Strand breaks in whole plasmid DNA produced by the decay of I-125 in a triplex-forming oligonucleotide

DNA strand breaks produced by the decay of I-125 positioned against a speci fic site in plasmid DNA via a triplex-forming oligonucleotide were studied both in the immediate vicinity of the site of the decay with a single nucle otide resolution and in the whole plasmid by measuring the percentages of s upercoiled, open-circular and linear forms. The localized breaks are distri buted within 10 bp in each direction from the decay site with maxima in bot h strands just opposite the I-125-dC residue in the triplex-forming oligonu cleotide. The distributions of breaks in the two DNA strands are almost sym metrical, in agreement with the geometry of the pyrimidine motif triplex. W e found that about 25% of the double-strand breaks were located outside the 90-bp fragment containing the triplex-forming oligonucleotide binding sequ ence. The ratio of single-to double-strand breaks in the whole plasmid was 11 for bound triplex-forming oligonucleotide compared to 26 when the triple x-forming oligonucleotide was free in solution. The number of double-strand breaks per decay of I-125 was 0.46 for bound triplex-forming oligonucleoti de and 0.17 for free triplex-forming oligonucleotide. Comparing the data on the localized damage and those for the whole plasmid, we concluded that, i n addition to DNA breaks that are confined to a helical turn around the I-1 25 atom, the decay can produce breaks hundreds of base pairs away in the pl asmid molecule. This linear plasmid molecule containing radiation-induced d amage at a specific DNA site should be useful in studies of the molecular m echanisms of DNA repair. (C) 2001 by Radiation Research Society.