DNA strand breaking by the hydroxyl radical is governed by the accessible surface areas of the hydrogen atoms of the DNA backbone

Abstract

Despite extensive study, there is little experimental information available as to which of the deoxyribose hydrogen atoms of duplex DNA reacts most with the hydroxyl radical. To investigate this question, we prepared a set of double-stranded DNA molecules in which deuterium had been incorporated specifically at each position in the deoxyribose of one of the four nucleotides. We then measured deuterium kinetic isotope effects on the rate of cleavage of DNA by the hydroxyl radical. These experiments demonstrate that the hydroxyl radical reacts with the various hydrogen atoms of the deoxyribose in the order 5 H > 4 H > 3 H Ϸ 2 H Ϸ 1 H. This order of reactivity parallels the exposure to solvent of the deoxyribose hydrogens. Our work therefore reveals the structural basis of the reaction of the hydroxyl radical with DNA. These results also provide information on the mechanism of DNA damage caused by ionizing radiation as well as atomic-level detail for the interpretation of hydroxyl radical footprints of DNA-protein complexes and chemical probe experiments on the structure of RNA and DNA in solution.The hydroxyl radical (⅐OH), the quintessential reactive oxygen species, is the mediator of much of the DNA damage caused by ionizing radiation (1). This damage includes strand breaks, which are initiated by abstraction of a deoxyribose hydrogen atom by the hydroxyl radical. DNA strand breaks induced by the hydroxyl radical also form the basis of a widely used method for making footprints of DNA-protein complexes (2, 3) and for studying the structure of DNA (4) and RNA (5) in solution. The key experimental advantage of the hydroxyl radical as a chemical probe is that it effects DNA cleavage with no base-or sequence-specificity (6-8). The hydroxyl radical produces highly detailed footprints that yield information about DNA structure (4, 7) and protein-DNA interactions (3, 8, 9) at single-nucleotide resolution.Mechanistic information on the reaction of the hydroxyl radical with nucleic acids will benefit our understanding of radiation damage to DNA as well as the interpretation of chemical probe experiments. The extensive literature on the radiation chemistry of DNA (1) is a rich source of mechanistic possibilities. Not surprisingly, because of the high reactivity of the hydroxyl radical, a wide spectrum of products has been detected on treatment of the constituents of DNA (nucleic bases, nucleosides, nucleotides, or simple-sequence singlestranded DNA, for example) with ionizing radiation (1). It has been more difficult to conduct similarly detailed experiments on the biologically relevant duplex form of DNA. It is not hard to conceive, though, that the hydroxyl radical might react in a different manner with double-stranded DNA compared with simpler nucleic acid systems because the shape of the double helix would strongly influence the accessibility of the various COH bonds in DNA.Until now, the extent of cleavage at a particular nucleotide in a hydroxyl radical footprinting experiment only could be interpreted at th...

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