RESUMEN
The present work is aimed at gaining conclusive mechanistic insights into the radiation-induced formation of the 5'R and 5'S diastereomers of both adenine and guanine 5',8-cyclo-2'-deoxyribonucleosides, with emphasis on the delineation of the inhibitory effect of O(2) in isolated and cellular DNA. The levels of purine 5',8-cyclo-2'-deoxyribonucleosides as assessed by HPLC-MS/MS were found to decrease steadily with the increase of O(2) concentration, the 5',8-cyclo-2'-deoxyguanosine being produced more efficiently than the 5',8-cyclo-2'-deoxyadenosine for low O(2) concentrations. A high stereoselectivity was observed in the intramolecular addition of the C5' radical to the C8 of the purine leading, after the creation of the C5'-C8 bond and a subsequent oxidation step, to the predominant formation of the 5'R diastereomer for both purine 5',8-cyclonucleosides. The reduced formation yield of the 4 tandem lesions in the presence of O(2) explains, at least partly, the low efficiency of radiation-induced yields of the purine 5',8-cyclo-2'-deoxyribonucleosides in cellular DNA, which are about two orders of magnitude lower than the previously reported data obtained from HPLC-MS analysis.
Asunto(s)
ADN/metabolismo , Rayos gamma , Oxígeno/farmacología , Nucleósidos de Purina/metabolismo , Animales , Bovinos , Ciclización , ADN/genética , Daño del ADN , Reparación del ADN/efectos de los fármacos , Reparación del ADN/efectos de la radiación , Humanos , Radical Hidroxilo/metabolismo , Modelos Moleculares , Conformación Molecular , Monocitos/metabolismo , Monocitos/efectos de la radiación , Nucleósidos de Purina/química , Estereoisomerismo , Especificidad por Sustrato , Agua/químicaRESUMEN
The factors that influence the reactivity of C5' radicals in purine moieties under aerobic conditions are unknown not only in DNA, but also in simple nucleosides. 5',8-Cyclopurine lesions are the result of a rapid C5' radical attack to the purine moieties before the reaction with oxygen. These well-known lesions among the DNA modifications were suppressed by the presence of molecular oxygen in solution. Here we elucidate the chemistry of three purine-substituted C5' radicals (i.e., 2'-deoxyadenosin-5'-yl, 2'-deoxyinosin-5'-yl, and 2'-deoxyguanosin-5'-yl) under oxidative conditions using gamma-radiolysis coupled with product studies. 2'-Deoxyadenosin-5'-yl and 2'-deoxyinosin-5'-yl radicals were selectively generated by the reaction of hydrated electrons (e(aq)(-)) with 8-bromo-2'-deoxyadenosine and 8-bromo-2'-deoxyinosine followed by a rapid radical translocation from the C8 to the C5' position. Trapping these two C5' radicals with Fe(CN)6(3-) gave corresponding hydrated 5'-aldehydes in good yields that were isolated and fully characterized. When an oxygen concentration in the range of 13-266 microM (typical oxygenated tissues) is used, the hydrated 5'-aldehyde is accompanied by the 5',8-cyclopurine nucleoside. The formation of 5',8-cyclopurines is relevant in all experiments, and the yields increased with decreasing O2 concentration. The reaction of HO(*) radicals with 2'-deoxyadenosine and 2'-deoxyguanosine under normoxic conditions was also investigated. The minor path of C5' radicals formation was found to be ca. 10% by quantifying the hydrated 5'-aldehyde in both experiments. Rate constants for the reactions of the 2'-deoxyadenosin-5'-yl with cysteine and glutathione in water were determined by pulse radiolysis to be (2.1 +/- 0.5) x 10(7) and (4.9 +/- 0.6) x 10(7) M(-1) s(-1) at 22 degrees C, respectively.
Asunto(s)
Radicales Libres/química , Oxígeno/química , Nucleósidos de Purina/química , Desoxiadenosinas , Desoxiguanosina , Radical HidroxiloRESUMEN
Electrochemical study of oxetanes mimicking DNA (6-4) photoproducts gives new insight into the repair mechanism by (6-4) photolyase. Both electrochemical oxidation and electrochemical reduction at carbon electrodes lead to the cleavage of the oxetanes in a retro-Paterno-Büchi sequence. Within the family of compounds investigated and the range of driving forces offered, transient formation of unstable radical ions is observed, for both oxidative and reductive cleavage. Taking advantage of the electrochemical signature of these mimics, enzymatic assay with Escherichia coli CPD photolyase coupled to electrochemical monitoring of the reaction brings evidence that enzymatic repair of (6-4) DNA photoproducts does involve a catalytic dissociative electron-transfer mechanism at the level of an oxetane intermediate.
Asunto(s)
ADN/química , Electroquímica/métodos , Éteres Cíclicos/química , Luz , Estructura MolecularRESUMEN
Cyclic voltammetry was used to study the reduction and oxidation behaviour of several pyrimidine cyclobutane dimers mimicking UV induced lesion in DNA strands in polar solvents (N,N-dimethylformamide and acetonitrile). Both electron injection and removal to and from the dimers, respectively, lead to their cleavage and reformation of the monomeric base. The influence of stereochemistry and substitution pattern at the cyclobutane motif on the reactivity has been studied. It appears that the repair process always proceeds in a sequential fashion with initial formation of a dimer ion radical intermediate, which then undergoes ring opening by homolytic cleavage of the two C-C bonds. Standard redox potentials for the formation of both radical anion and radical cation state of the dimers were determined. Quantum calculations on simplified model compounds reveal the reason for the finding that the exergonic homolytic cleavages of the carbon-carbon bonds are endowed with sizeable activation barriers. The consequences of these mechanistic studies on the natural enzymatic repair by photolyase enzyme are discussed.