Oxidative DNA damage is an inevitable consequence of cellular metabolism, with a propensity for increased levels following toxic insult. Although more than 20 base lesions have been identified, only a fraction of these have received appreciable study, most notably 8-oxo-2'deoxyguanosine. This lesion has been the focus of intense research interest and been ascribed much importance, largely to the detriment of other lesions. The present work reviews the basis for the biological significance of oxidative DNA damage, drawing attention to the multiplicity of proteins with repair activities along with a number of poorly considered effects of damage. Given the plethora of (often contradictory) reports describing pathological conditions in which levels of oxidative DNA damage have been measured, this review critically addresses the extent to which the in vitro significance of such damage has relevance for the pathogenesis of disease. It is suggested that some shortcomings associated with biomarkers, along with gaps in our knowledge, may be responsible for the failure to produce consistent and definitive results when applied to understanding the role of DNA damage in disease, highlighting the need for further studies.

Oxidative DNA damage: mechanisms, mutation, and disease

The generation of reactive oxygen species may be both beneficial to cells, performing a function in inter- and intracellular signalling, and detrimental, modifying cellular biomolecules, accumulation of which has been associated with numerous diseases. Of the molecules subject to oxidative modification, DNA has received the greatest attention, with biomarkers of exposure and effect closest to validation. Despite nearly a quarter of a century of study, and a large number of base- and sugar-derived DNA lesions having been identified, the majority of studies have focussed upon the guanine modification, 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-OH-dG). For the most part, the biological significance of other lesions has not, as yet, been investigated. In contrast, the description and characterisation of enzyme systems responsible for repairing oxidative DNA base damage is growing rapidly, being the subject of intense study. However, there remain notable gaps in our knowledge of which repair proteins remove which lesions, plus, as more lesions identified, new processes/substrates need to be determined. There are many reports describing elevated levels of oxidatively modified DNA lesions, in various biological matrices, in a plethora of diseases; however, for the majority of these the association could merely be coincidental, and more detailed studies are required. Nevertheless, even based simply upon reports of studies investigating the potential role of 8-OH-dG in disease, the weight of evidence strongly suggests a link between such damage and the pathogenesis of disease. However, exact roles remain to be elucidated.

Oxidative DNA damage and disease: induction, repair and significance

Electrostatic Basis for Enzyme Catalysis

DNA is one of the prime molecules, and its stability is of utmost importance for proper functioning and existence of all living systems. Genotoxic chemicals and radiations exert adverse effects on genome stability. Ultraviolet radiation (UVR) (mainly UV-B: 280–315 nm) is one of the powerful agents that can alter the normal state of life by inducing a variety of mutagenic and cytotoxic DNA lesions such as cyclobutane-pyrimidine dimers (CPDs), 6-4 photoproducts (6-4PPs), and their Dewar valence isomers as well as DNA strand breaks by interfering the genome integrity. To counteract these lesions, organisms have developed a number of highly conserved repair mechanisms such as photoreactivation, base excision repair (BER), nucleotide excision repair (NER), and mismatch repair (MMR). Additionally, double-strand break repair (by homologous recombination and nonhomologous end joining), SOS response, cell-cycle checkpoints, and programmed cell death (apoptosis) are also operative in various organisms with the expense of specific gene products. This review deals with UV-induced alterations in DNA and its maintenance by various repair mechanisms.

https://downloads.hindawi.com/journals/jna/2010/592980.pdf

Molecular Mechanisms of Ultraviolet Radiation-Induced DNA Damage and Repair

In this survey, emphasis was placed on the main photoreactions of nucleic acid components, involving both direct and indirect effects. The main UVB- and UVA-induced DNA photoproducts, together with the mechanisms of their formation, are described. Information on the photoproduct distribution within cellular DNA is also provided, taking into account the limitations of the different analytical methods applied to monitor the formation of the DNA damage. Thus, the formation of the main DNA dimeric pyrimidine lesions produced by direct absorption of UVB photons was assessed using a powerful HPLC-tandem mass spectrometry assay. In addition, it was found that UVA photooxidation damage mostly involves the guanine residues of cellular DNA as the result of singlet oxygen generation by still unknown endogenous photosensitizers.

Direct and indirect effects of UV radiation on DNA and its components.

The formation of monomeric products in DNA upon exposure to UV radiation was investigated. Three novel products were identified in DNA in aqueous solution upon exposure to UV radiation at 254 nm in a dose range from 100 to 10 000 J/m{sup 2}. These were 4,6-diamino-5-formamidopyrimidine, 2,6-diamino-4-hydroxy-5-formamidopyrimidine, and 5-hydroxy-5,6-dihydrothymine. These three products are known to be substrates for base excision repair enzymes involved in the reversal of oxidative DNA damage. The dependence of the yields of formamidopyrimidines on UV radiation dose was nonlinear, whereas the yield of 5-hydroxy-5,6-dihydrothymine was increased linearly in the entire dose range. Of these products, 4,6-diamino-5-formamidopyrimidine was the only compound produced in appreciable amounts at 310 nm. At the highest dose used, the formation of other pyrimidine- and purine-derived products was also observed. Their amounts, however, were increased above control levels up to 2-fold only. The hydroxyl radical scavenger dimethyl sulfoxide has no effect on product yields excluding the involvement of hydroxyl radical in product formation. 4,6-Diamino-5-formamidopyrimidine and 2,6-diamino-4-hydroxy-5-formamidopyrimidine may be produced by hydration of adenine and guanine, respectively, across the N(7)-C(8) double bond by mechanisms similar to those proposed previously for well-known formation of pyrimidine hydrates with the hydroxyl group located at C(6). Formation ofmore » 5-hydroxy-5,6-dihydrothymine indicates that hydration of thymine with the hydroxyl group located at C(5) of the pyrimidine ring also occurs. The results of these studies indicate that these monomeric base damage products could be a biologically important component of the photoproducts that are responsible for the deleterious effects of UV radiation. 50 refs., 5 figs.« less

Monomeric base damage products from adenine, guanine, and thymine induced by exposure of DNA to ultraviolet radiation.

Conformational transitions and structural deformability of EcoRV endonuclease revealed by crystallographic analysis.

Natural-abundance 13C-NMR spectra of [d(TCGCG)] (1), [d(CGCGCG)]2 (2), and [d(GGTATACC)]2 (3) were measured at 906 MHz to obtain 13C-1H NOEs and T1 relaxation times; relaxation data were also measured at 1257 MHz for 1 and 2 and at 629 MHz for 1 Analysis of the relaxation data was performed in the context of the "model-free" approach of Lipari and Szabo [Lipari, G, & Szabo, A (1982) J Am Chem Soc 104, 4546-4559], leading to the following conclusions: (i) Optimized values for the overall correlation times of 09 ns for 1 and 14 ns for 2 are close to those predicted by light-scattering results on similar molecules [Eimer et al (1990) Biochemistry 29, 799-811] (ii) For the nonterminal residues, the "order parameter", S2, is around 08 for the protonated base carbons and 06 for the sugar carbons, indicating less spatial restriction on the sugar carbons (in the model-free approach, the order parameter is 1 for a rigid body and 0 for a system with completely unrestricted internal motion) (iii) The order parameters for the terminal residues vary over a wide range with the smallest values around 02-03 for the HO-13C5' and the 13C3'-OH; rational trends are seen in the variation of S2 with chain position in the terminal residues (iv) The analysis shows that the order parameters are accurate within 15% (v) The "effective internal correlation time", tau e, is very short for the sugar carbons (30-300 ps) and less well-defined, but probably also short, for the bases (vi) The analysis indicates that most of the relaxation in DNA is accounted for by S2 and the tau e is so short that a good approximation to any relaxation property, P (eg, T1, T2, 13C-1H NOE, 1H-1H cross-relaxation rate), is P = S2Prigid, where Prigid is the value for the property in a system without internal motion (the analysis assumes the same isotropic overall motion for both the rigid and flexible bodies)

13C-NMR Relaxation in Three DNA Oligonucleotide Duplexes: Model-Free Analysis of Internal and Overall Motion

The complete catalytic cycle of EcoRV endonuclease has been observed by combining fluorescence anisotropy with fluorescence resonance energy transfer (FRET) measurements. Binding, bending, and cleavage of substrate oligonucleotides were monitored in real time by rhodamine-x anisotropy and by FRET between rhodamine and fluorescein dyes attached to opposite ends of a 14-mer DNA duplex. For the cognate GATATC site binding and bending are found to be nearly simultaneous, with association and bending rate constants of (1.45-1.6) x 10(8) M(-1) s(-1). On the basis of the measurement of k(off) by a substrate-trapping approach, the equilibrium dissociation constant of the enzyme-DNA complex in the presence of inhibitory calcium ions was calculated as 3.7 x 10(-12) M from the kinetic constants. Further, the entire DNA cleavage reaction can be observed in the presence of catalytic Mg(2+) ions. These measurements reveal that the binding and bending steps occur at equivalent rates in the presence of either Mg(2+) or Ca(2+), while a slow decrease in fluorescence intensity following bending corresponds to k(cat), which is limited by the cleavage and product dissociation steps. Measurement of k(on) and k(off) in the absence of divalent metals shows that the DNA binding affinity is decreased by 5000-fold to 1.4 x 10(-8) M, and no bending could be detected in this case. Together with crystallographic studies, these data suggest a model for the induced-fit conformational change in which the role of divalent metal ions is to stabilize the sharply bent DNA in an orientation suitable for accessing the catalytic transition state.

Simultaneous DNA Binding and Bending by EcoRV Endonuclease Observed by Real-Time Fluorescence †

Specific recognition by EcoRV endonuclease of its cognate, sharply bent GATATC site at the center TA step occurs solely via hydrophobic interaction with thymine methyl groups Mechanistic kinetic analyses of base analog-substituted DNAs at this position reveal that direct readout provides 5 kcal mol–1 toward specificity, with an additional 6–10 kcal mol–1 arising from indirect readout Crystal structures of several base analog complexes show that the major-groove hydrophobic contacts are crucial to forming required divalent metal-binding sites, and that indirect readout operates in part through the sequence-dependent free-energy cost of unstacking the center base-pair step of the DNA

Amy M. Martin

Papers

Monomeric base damage products from adenine, guanine, and thymine induced by exposure of DNA to ultraviolet radiation.

Conformational transitions and structural deformability of EcoRV endonuclease revealed by crystallographic analysis.

13C-NMR Relaxation in Three DNA Oligonucleotide Duplexes: Model-Free Analysis of Internal and Overall Motion

Simultaneous DNA Binding and Bending by EcoRV Endonuclease Observed by Real-Time Fluorescence †

Structural and energetic origins of indirect readout in site-specific DNA cleavage by a restriction endonuclease.