Detection of oxidative base DNA damage by a new biochemical assay

Reactive oxygen species (ROS) damage DNA which appears to represent the maj or target involved in mutagenesis, carcinogenesis, and aging cell responses . Various DNA modifications are generated by ROS, but 8-hydroxy-2'-deoxygua nosine (8-oxoG) has retained a lot of attention in the last few years. Ther efore, numerous methods have been developed to detect and quantify the exte nt of 8-oxoG in DNA, most of them requiring a significant amount of DNA tha t might be limiting in the case of biological samples. 8-oxoG is repaired i n Escherichia coli by a specific glycosylase, the Fpg (formamidopyrimidine DNA glycosylase) protein, in a reaction that requires a covalent intermedia te favored under reducing conditions. We set up a new assay based on the ca pture of plasmid DNA into sensitized microplate wells. DNA damaged by photo activation of methylene blue was adsorbed on a polylysine-treated plastic w ell. Then the Fpg protein was added, allowed to fix on the damage by taking advantage of minimized glycosylase activity at low temperature and the red uctive trapping of the covalent intermediate, yielding to a stable DNA-prot ein interaction. The trapped protein was subsequently recognized by a speci fic antibody. A secondary antibody coupled with horseradish peroxidase was used to detect the complex and the measurement was carried out by chemilumi nescence, This new assay offers various potentialities, specifically in the field of technology of ROS producers. (C) 2000 Academic Press.