SINGLE-STRAND AND DOUBLE-STRAND PHOTOCLEAVAGE OF DNA BY YO, YOYO AND TOTO

Photocleavage of dsDNA by the fluorescent DNA stains oxazole yellow (Y O), its dimer (YOYO) and the dimer TOTO of thiazole orange (TO) has be en investigated as a function of binding ratio, On visible illuminatio n, both YO and YOYO cause single-strand cleavage, with an efficiency t hat varies with the dye/DNA binding ratio in a manner which can be rat ionalized in terms of free dye being an inefficient photocleavage reag ent and externally bound dye being more efficient than intercalated dy e, Moreover, the photocleavage mechanism changes with binding mode, Ph otocleavage by externally bound dye is, at least partly, oxygen depend ent with scavenger studies implicating singlet oxygen as the activated oxygen intermediate, Photocleavage by intercalated dye is essentially oxygen-independent but can be inhibitied by moderate concentrations o f beta-mercaptoethanol-direct attack on the phosphoribose backbone is a possible mechanism, TOTO causes single-strand cleavage approximately five times less efficiently than YOYO. No direct double-strand breaks (dsb) are detected with YO or YOYO, but in both cases single-strand b reaks (ssb) are observed to accumulate to eventually produce double-st rand cleavage, With intercalated YO the accumulation occurs in a manne r consistent with random generation of strand lesions, while with bisi ntercalated YOYO the yield of double-strand cleavage (per ssb) is 5-fo ld higher, A contributing factor is the slow dissociation of the bis-i ntercalated dimer, which allows for repeated strand-attack at the same binding site, but the observation that the dsb/ssb yield is considera bly lower for externally bound than for bis-intercalated YOYO at low d ye/DNA ratios indicates that the binding geometry and/or the cleavage mechanism are also important for the high dsb-efficiency. In fact, dou ble-strand cleavage yields with bis-intercalated YOYO are higher than those predicted by simple models, implying a greater than statistical probability for a second cleavage event to occur adjacent to the first (i.e, to be induced by the same YOYO molecule), With TOTO the efficie ncy of the ssb-accumulation is comparable to that observed with YOYO.