Detection of oxidative base DNA damage by a new biochemical assay.
Reactive oxygen species (ROS) damage DNA which appears to represent the major target involved in mutagenesis, carcinogenesis, and aging cell responses. Various DNA modifications are generated by ROS, but 8-hydroxy-2'-deoxyguanosine (8-oxoG) has retained a lot of attention in the last few years. Therefore, numerous methods have been developed to detect and quantify the extent of 8-oxoG in DNA, most of them requiring a significant amount of DNA that might be limiting in the case of biological samples. 8-oxoG is repaired in Escherichia coli by a specific glycosylase, the Fpg (formamidopyrimidine DNA glycosylase) protein, in a reaction that requires a covalent intermediate favored under reducing conditions. We set up a new assay based on the capture of plasmid DNA into sensitized microplate wells. DNA damaged by photoactivation of methylene blue was adsorbed on a polylysine-treated plastic well. Then the Fpg protein was added, allowed to fix on the damage by taking advantage of minimized glycosylase activity at low temperature and the reductive trapping of the covalent intermediate, yielding to a stable DNA-protein interaction. The trapped protein was subsequently recognized by a specific antibody. A secondary antibody coupled with horseradish peroxidase was used to detect the complex and the measurement was carried out by chemiluminescence. This new assay offers various potentialities, specifically in the field of technology of ROS producers.
Sattler
U
U
Institut de Pharmacologie et de Biologie Structurale, CNRS UPR 9062, 205 route de Narbonne, Toulouse Cedex, 31077, France.
Calsou
P
P
Boiteux
S
S
Salles
B
B
D000080242:8-Hydroxy-2'-Deoxyguanosine; D004249:DNA Damage; D004260:DNA Repair; D004269:DNA, Bacterial / Q000032:analysis / Q000737:chemistry / Q000187:drug effects; D043382:DNA-Formamidopyrimidine Glycosylase; D003849:Deoxyguanosine / Q000031:analogs & derivatives* / Q000032:analysis; D004926:Escherichia coli / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D029968:Escherichia coli Proteins; D005069:Evaluation Studies as Topic; D008163:Luminescent Measurements; D008722:Methods; D008751:Methylene Blue; D009699:N-Glycosyl Hydrolases / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D012680:Sensitivity and Specificity; D013379:Substrate Specificity
D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D017382:Reactive Oxygen Species; D000080242:8-Hydroxy-2'-Deoxyguanosine; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli; D003849:Deoxyguanosine; D008751:Methylene Blue