The specific recognition of DNA modifications by repair endonucleases
was used to characterize the DNA damage induced by photosensitizers in
the presence of visible light. Under cell-free conditions, chemically
unrelated photosensitizers (methylene blue, acridine orange, proflavi
n, riboflavin, hematoporphyrin) induce the same type of DNA damage. It
is characterized by a high number of base modifications sensitive to
the repair endonuclease FPG protein (formamidopyrimidine-DNA glycosyla
se), while both the number of DNA strand breaks and the number of site
s of base loss (sensitive to exonuclease III or endonuclease IV) is lo
w. Therefore the damage is markedly different from that induced by hyd
roxyl radicals. Mechanistically, the generation of the base modificati
ons sensitive to FPG protein involves singlet oxygen in some, but poss
ibly not all cases, as substituting D2O for H2O increases the reaction
yield six-fold in the case of methylene blue, but only 1.4-fold in th
e case of acridine orange. In plasmids from Salmonella typhimurium str
ains treated with methylene blue or acridine orange plus light and fro
m Escherichia coli strains treated with acridine orange or proflavin p
lus light, the same type of damage was observed as under cell-free con
ditions. In L1210 mouse leukemia cells exposed to acridine orange plus
light, the numbers of modifications sensitive to FPG protein and exon
uclease Ill were quantified, in addition to strand breaks, by a modifi
ed alkaline elution assay. Again, the number of base modifications sen
sitive to FPG protein was found to be several-fold higher than the num
ber of strand breaks and sites of base loss. It has to be concluded th
at the DNA damage in the intact cells is not mediated by hydroxyl radi
cals or cellular nucleases, but by the same mechanism as operates unde
r cell-free conditions with these agents.