Localization of Fe(2+) at an RTGR sequence within a DNA duplex explains preferential cleavage by Fe(2+) and H2O2.
Nicking of duplex DNA by the iron-mediated Fenton reaction occurs preferentially at a limited number of sequences. Of these, purine-T-G-purine (RTGR) is of particular interest because it is a required element in the upstream regulatory regions of many genes involved in iron and oxidative-stress responses. In order to study the basis of this preferential nicking, NMR studies were undertaken on the RTGR-containing duplex oligonucleotide, d(CGCGATATGACACTAG)/d(CTAGTGTCATATCGCG). One-dimensional and two-dimensional 1H NMR measurements show that Fe(2+) interacts preferentially and reversibly at the ATGA site within the duplex at a rate that is rapid relative to the chemical-shift timescale, while selective paramagnetic NMR line-broadening of the ATGA guanine H8 suggests that Fe(2+) interacts with the guanine N7 moiety. Localization at this site is supported by Fe(2+) titrations of a duplex containing a 7-deazaguanine substitution in place of the guanine in the ATGA sequence. The addition of a 100-fold excess of Mg(2+) over Fe(2+) does not affect the Fe(2+)-dependent broadening. When the ATGA site in the duplex is replaced by ATGT, an RTGR site (GTGA) is created on the opposite strand. Preferential iron localization then takes place at the 3' guanine in GTGA but no longer at the guanine in ATGT, consistent with the lack of preferential cleavage of ATGT sites relative to ATGA sites.
Rai
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P
Biophysics Graduate Group, University of California, Berkeley, CA 94720-3206, USA.
Cole
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TD
Wemmer
D E
DE
Linn
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D020029:Base Pairing; D001483:Base Sequence; D002413:Cations, Divalent / Q000378:metabolism* / Q000494:pharmacology; D003035:Cobalt / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects / Q000235:genetics; D006861:Hydrogen Peroxide / Q000378:metabolism* / Q000494:pharmacology; D007501:Iron / Q000378:metabolism* / Q000494:pharmacology; D009682:Magnetic Resonance Spectroscopy; D008345:Manganese / Q000378:metabolism; D008958:Models, Molecular; D018384:Oxidative Stress / Q000187:drug effects; D013379:Substrate Specificity; D013816:Thermodynamics
D002413:Cations, Divalent; C045076:Fenton's reagent; D003035:Cobalt; D008345:Manganese; D004247:DNA; D006861:Hydrogen Peroxide; D007501:Iron