A NEW METHOD FOR INTRODUCING DOUBLE-STRAND BREAKS INTO CELLULAR DNA

A novel method is used to introduce double-strand breaks into cellular DNA containing controlled levels of 5-bromo-2'-deoxyuridine (BrdU). Chinese hamster V79 cells substituted with BrdU are treated with Hoechst dye 33258 and then exposed to UVA light. Using neutral elution (pH 7.2) the yield of DNA double-strand breaks is found to be linearly dependent on the level of BrdU substitution (0.36-7.5%), concentration of Hoechst dye (0-100 μg cm- 3), and fluence of UVA light (0.2-8 kJ m-2). The yield of double-strand breaks produced by this photolysis treatment is 5.1 x 10-6 breaks/BrdU residue/kJ m-2, regardless of whether one or both strands of the DNA polymer contain BrdU. No double-strand breaks are detected in the absence of Hoechst dye, BrdU, or UVA light. The formation of double-strand breaks appears to involve strand cleavage at a BrdU site on one strand with cleavage in the opposite strand not necessarily requiring the presence of BrdU. The utility of this photolytic regimen in modeling the biological significance of double-strand break lesions and some putative mechanisms for their formation are discussed.