Localization of Fe2+ at an RTGR sequence within a DNA duplex explains preferential cleavage by Fe2+ and H2O2

Nicking of duplex DNA by the iron-mediated Fenton reaction occurs preferent ially 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 resp onses. In order to study the basis of this preferential nicking, NMR studie s were undertaken on the RTGR-containing duplex oligonucleotide, d(CGCGATAT GACACTAG)/d(CTAGTGTCATATCGCG). One-dimensional and two-dimensional H-1 NMR measurements show that Fe2+ interacts preferentially and reversibly at the ATGA site within the duplex at a rate that is rapid relative to the chemica l-shift timescale, while selective paramagnetic NMR line-broadening of the T ATGA guanine H8 suggests that Fe2+ interacts with the guanine N7 moiety. Localization at this site is supported by Fe2+ titrations of a duplex conta ining a 7-deazaguanine substitution in place of the guanine in the ATGA seq uence. The addition of a 100-fold excess of Mg2+ over Fe2+ does not affect the Fe2+-dependent broadening. When the ATGA site in the duplex is replaced by ATGT, an RTGR site (GTGA) is created on the opposite strand. Preferenti al iron localization then takes place at the 3' guanine in GTGA but no long er at the guanine in ATGT, consistent with the lack of preferential cleavag e of ATGT sites relative to ATGA sites. (C) 2001 Academic Press.