oxidative DNA damage in vitro: A tool for screening antioxidative compounds

Reactive oxygen species (ROS) provoke the formation of base DNA alterations that are processed by an excision step of the lesion followed by a repair synthesis and ligation step to restore the strand continuity. We have repor ted previously the detection of DNA adducts by an ill vitro chemiluminescen ce DNA repair synthesis assay (Salles er al.. 1995) which allows the measur ement of repair synthesis by cell-free extracts in damaged plasmid DNA adso rbed on sensitized microplate wells. The 3D (DNA damage detection) assay wa s performed in the presence of biotin-dUTP which was incorporated during th e repair synthesis step. The extent of repair synthesis was measured in an ELISA reaction with ExtrAvidin-horse radish peroxidase and chemiluminescenc e detection. The 3D assay allows detection of any type of base alterations including base oxidation. Interestingly, under controlled production of ROS a screening procedure of antioxidants might be carried out with the 3D ass ay. By taking advantage of plasmid DNA adsorption, oxidative base damage ca n be recognized by the Escherichia coli Fpg protein which was detected in a n ELISA reaction with specific antibody and chemiluminescence measurement ( 4D assay). With the sceening procedure of antioxidative compounds in mind, the development of such assays and their drawbacks are discussed. (C) 1999 Elsevier Science Ltd. All rights, reserved.