Radical-induced purine lesion formation is dependent on DNA helical topology

Abstract

Herein we report the quantification of purine lesions arising from gamma-radiation sourced hydroxyl radicals (HO · ) on tertiary dsDNA helical forms of supercoiled (SC), open circular (OC), and linear (L) conformation, along with single-stranded folded and non-folded sequences of guanine-rich DNA in selected G-quadruplex structures. We identify that DNA helical topology and folding plays major, and unexpected, roles in the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) and 8-oxo-7,8-dihydro-2'-deoxyadenosine (8-oxo-dA), along with tandem-type purine lesions 5 (mutTel24) were exposed to HO · radicals and purine lesions were then quantified via stable isotope dilution LC-MS/MS analysis. Purine oxidation in dsDNA follows L > OC ) SC indicating greater damage towards the extended B-DNA topology. Conversely, G-quadruplex sequences were significantly more resistant toward purine oxidation in their unfolded states as compared with G-tetrad folded topologies; this effect is confirmed upon comparative analysis of Tel22 ($50% solution folded) and mutTel24 ($90% solution folded). In an effort to identify the accessibly of hydroxyl radicals to quadruplex purine nucleobases, G-quadruplex solvent cavities were then modeled at 1.33 Å with evidence suggesting that folded G-tetrads may act as potential oxidant traps to protect against chromosomal DNA damage. ARTICLE HISTORY

A phosphate-targeted dinuclear Cu(II) complex combining major groove binding and oxidative DNA cleavage

“…To shed further light on the DNA oxidation pathway, an enzyme-linked immunosorbent assay (ELISA) for the quantitation of 8-oxo-dG lesions was employed. 8-oxo-dG is an important marker for identifying diffusible hydroxyl radicals, which are known to preferentially attack guanosine ( 2 , 69 ) and previous studies have established correlation between DNA oxidation by copper bis -1,10-phenanthroline complexes and 8-oxo-dG formation ( 44 , 70 ). In this study, pUC19 DNA exposed to Cu 2 TPNap in the presence of added reductant (used to accelerate DNA oxidation, cf .…”

Membrane Lipidome Reorganization and Accumulation of Tissue DNA Lesions in Tumor-Bearing Mice: An Exploratory Study

“…50 μg isolated DNA were dissolved in 100 μL of Ar flushed 10 mM Tris-HCl (pH 7.9), containing 10 mM MgCl 2 , 50 mM NaCl, 0.2 mM pentostatin, 5 μM BHT and 3 mM deferoxamine and the internal standards were added ([ 15 N 5 ]-5′ S -cdA, [ 15 N 5 ]-5′ R -cdA, [ 15 N 5 ]-5′ S -cdG, [ 15 N 5 ]-5′R-cdG and [ 15 N 5 ]-8-oxo-dA) as previously described [21,22,67,68] (see Figure S1). 3 U of benzonase (in 20 mM Tris-HCl pH 8.0, 2 mM MgCl 2 and 20 mM NaCl), 4 mU phosphodiesterase I, 3 U DNAse I, 2 mU of phosphodiesterase II and 2 U of alkaline phosphatase were added and the mixture was incubated at 37 °C.…”

“…HPLC-UV clean-up and enrichment of the enzyme free samples were performed on a 4.6 mm × 150 mm Atlantis ® dC18 100 Å column (5 μm particle size, Waters, Milford, MA, USA), loaded with a 4.6 mm × 20 mm Guard Column 2 pK (Atlantis ® dC18 5 μm) on a Waters Alliance ® HPLC System (Waters e2695Separations Module, including a Waters 2998 Photodiode Array (PDA) detector as previously described [22]. The gradient program used an eluent composed by 2 mM ammonium formate, acetonitrile and methanol, while the fractions containing the lesions were collected, freeze-dried, pooled, freeze-dried again, and redissolved in Milli-Q water before been injected for LC-MS/MS analysis [21,67,68].…”

COST Action CM1201 “Biomimetic Radical Chemistry”: free radical chemistry successfully meets many disciplines

“…The rate constants for a unimolecular pathway were estimated to be ca. 5 Â 10 4 s À1 for both 2 0 -deoxyadenosin-5 0 -yl and 2 0 -deoxyguanosin-5 0 -yl radicals in ss-DNA, and about halved (2-3 Â10 4 s À1 ) in ds-DNA [44,45]. The same purine lesions were also quantified in order to individuate the influence given by different DNA helical topology and folding on the purine exposure to the damage [46].…”

“…A cost-effective and efficient protocol for the quantification of the four purine 5 0 ,8-cyclo-2 0 -deoxynucleosides together with the two 8-oxo derivatives (8-oxo-dA and 8-oxo-dG) in oxidized DNA was obtained [43] and applied to radiation-induced formation of purine lesions [44][45][46]. In single-and double-stranded DNA, the overall lesion formation follows the order: [44].…”