PHOTOCHEMICAL PRODUCTION OF URACIL QUANTIFIED IN BROMODEOXYURIDINE-SUBSTITUTED SV40 DNA BY URACIL DNA GLYCOSYLASE AND A LYSYL-TYROSYL-LYSINE TRIPEPTIDE

Exposure to UVA radiation of SV40 DNA substituted with bromodeoxyuridi ne (BrdU) in the presence of Hoechst dye 33258 results in the producti on of uracil. The yield of uracil was determined by measuring the incr ease in the single-strand break (SSB) yield after incubation of the ph otolyzed DNA with uracil-DNA glycosylase (UDG) in the presence of the tripeptide lysyl-tyrosyl-lysine (KYK). UDG removes uracil to leave an abasic site which is then cleaved to a SSB by KYK. The SSB yield was q uantified by digital video imaging of ethidium fluorescence after sepa ration of the I, II and III forms of SV40 DNA by agarose gel electroph oresis. Uracil is not detected when photolysis is carried out in the a bsence of the dye nor when unsubstituted DNA is used as the substrate, Without UDG or KYK treatment, the F-0 for the loss of form I DNA is 1 00 J/m(2). This falls to 13 J/m(2) after incubation with UDG and KYK, indicating that uracil formation is similar to 5-fold more efficient t han SSB formation, Formation of uracil suggests a mechanism for the hi gh cellular toxicity of the dye-BrdU-UVA treatment.