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

Priyamvada Rai, Timothy D. Cole, David E. Wemmer, Stuart Linn

Research output: Contribution to journalArticlepeer-review

56 Scopus citations

Abstract

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 Fe2+ 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 Fe2+ interacts with the guanine N7 moiety. Localization at this site is supported by Fe2+ 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 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. 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.

Original languageEnglish (US)
Pages (from-to)1089-1101
Number of pages13
JournalJournal of molecular biology
Volume312
Issue number5
DOIs
StatePublished - Oct 5 2001
Externally publishedYes

Keywords

  • H NMR
  • DNA nicking
  • Fe localization
  • Fenton reaction
  • Paramagnetic signal broadening

ASJC Scopus subject areas

  • Virology

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