Oxygen Independent DNA Interstrand Cross-Link Formation by a Nucleotide Radical

Abstract

A 5-(2′-Deoxyuridinyl)methyl radical (1) was independently generated from three photochemical precursors and is the first example of a DNA radical that forms interstrand cross-links. Oxygen labeling experiments support generation of 1 by all precursors. Interstrand cross-links are produced upon irradiation of DNA containing any of the precursors. Cross-linking occurs via reaction with the opposing 2′-deoxyadenosine and is independent of O 2 . The independence of cross-link formation on O 2 is explained by kinetic analysis, which shows that the radical reacts reversibly with O 2 . Examination of the effects of glutathione on cross-link formation under anaerobic conditions suggests that adoption of the syn-conformation by 1 is the rate-limiting step in the process. Interstrand crosslink formation is reversible in the presence of a good nucleophile. The stability of the interstrand cross-link suggests that the isolated molecule is a rearrangement product of that formed in solution. The rearrangement is a consequence of the isolation procedure but also occurs slowly in solution. Oxygen independent cross-link formation may be useful for the purposeful damage of DNA in hypoxic tumor cells, where O 2 is deficient.The biological importance of DNA damage is evident in many ways. The double sword nature of these chemical transformations is illustrated by their involvement in the etiology and treatment of cancer. 1-3 In addition, there is a growing appreciation of the importance of DNA damage in aging. 4-6 DNA radicals are an important family of reactive intermediates that give rise to modified intact polymers and also lead to single-and double-strand breaks. 7-9 Recently, we reported that an independently generated 5-(2′-deoxyuridinyl)methyl radical (1) produced interstrand cross-links (ISCs) with an opposing 2′-deoxyadenosine (dA) (Scheme 1). 10 The radical is derived from formal hydrogen atom abstraction from the methyl group of thymidine and is not believed to be formed in large amounts by ionizing radiation despite the low bond dissociation energy (BDE) of the carbon-hydrogen bonds and their accessibility in the major groove. The preliminary report concerning 1 was the first explicit example of DNA interstrand cross-link (ISC) formation through a nucleic acid radical. ISCs are often responsible for the cytotoxic effects of DNA damaging agents. 11,12 Hence, ISC formation by a radical produced in even small amounts (e.g., 1) by agents such as γ-radiolysis could be biologically significant. Our interest in the radical-mediated cross-link formation is enhanced by the observation that the process involving 1 is independent of O 2 . This suggests that, in addition to being chemically novel, the formation of interstrand cross-links from 1 may be useful in hypoxic cells, such as those present in tumors. In addition to arising from direct hydrogen atom abstraction, the 5-(2′-deoxyuridinyl)methyl radical (1) can be formed from a two-step process, such as that induced by the direct effect of γ-radiolysis (Scheme 1). In a...

Replication termination mechanism as revealed by Tus-mediated polar arrest of a sliding helicase

“…Phenyl-selenide oligos were prepared as described (29,30,38). For all of the oligos used in this work and the details of the substrate construction see Table S1 and SI Text.…”

“…Using such a substrate one could ask the question as to whether DnaB would slide unimpeded over the interstrand cross-links and be arrested by the Tus-Ter complex in a polar mode. To perform this experiment, we synthesized a 24-mer oligo containing phenyl-selenide-dTTP (24⌽-Se B ) (29)(30)(31) at the positions corresponding to the T residues at coordinates 4 and 5 of the bottom strand of Ter. This oligo was used to construct the duplex 99B-24⌽-Se B (Fig.…”

5-Phenylselenyl- and 5-methylselenyl-methyl-2′-deoxyuridine induce oxidative stress, DNA damage, and caspase-2-dependent apoptosis in cancer cells

“…The olive tail moment (OTM), defined as the product of the percentage DNA in the tail and the displacement between the position of the mean centers of mass in the heads and tails, was used as a measure of DNA damage. For the alkaline comet assay, prior to electrophoresis the slides were immersed in freshly prepared alkaline solution (pH [ 13).…”

“…Previous studies have shown that phenylselenide derivative 5-phenylselenyl-methyl-2 0 -deoxyuridine (PhSe-T) can be incorporated into DNA by DNA polymerases and can produce ICLs with the opposing strand 2 0 -deoxyadenosine (dA) [10][11][12][13][14]. The nucleoside derivative 5-methylselenylmethyl-25-phenylselenyl-methyl-2 0 -deoxyuridine-deoxyuridine (MeSe-T) was recently synthesized following a similar synthetic method as PhSe-T, and its molecular structure is identical to that of PhSe-T, except for replacement of the phenyl group with a methyl group [15] (Fig.…”

Synthesis of Reactive Oligonucleotides for Gene Targeting and Their Application to Gene Expression Regulation

“…45,46 In addition, the oxidation of phenylselenide derivatives has induced crosslinking reactions with dA via [2,3]-sigmatropic rearrangement. 47,48 2¤-Deoxycytidine analogs have also formed crosslinks with the opposing deoxyguanosine (dG) under the same conditions. 49 TFO containing phenylselenide-substituted thymidine derivatives and 5-methyldeoxycytidine at terminal position have also been reported to undergo cross-linking reaction with dA and dG in the homopurine strand ( Figure 8).…”