Preferential cleavage sites have been determined for Fe 2؉ /H 2 O 2 -mediated oxidations of DNA. In 50 mM H 2 O 2 , preferential cleavages occurred at the nucleoside 5 to each of the dG moieties in the sequence RGGG, a sequence found in a majority of telomere repeats. Within a plasmid containing a (TTAGGG) 81 human telomere insert, 7-fold more strand breakage occurred in the restriction fragment with the insert than in a similar-sized control fragment. This result implies that telomeric DNA could protect coding DNA from oxidative damage and might also link oxidative damage and iron load to telomere shortening and aging. In micromolar H 2 O 2 , preferential cleavage occurred at the thymidine within the sequence RTGR, a sequence frequently found to be required in promoters for normal responses of many procaryotic and eucaryotic genes to iron or oxygen stress. Computer modeling of the interaction of Fe 2؉ with RTGR in B-DNA suggests that due to steric hindrance with the thymine methyl, Fe 2؉ associates in a specific manner with the thymine flipped out from the base stack so as to allow an octahedrally-oriented coordination of the Fe 2؉ with the three purine N 7 residues. Fe 2؉ -dependent changes in NMR spectra of duplex oligonucleotides containing ATGA versus those containing AUGA or A 5m CGA were consistent with this model.

Exposure of Escherichia coli to H202 leads to two kinetically distinguishable modes of killing: mode I killing occurs maximally near 2 mM H202, whereas mode II killing is essentially independent of H202 concentrations up to 20 mM.A major portion of H202 toxicity is attributed to DNA damage caused by the iron-mediated Fenton reaction. By studying DNA damage during Fenton reactions in vitro, the same complex kinetics were observed and three types of oxidants were distinguished based upon their reactivities toward H202 and alcohols and upon iron-chelator effects. but the unusual biphasic dose-response results to H202 observed for both killing and DNA nicking could indicate that there might be at least two types of oxidants. The objective of this study is to further investigate this possibility.MATERIALS AND METHODS H202 (30% solution) and 2-methyl-2-propanol ("t-butanol") were from Fisher Scientific. FeSO4, 1,10-phen, 4,7-phenanthroline (4,7-phen), 2,9-dimethyl-1,10-phenanthroline (dmphen), and 2,2'-dipy were from Aldrich. Ethanol was from Quantum Chemical (Tuscola, IL). Bacteriophage PM2 DNA labeled with [methyl-3H]thymidine (5) was isolated by detergent and phenol extraction, followed by density gradient centrifugation. The DNA was dialyzed against EDTA for at least 2 days and was not sensitive to H202 alone.Killing and Mutagenesis Analysis of E. coli. Strain AB1157 (WT) was exposed to H202 for 15 min and then plated. Where indicated, 1 mM 1,10-phen or 1 mM 2,2'-dipy was added to the cells 5 min prior to the challenge with H202.DNA Nicking Determination. PM2 [3H]DNA (17 AM nucleotide residues) was mixed with 0.8% NaCl/80 nM FeSO4 and 320 nM chelator ethanol and/or H202 as indicated. Reactions were stopped after 7.5 min at 25°C by the addition of catalase at 2 ,g/ml. Nicking of the DNA was quantitated by nitrocellulose filter binding as described (6).Other Methods. H202 concentrations were measured by UV absorption at 240 nm by assuming 8240 = 39.4 M-1 cm-1 (7) or by a peroxidase dye assay (8). Ferrous concentrations were determined by colorimetry with 1,10-phen by assuming that the Fe2+(1,10-phen)3 complex has an E510 = 1.11 X 104 M-1 cm-1 (9). The concentration of PM2 DNA was determined by UV absorption at 260 nm by assuming -260 = 6.5 mM-'-cm-'.RESULTS AND DISCUSSION Classification of Oxidants Formed by the Fenton Reaction in the Presence of DNA. In the presence of Fe2+ and H202, DNA nicking is maximal at 0.05 mM H202 then drops to about one-third that level at 3 mM H202, beyond which it remains relatively constant (Fig. 1). This dose-response result suggests that at least some species of the oxidant(s) is sensitive to H202.We were able to ascribe inhibition of aniline dye bleaching by higher concentrations of H202 to simple competition between H202 and the dye for -OH (4) in which H202 reacts with -OH to form the less active 'HO2 radical:H202 + 'OH -* 'HO2 + H20.[2]