ABSTRACT
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 reported previously the detection of DNA adducts by an in vitro chemiluminescence DNA repair synthesis assay (Salles et al., 1995) which allows the measurement of repair synthesis by cell-free extracts in damaged plasmid DNA adsorbed on sensitized microplate wells. The 3D (DNA damage detection) assay was performed in the presence of biotin-dUTP which was incorporated during the repair synthesis step. The extent of repair synthesis was measured in an ELISA reaction with ExtrAvidin-horse radish peroxidase and chemiluminescence 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 assay. By taking advantage of plasmid DNA adsorption, oxidative base damage can be recognized by the Escherichia coli Fpg protein which was detected in an 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.
Subject(s)
Antioxidants/pharmacology , DNA Repair , Escherichia coli Proteins , DNA Adducts/analysis , DNA Damage , DNA-Formamidopyrimidine Glycosylase , Enzyme-Linked Immunosorbent Assay , Methods , N-Glycosyl Hydrolases , Plasmids , Reactive Oxygen Species/metabolismABSTRACT
Inhibition of the nucleotide excision repair (NER) process is believed to cause the potentiation of the genotoxic and mutagenic effects of DNA damaging agents like UV-light or cisplatin by metal ions. However, the precise underlying molecular mechanism of this phenomenon is still unknown. Using in vitro assays, we have determined the potential interference of several metal (II) ions with the lesion recognition and strand incision/displacement steps of the NER mechanism, independently from the DNA polymerization step. When combinations of an optimal Mg2+ concentration and concentrations of various metal ions in a range from 0.1 to 1 mM were tested, all combinations, with Mn2+ and Ni2+ excepted, inhibited specifically the incision repair activity by human protein extracts. There was a good correlation for Cd2+, Co2+, Fe2+, Cu2+, Hg2+, Pb2+ and Zn2+ between an inhibiting effect on the incision activity and a reduced protein binding activity to a damaged DNA probe as assessed by gel mobility shift assay.
