Nucleic Acids Research, Vol 24, Issue 6 1080-1090, Copyright © 1996 by Oxford University Press
B Akerman and E Tuite
Photocleavage of dsDNA by the fluorescent DNA stains oxazole yellow (YO),
its dimer YOYO) and the dimer TOTO of thiazole orange (TO) has been
investigated as a function of binding ratio. On visible illumination, both
YO and YOYO cause single-strand cleavage, with an efficiency that varies
with the dye/DNA binding ratio in a manner which can be rationalized in
terms of free dye being an inefficient photocleavage reagent and externally
bound dye being more efficient than intercalated dye. Moreover, the
photocleavage mechanism changes with binding mode. Photocleavage by
externally bound dye is, at least partly, oxygen dependent with scavenger
studies implicating singlet oxygen as the activated oxygen intermediate.
Photocleavage by intercalated dye is essentially oxygen-independent but can
be inhibited by moderate concentrations of 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 breaks (ssb) are observed to accumulate to
eventually produce double-strand cleavage. With intercalated YO the
accumulation occurs in a manner consistent with random generation of strand
lesions, while with bisintercalated YOYO the yield of double- strand
cleavage (per ssb) is 5-fold higher. A contributing factor is the slow
dissociation of the bis-intercalated dimer, which allows for repeated
strand-attack at the same binding site, but the observation that the
dsb/ssb yield is considerably lower for externally bound than for
bis-intercalated YOYO at low dye/DNA ratios indicates that the binding
geometry and/or the cleavage mechanism are also important for the high
dsb-efficiency. In fact, double-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 efficiency of the ssb-accumulation is comparable
to that observed with YOYO.
ARTICLES
Single- and double-strand photocleavage of DNA by YO, YOYO and TOTO
Department of Physical Chemistry, Chalmers University of Technology, Goteborg, Sweden.
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