4]] Localization of strand breaks in plasmid DNA treated with reactive oxygen species

Abstract

Publisher Summary This chapter describes a procedure for determining the localization of single-strand breaks in plasmid DNA. The damaged strand of DNA is selectively end-labeled; its size is defined on a denaturing polyacrylamide gel. Damage to DNA other than strand breaks, such as base modifications that may interfere with other methods, do not usually affect the result. The intensity of the signals obtained allows estimating the proportion of strands broken at each site. This method is used to show that strand breaks induced by singlet oxygen in a metallothionein promoter contained in a plasmid occur selectively at guanosine residues, with similar frequency at different positions in the sequence. The method is also suitable for examining other agents that damage DNA and is applicable to sequences other than the metallothionein promoter. The plasmid vector pBluescriptII is treated with the activated oxygen species of choice, the damaging agent is removed, and the amount and type of damage inflicted are examined by agarose gel electrophoresis.