PHOTOSENSITIZED DAMAGE TO SUPERCOILED PLASMID DNA INDUCED BY 334‐nm RADIATION IN THE PRESENCE OF 2‐THIOURACIL CONSISTS OF ALKALI‐ and PIPERIDINE‐LABILE SITES AS WELL AS FRANK STRAND BREAKS

Abstract

A covalently closed, supercoiled plasmid was irradiated with 334-nm ultraviolet radiation in the presence of the naturally occurring photosensitizer 2-thiouracil (s2-Ura). After irradiation, some DNA samples were treated to reveal labile sites. Agarose gel electrophoresis was then used to resolve the unrelaxed supercoils from the relaxed forms, and the DNA bands were quantitated by fluorescence scanning. Irradiation of the plasmid in the absence of s2Ura induced small numbers of frank DNA strand breaks (FSB), alkali-labile sites (ALS), and piperidine-labile sites (PLS). The induction of each of these lesions was enhanced 30 times when s2Ura was present during aerobic irradiation. Anoxia, as well as the hydroxyl radical scanvergers acetate and formate, inhibited the formation of all three lesion types. The relative proportions of the three lesion types produced by several DNA damaging treatments were measured. Hydrogen peroxide, .gamma.-irradiation, and s2Ura photosensitization produced nearly identical damage proportions, with PLS:FSB ratios of 1.25:1, 0.78:1, and 0.84:1, respectively. Treatment with singlet oxygen [data from Blazek et al. (1989) Photochem. Photobiol. 48, 607-613] produced much different proportions, with a PLS:FSB ratio of 4:1:1. These results may indicate a role for hydroxyl radical in s2Ura-photosensitized DNA damage.