Quantification of DNA repair capacity towards oxidatively damaged DNA in subcellular and cellular systems by a nonradioactive cleavage assay

Abstract

The identification of appropriate biomarkers for oxidative stress is one major aim in molecular epidemiology. Besides the quantifi cation of specifi c DNA lesions such as of 8-oxoguanine (8oxoG), another approach consists in the assessment of the repair capacity towards 8oxoG, mediated predominantly by the human 8-oxoguanine DNA glycosylase 1 (hOGG1); further processing of base excision repair involves AP endonuclease 1 (APE1). Thus, during the last few years the so-called cleavage assays have been described, investigating the incision capacity of cell extracts towards 32 P-labelled and 8oxoG damaged oligonucleotides. Here, we describe a sensitive nonradioactive test system based on Cy5-labelled oligonucleotides with hairpin-like structures, enabling the assessment of activities of the isolated hOGG1 and APE1 as well as their activities in extracts prepared from cultured cells or peripheral blood mononuclear cells (PBMC). This approach allows the sensitive quantifi cation of modulating exposures, such as inhibitory metal compounds, and also the determination of interindividual differences in DNA repair capacities. The method is as sensitive and even faster as compared to the use of radioactively labelled oligonucleotides and additionally offers the advantage of reduced costs and low health risk.