Search Results (13)

A branch of the nucleotide excision repair (NER) pathway, transcription-coupled repair (TCR or TC-NER) specifically operates on the template DNA strand of actively transcribed genes. Initiated by stalling of elongating RNA polymerase complexes at damaged sites, TC-NER has historically been viewed as “accelerated repair”, arguably necessary for the maintenance of vital transcription function. Conversely, the conventional “global genome” (GG-NER) mechanism, operating throughout the genome, is usually regarded as a much slower process, even though it has long been found that differences in repair kinetics between transcribed DNA and the rest of the genome are not manifested for all structural types of DNA damage. Considering that damage detection is the rate-limiting step of overall repair reactions in most cases and that the mechanisms of the initial recognition of modified DNA structure are fundamentally different between TC-NER and GG-NER, it is suggestive to attribute the observed kinetic differences to different damage spectra recognized by the two pathways. This review summarizes current knowledge on the differential requirements of TC-NER and GG-NER towards specific damage types, based on their structural rather than spatial characteristics, and highlights some common features of DNA modifications repaired preferentially or exclusively by TC-NER, while evading other repair mechanisms. Full article

Most DNA damage caused by oxidative metabolism consists of single lesions that can accumulate in tissues. This review focuses on two classes of lesions: the two 8-oxopurine (8-oxo-Pu) lesions that are repaired by the base excision repair (BER) enzyme and the four 5′,8-cyclopurine (cPu) lesions that are repaired exclusively by the nucleotide excision repair (NER) enzyme. The aim is to correlate the simultaneous quantification of these two classes of lesions in the context of neurological disorders. The first half is a summary of reactive oxygen species (ROS) with particular attention to the pathways of hydroxyl radical (HO^•) formation, followed by a summary of protocols for the quantification of six lesions and the biomimetic chemistry of the HO^• radical with double-stranded oligonucleotides (ds-ODN) and calf thymus DNA (ct-DNA). The second half addresses two neurodegenerative diseases: xeroderma pigmentosum (XP) and Cockayne syndrome (CS). The quantitative data on the six lesions obtained from genomic and/or mitochondrial DNA extracts across several XP and CS cell lines are discussed. Oxidative stress contributes to oxidative DNA damage by resulting in the accumulation of cPu and 8-oxo-Pu in DNA. The formation of cPu is the postulated culprit inducing neurological symptoms associated with XP and CS. Full article

In recent years, nucleic acids have emerged as powerful biomaterials, revolutionizing the field of biomedicine. This review explores the multifaceted applications of nucleic acids, focusing on their pivotal role in various biomedical applications. Nucleic acids, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), possess unique properties such as molecular recognition ability, programmability, and ease of synthesis, making them versatile tools in biosensing and for gene regulation, drug delivery, and targeted therapy. Their compatibility with chemical modifications enhances their binding affinity and resistance to degradation, elevating their effectiveness in targeted applications. Additionally, nucleic acids have found utility as self-assembling building blocks, leading to the creation of nanostructures whose high order underpins their enhanced biological stability and affects the cellular uptake efficiency. Furthermore, this review delves into the significant role of oligonucleotides (ODNs) as indispensable tools for biological studies and biomarker discovery. ODNs, short sequences of nucleic acids, have been instrumental in unraveling complex biological mechanisms. They serve as probes for studying gene expression, protein interactions, and cellular pathways, providing invaluable insights into fundamental biological processes. By examining the synergistic interplay between nucleic acids as powerful biomaterials and ODNs as indispensable tools for biological studies and biomarkers, this review highlights the transformative impact of these molecules on biomedical research. Their versatile applications not only deepen our understanding of biological systems but also are the driving force for innovation in diagnostics and therapeutics, ultimately advancing the field of biomedicine. Full article

Mitochondrial (mt) DNA and nuclear (n) DNA have known structures and roles in cells; however, they are rarely compared under specific conditions such as oxidative or degenerative environments that can create damage to the DNA base moieties. Six purine lesions were ascertained in the mtDNA of wild type (wt) CSA (CS3BE–wtCSA) and wtCSB (CS1AN–wtCSB) cells and defective counterparts CS3BE and CS1AN in comparison with the corresponding total (t) DNA (t = n + mt). In particular, the four 5′,8–cyclopurine (cPu) and the two 8–oxo–purine (8–oxo–Pu) lesions were accurately quantified by LC–MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. The 8–oxo–Pu levels were found to be in the range of 25–50 lesions/10⁷ nucleotides in both the mtDNA and tDNA. The four cPu were undetectable in the mtDNA both in defective cells and in the wt counterparts (CSA and CSB), contrary to their detection in tDNA, indicating a nonappearance of hydroxyl radical (HO^•) reactivity within the mtDNA. In order to assess the HO^• reactivity towards purine nucleobases in the two genetic materials, we performed γ–radiolysis experiments coupled with the 8–oxo–Pu and cPu quantifications on isolated mtDNA and tDNA from wtCSB cells. In the latter experiments, all six purine lesions were detected in both of the DNA, showing a higher resistance to HO^• attack in the case of mtDNA compared with tDNA, likely due to their different DNA helical topology influencing the relative abundance of the lesions. Full article

Ionizing radiation is a factor that seriously damages cellular mechanisms/macromolecules, e.g., by inducing damage in the human genome, such as 5′,8-cyclo-2′-deoxypurines (cdPus). CdPus may become a component of clustered DNA lesions (CDL), which are notably unfavorable for the base excision repair system (BER). In this study, the influence of 5′S and 5′R diastereomers of 5′,8-cyclo-2′-deoxyadenosine (cdA) and 5′,8-cyclo-2′-deoxyguanosine (cdG) on the uracil-DNA glycosylase (UDG) and human AP site endonuclease 1 (hAPE1) activity has been taken under consideration. Synthetic oligonucleotides containing 2′-deoxyuridine (dU) and cdPu were used as a model of single-stranded CDL. The activity of the UDG and hAPE1 enzymes decreased in the presence of RcdG compared to ScdG. Contrary to the above, ScdA reduced enzyme activity more than RcdA. The presented results show the influence of cdPus lesions located within CDL on the activity of the initial stages of BER dependently on their position toward dU. Numerous studies have shown the biological importance of cdPus (e.g., as a risk of carcinogenesis). Due to that, it is important to understand how to recognize and eliminate this type of DNA damage from the genome. Full article

Nucleotide excision repair (NER) is the most versatile DNA repair pathway, which can remove diverse bulky DNA lesions destabilizing a DNA duplex. NER defects cause several autosomal recessive genetic disorders. Xeroderma pigmentosum (XP) is one of the NER-associated syndromes characterized by low efficiency of the removal of bulky DNA adducts generated by ultraviolet radiation. XP patients have extremely high ultraviolet-light sensitivity of sun-exposed tissues, often resulting in multiple skin and eye cancers. Some XP patients develop characteristic neurodegeneration that is believed to derive from their inability to repair neuronal DNA damaged by endogenous metabolites. A specific class of oxidatively induced DNA lesions, 8,5′-cyclopurine-2′-deoxynucleosides, is considered endogenous DNA lesions mainly responsible for neurological problems in XP. Growing evidence suggests that XP is accompanied by defective mitophagy, as in primary mitochondrial disorders. Moreover, NER pathway is absent in mitochondria, implying that the mitochondrial dysfunction is secondary to nuclear NER defects. In this review, we discuss the current understanding of the NER molecular mechanism and focuses on the NER linkage with the neurological degeneration in patients with XP. We also present recent research advances regarding NER involvement in oxidative DNA lesion repair. Finally, we highlight how mitochondrial dysfunction may be associated with XP. Full article

In this work, we used ωB97XD density functional and 6-31++G** basis set to study the structure, electron affinity, populations via Boltzmann distribution, and one-electron reduction potentials (E°) of 2′-deoxyribose sugar radicals in aqueous phase by considering 2′-deoxyguanosine and 2′-deoxythymidine as a model of DNA. The calculation predicted the relative stability of sugar radicals in the order C4′^• > C1′^• > C5′^• > C3′^• > C2′^•. The Boltzmann distribution populations based on the relative stability of the sugar radicals were not those found for ionizing radiation or OH-radical attack and are good evidence the kinetic mechanisms of the processes drive the products formed. The adiabatic electron affinities of these sugar radicals were in the range 2.6–3.3 eV which is higher than the canonical DNA bases. The sugar radicals reduction potentials (E°) without protonation (−1.8 to −1.2 V) were also significantly higher than the bases. Thus the sugar radicals will be far more readily reduced by solvated electrons than the DNA bases. In the aqueous phase, these one-electron reduced sugar radicals (anions) are protonated from solvent and thus are efficiently repaired via the “electron-induced proton transfer mechanism”. The calculation shows that, in comparison to efficient repair of sugar radicals by the electron-induced proton transfer mechanism, the repair of the cyclopurine lesion, 5′,8-cyclo-2′-dG, would involve a substantial barrier. Full article

Cockayne Syndrome (CS) is an autosomal recessive neurodegenerative premature aging disorder associated with defects in nucleotide excision repair (NER). Cells from CS patients, with mutations in CSA or CSB genes, present elevated levels of reactive oxygen species (ROS) and are defective in the repair of a variety of oxidatively generated DNA lesions. In this study, six purine lesions were ascertained in wild type (wt) CSA, defective CSA, wtCSB and defective CSB-transformed fibroblasts under different oxygen tensions (hyperoxic 21%, physioxic 5% and hypoxic 1%). In particular, the four 5′,8-cyclopurine (cPu) and the two 8-oxo-purine (8-oxo-Pu) lesions were accurately quantified by LC-MS/MS analysis using isotopomeric internal standards after an enzymatic digestion procedure. cPu levels were found comparable to 8-oxo-Pu in all cases (3–6 lesions/10⁶ nucleotides), slightly increasing on going from hyperoxia to physioxia to hypoxia. Moreover, higher levels of four cPu were observed under hypoxia in both CSA and CSB-defective cells as compared to normal counterparts, along with a significant enhancement of 8-oxo-Pu. These findings revealed that exposure to different oxygen tensions induced oxidative DNA damage in CS cells, repairable by NER or base excision repair (BER) pathways. In NER-defective CS patients, these results support the hypothesis that the clinical neurological features might be connected to the accumulation of cPu. Moreover, the elimination of dysfunctional mitochondria in CS cells is associated with a reduction in the oxidative DNA damage. Full article

Xeroderma Pigmentosum (XP) is a DNA repair disease characterized by nucleotide excision repair (NER) malfunction, leading to photosensitivity and increased incidence of skin malignancies. The role of XP-A in NER pathways has been well studied while discrepancies associated with ROS levels and the role of radical species between normal and deficient XPA cell lines have been observed. Using liquid chromatography tandem mass spectrometry we have determined the four 5’,8-cyclopurines (cPu) lesions (i.e., 5′R-cdG, 5′S-cdG, 5′R-cdA and 5′S-cdA), 8-oxo-dA and 8-oxo-dG in wt (EUE-pBD650) and XPA-deficient (EUE-siXPA) human embryonic epithelial cell lines, under different oxygen tension (hyperoxic 21%, physioxic 5% and hypoxic 1%). The levels of Fe and Cu were also measured. The main findings of our study were: (i) the total amount of cPu (1.82–2.52 lesions/10⁶ nucleotides) is the same order of magnitude as 8-oxo-Pu (3.10–4.11 lesions/10⁶ nucleotides) in both cell types, (ii) the four cPu levels are similar in hyperoxic and physioxic conditions for both wt and deficient cell lines, whereas 8-oxo-Pu increases in all cases, (iii) both wt and deficient cell lines accumulated high levels of cPu under hypoxic compared to physioxic conditions, whereas the 8-oxo-Pu levels show an opposite trend, (iv) the diastereoisomeric ratios 5′R/5′S are independent of oxygen concentration being 0.29 for cdG and 2.69 for cdA for EUE-pBD650 (wt) and 0.32 for cdG and 2.94 for cdA for EUE-siXPA (deficient), (v) in deficient cell lines Fe levels were significantly higher. The data show for the first time the connection of oxygen concentration in cells with different DNA repair ability and the levels of different DNA lesions highlighting the significance of cPu. Membrane lipidomic data at 21% O₂ indicated differences in the fatty acid contents between wild type and deficient cells, envisaging functional effects on membranes associated with the different repair capabilities, to be further investigated. Full article

The reaction of hydroxyl radical (HO^•) with DNA produces many primary reactive species and many lesions as final products. In this study, we have examined the optical spectra of intermediate species derived from the reaction of HO^• with a variety of single- and double-stranded oligodeoxynucleotides and ct-DNA in the range of 1 μs to 1 ms by pulse radiolysis using an Intensified Charged Coupled Device (ICCD) camera. Moreover, we applied our published analytical protocol based on an LC-MS/MS system with isotopomeric internal standards to enable accurate and precise measurements of purine lesion formation. In particular, the simultaneous measurement of the four purine 5′,8-cyclo-2′-deoxynucleosides (cPu) and two 8-oxo-7,8-dihydro-2′-deoxypurine (8-oxo-Pu) was obtained upon reaction of genetic material with HO^• radicals generated either by γ-radiolysis or Fenton-type reactions. Our results contributed to the debate in the literature regarding absolute level of lesions, method of HO^• radical generation, 5′R/5′S diastereomeric ratio in cPu, and relative abundance between cPu and 8-oxo-Pu. Full article

DNA lesions are formed continuously in each living cell as a result of environmental factors, ionisation radiation, metabolic processes, etc. Most lesions are removed from the genome by the base excision repair system (BER). The activation of the BER protein cascade starts with DNA damage recognition by glycosylases. Uracil-DNA glycosylase (UDG) is one of the most evolutionary preserved glycosylases which remove the frequently occurring 2′-deoxyuridine from single (ss) and double-stranded (ds) oligonucleotides. Conversely, the unique tandem lesions (5′R)- and (5′S)-5′,8-cyclo-2′-deoxyadenosine (cdA) are not suitable substrates for BER machinery and are released from the genome by the nucleotide excision repair (NER) system. However, the cyclopurines appearing in a clustered DNA damage structure can influence the BER process of other lesions like dU. In this article, UDG inhibition by 5′S- and 5′R-cdA is shown and discussed in an experimental and theoretical manner. This phenomenon was observed when a tandem lesion appears in single or double-stranded oligonucleotides next to dU, on its 3′-end side. The cdA shift to the 5′-end side of dU in ss-DNA stops this effect in both cdA diastereomers. Surprisingly, in the case of ds-DNA, 5′S-cdA completely blocks uracil excision by UDG. Conversely, 5′R-cdA allows glycosylase for uracil removal, but the subsequently formed apurinic/apyrimidinic (AP) site is not suitable for human AP-site endonuclease 1 (hAPE1) activity. In conclusion, the appearance of the discussed tandem lesion in the structure of single or double-stranded DNA can stop the entire base repair process at its beginning, which due to UDG and hAPE1 inhibition can lead to mutagenesis. On the other hand, the presented results can cast some light on the UDG or hAPE1 inhibitors being used as a potential treatment. Full article

Purine 5′,8-cyclo-2′-deoxynucleosides (cPu) are tandem-type lesions observed among the DNA purine modifications and identified in mammalian cellular DNA in vivo. These lesions can be present in two diasteroisomeric forms, 5′R and 5′S, for each 2′-deoxyadenosine and 2′-deoxyguanosine moiety. They are generated exclusively by hydroxyl radical attack to 2′-deoxyribose units generating C5′ radicals, followed by cyclization with the C8 position of the purine base. This review describes the main recent achievements in the preparation of the cPu molecular library for analytical and DNA synthesis applications for the studies of the enzymatic recognition and repair mechanisms, their impact on transcription and genetic instability, quantitative determination of the levels of lesions in various types of cells and animal model systems, and relationships between the levels of lesions and human health, disease, and aging, as well as the defining of the detection limits and quantification protocols. Full article

5’,8-Cyclo-2’-deoxyadenosine (cdA), in the 5’R and 5’Sdiastereomeric forms, are typical non strand-break oxidative DNA lesions, induced by hydroxyl radicals, with emerging importance as a molecular marker. These lesions are exclusively repaired by the nucleotide excision repair (NER) mechanism with a low efficiency, thus readily accumulating in the genome. Poly(ADP-ribose) polymerase1 (PARP1) acts as an early responder to DNA damage and plays a key role as a nick sensor in the maintenance of the integrity of the genome by recognizing nicked DNA. So far, it was unknown whether the two diastereomeric cdA lesions could induce specific PARP1 binding. Here, we provide the first evidence of PARP1 to selectively recognize the diastereomeric lesions of 5’S-cdA and 5’R-cdA in vitro as compared to deoxyadenosine in model DNA substrates (23-mers) by using circular dichroism, fluorescence spectroscopy, immunoblotting analysis, and gel mobility shift assay. Several features of the recognition of the damaged and undamaged oligonucleotides by PARP1 were characterized. Remarkably, PARP1 exhibits different affinities in binding to a double strand (ds) oligonucleotide, which incorporates cdA lesions in R and S diastereomeric form. In particular, PARP1 proved to bind oligonucleotides, including a 5’S-cdA, with a higher affinity constant for the 5’S lesion in a model of ds DNA than 5’R-cdA, showing different recognition patterns, also compared with undamaged dA. This new finding highlights the ability of PARP1 to recognize and differentiate the distorted DNA backbone in a biomimetic system caused by different diastereomeric forms of a cdA lesion. Full article