Assay conditions for estimating differences in base excision repair activity with Fpg-modified comet assay.

Abstract

DNA repair is an essential agent in cancer development, progression, prognosis, and response to therapy. We have adapted a cellular repair assay based on the formamidopyrimidine DNA glycosylase (Fpg)-modified comet assay to assess DNA repair kinetics. The removal of oxidized nucleobases over time (0-480 min) was analyzed in peripheral blood mononuclear cells (PBMCs) and 8 cell lines. DNA damage was induced by exposure to either Ro19-8022 plus visible light or potassium bromate (KBrO₃). The initial amount of damage induced by Ro 19-8022 plus light varied between cell lines, and this was apparently associated with the rate of repair. However, the amount of DNA damage induced by KBrO₃ varied less between cell types, so we used this agent to study the kinetics of DNA repair. We found an early phase of ca. 60 min with fast removal of Fpg-sensitive sites, followed by slower removal over the following 7 h. In conclusion, adjusting the initial damage at T₀ to an equal level can be achieved by the use of KBrO₃, which allows for accurate analysis of subsequent cellular DNA repair kinetics in the first hour after exposure.