2‐Deoxyribonolactone Lesions in X‐ray‐Irradiated DNA: Quantitative Determination by Catalytic 5‐Methylene‐2‐furanone Release

Abstract

KeywordsDNA cleavage; DNA; oligonucleotides; X-rays The C1′-oxidized abasic DNA lesion 2-deoxyribonolactone (dL, 1) has attracted significant attention in recent years owing to its potential mutagenicity, which is associated with the ability of lesion 1 to misincorporate nucleotides during DNA synthesis, its resistance to base-excisionrepair (BER) enzymes, and its ability to form cross-links with BER enzymes efficiently. [1] Lesion 1 is produced in model systems by the action of a number of DNA-damaging agents such as copper(I)phenanthroline, [2] other metal complexes, [3] enediyne antibiotics, [4] as well as by UV light, [5] photosensitization, [6] and ionizing radiation. [7] However, the formation of 1 in vivo and its biological effects have been poorly documented. Until recently, lesion 1 in model systems has been identified primarily by detection of its low-molecular decomposition product 5-methylene-2-furanone (5-MF, 3). [2b,8] Only recently has a method for the selective detection of 1 been developed based on its reactivity with biotinylated cysteine. [9] Lesion 1 is relatively unstable; decomposition of 1, which is significantly accelerated by heating or alkaline treatment, results in strand scission and release of 3 (Scheme 1). [10][11][12] Decomposition of 1 is a two-step process: β elimination of the 3′-phosphate fragment forms butenolide 2, and δ elimination of the 5′-phosphate fragment produces 3 as the final product. The product 3 is unstable at basic pH values and hence cannot be detected upon alkaline decomposition of 1.In our recent paper [8] we demonstrated that 3, a characteristic decomposition product of 1, is released from X-irradiated calf thymus DNA upon postirradiation treatment with DNAphosphate-binding catalysts such as spermine. We hypothesized that 1 is a precursor to 3 in irradiated DNA and suggested that the yield of 1 in DNA can be quantified by using the yield of released 3.Herein we report the isolation of dL-containing tetramers from X-irradiated d(CGCG) and d (pCGCG) films, with the dL lesions corresponding to all four positions of the d-(C 1 G 2 C 3 G 4 ) tetramer. We demonstrate that these dL-containing tetramers undergo catalytic decomposition with quantitative release of 3. These results provide proof that lesion 1 is the major, perhaps only, precursor to 3 in irradiated highly polymerized DNA.

Selective Radiation-Induced Generation of 2-Deoxyribonolactone Lesions in DNA Mediated by Aromatic Iodonium Derivatives

“…The position of the lactone moiety in 10 was established by converting this product into pGATCG (m/e 1583.09 found, 1583.01 calculated) after 15 min at 90ЊC in PBS buffer containing 1 mM spermine tetrahydrochloride. Product 11 was resistant to spermine treatment, which is typical of a 3Ј-terminal deoxyribonolactone lacking the phosphate end group (36). As in the case of d(CGCG), the variety of damage products was greater at 45ЊC (Fig.…”

“…Reference deoxyribonolactones 5-8 ( Fig. 1) were isolated and identified in the previous study (36). Calf thymus DNA was from Sigma; all other reagents and HPLC-grade solvents were used as received.…”

“…, an oligodeoxynucleotide (4 mg/ ml) and sodium formate (hydroxyl radical scavenger, 20 mM) in PBS (pH 7.4). The irradiation products were separated by HPLC on a Luna C18 5-m, 4.6 ϫ 250-mm column under the conditions described previously (36) with minor variations in the acetonitrile gradient and run time. Preliminary identification of some products was performed using the authentic deoxyribonolactone-containing deoxyoligonucleotides 5-8 ( Fig.…”

“…Determination of L in calf thymus DNA was based on their quantitative conversion into 5-methylenefuranone (9) in neutral solutions at 70ЊC in the presence of spermine as a catalyst (36). It was found that concentration of 9 maximizes after 30 min incubation at this temperature in PBS buffer (pH 7.4), where most of the experiments were performed.…”

“…Then 150 l of the solution was mixed with an equal volume of 2 mM aqueous spermine tetrahydrochloride, incubated 30 min at 70ЊC, chilled in ice and centrifuged. The concentrations of unaltered bases and 9 in the supernatant were determined by reverse-phase HPLC as described previously (36) using uracil (U) as an internal standard. The radiation-chemical yields of deoxyribonolactone lesions G(L) and unaltered base release G(B) were calculated from the initial slopes of the corresponding dose-response plots.…”