Molecular simulations reveal a common binding mode for glycosylase binding of oxidatively damaged DNA lesions

Cellular DNA is constantly exposed to oxidative stress from both exogenous and endogenous sources, creating lesions that lead to aging related diseases, including cancer. 8-Oxo-guanine (8OG) is one of the most common forms of oxidative DNA damage, and failure to repair this lesion results in G:C to T:A transversion. Another common lesion, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapydG), shares the same precursor as 8OG. In Escherichia coli, both lesions are recognized and excised by the DNA glycosylase Fpg. X-ray crystallographic studies have shown that FapydG and 8OG adopt different conformations in the active site of Fpg. Our simulations suggest that the different binding modes observed for 8OG and FapydG arise directly from response to the nonconserved E77 present in the thermophilic Fpg sequences used for the crystallography experiments. In simulations with consensus S77, these lesions adopt very similar binding modes.     

Possible cause of G-C-->C-G transversion mutation by guanine oxidation product, imidazolone

BACKGROUND: The genome is constantly assaulted by oxidation reactions which are likely to be associated with oxygen metabolism, and oxidative lesions are generated by many types of oxidants. Such genotoxin-induced alterations in the genomic message have been implicated in aging and in several pathophysiological processes, particularly those associated with cancer. The guanine base (G) in genomic DNA is highly susceptible to oxidative stress due to having the lowest oxidation potential. Therefore, G-C-->T-A and G-C-->C-G transversion mutations frequently occur under oxidative conditions. One typical lesion of G is 8-oxo-7,8-dihydro-guanine (8-oxoG), which can pair with A. This pairing may cause G-C-->T-A transversion mutations. Although the number of G-C-->C-G transversions is rather high under specific oxidation conditions such as riboflavin photosensitization, the molecular basis of G-C-->C-G transversions is not known. RESULTS: To determine which oxidative products are responsible for G-C-->C-G transversion mutations, we photooxidized 5'-d(AAAAAAGGAAAAAA)/5'-d(TTTTTTCCTTTTTT) using either riboflavin or anthraquinone (AQ) carboxylate under UV irradiation. Prolonged low-temperature (4 degrees C) enzymatic digestion of photoirradiated sample indicated that under both conditions the amount of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) initially increased with decreasing amounts of 2'-deoxyguanosine (dG), then decreased with the formation of 2-amino-5-[(2-deoxy-beta-D-erythro-pentofuranosyl)amino]-4H-imidazol-4-one (dIz), suggesting that nascent 8-oxoG was further oxidized to 2,5-diamino-4H-imidazol-4-one (Iz) in duplex DNA. Photoirradiation of an AQ-linked oligomer with a complementary strand containing 8-oxoG indicated that 8-oxoG residues were oxidized to Iz. These results indicate that Iz is formed from 8-oxoG through long-range hole migration. Primer extension experiments using a template containing Iz demonstrated that only dGTP is specifically incorporated opposite Iz suggesting that specific Iz-G base pairs are formed. The 'reverse' approach consisting of DNA polymerization using dIzTP showed that dIzTP is incorporated opposite G, further confirming the formation of a Iz-G base pair. CONCLUSIONS: HPLC product analysis demonstrated that Iz is a key oxidation product of G through 8-oxoG in DNA photosensitized with riboflavin or anthraquinone. Photoreaction of AQ-linked oligomer confirmed that Iz is formed from 8-oxoG through long-range hole migration. Two sets of primer extension experiments demonstrated that Iz can specifically pair with G in vitro. Specific Iz-G base pair formation can explain the G-C-->C-G transversion mutations that appear under oxidative conditions.     

Discovery and synthesis of new UV-induced intrastrand C(4-8)G and G(8-4)C photolesions

UV irradiation of cellular DNA leads to the formation of a number of defined mutagenic DNA lesions. Here we report the discovery of new intrastrand C(4-8)G and G(8-4)C cross-link lesions in which the C(4) amino group of the cytosine base is covalently linked to the C(8) position of an adjacent dG base. The structure of the novel lesions was clarified by HPLC-MS/MS data for UV-irradiated DNA in combination with chemical synthesis and direct comparison of the synthetic material with irradiated DNA. We also report the ability to generate the lesions directly in DNA with the help of a photoactive precursor that was site-specifically incorporated into DNA. This should enable detailed chemical and biochemical investigations of these lesions.     

Effects of covalent modification by 4‐hydroxy‐2‐nonenal on the noncovalent oligomerization of ubiquitin

When lipid membranes containing ω‐6 polyunsaturated fatty acyl chains are subjected to oxidative stress, one of the reaction products is 4‐hydroxy‐2‐nonenal (HNE)—a chemically reactive short chain alkenal that can covalently modify proteins. The ubiquitin proteasome system is involved in the clearing of proteins modified by oxidation products such as HNE, but the chemical structure, stability and function of ubiquitin may be impaired by HNE modification. To evaluate this possibility, the susceptibility of ubiquitin to modification by HNE has been characterized over a range of concentrations where ubiquitin forms non‐covalent oligomers. Results indicate that HNE modifies ubiquitin at only two of the many possible sites, and that HNE modification at these two sites alters the ubiquitin oligomerization equilibrium. These results suggest that any role ubiquitin may have in clearing proteins damaged by oxidative stress may itself be impaired by oxidative lipid degradation products. Copyright © 2016 John Wiley & Sons, Ltd.     

Base pairing and replicative processing of the formamidopyrimidine-dG DNA lesion

The 2,6-diamino-4-hydroxy-5-formamidopyrimidine of 2'-deoxyguanosine (FaPydG) is one of the major DNA lesions found after oxidative stress in cells. To clarify the base pairing and coding potential of this major DNA lesion with the aim to estimate its mutagenic effect, we prepared oligonucleotides containing a cyclopentane based analogue of the DNA lesion (cFaPydG). In addition, oligonucleotides containing the cyclopentane analogue of 2'-deoxyguanosine (cdG), and oligonucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) were synthesized. The thermodynamic stability of duplexes containing these building blocks and all canonical counterbases were determined by concentration dependent melting-point measurements (van't Hoff plots). The data reveal that cFaPydG greatly destabilizes a DNA duplex (DeltaDeltaG degrees (298K) approximately 2-4 kcal mol(-1)). The optimal base pairing partner for the cFaPydG lesion is dC. Investigation of duplexes containing dG and cdG shows that the effect of substituting the deoxyribose by a cyclopentane moiety is marginal. The data also provide strong evidence that the FaPydG lesion is unable to form a base pair with dA. Our computational studies indicate that the syn-conformation required for base pairing with dA is energetically unfavorable. This is in contrast to 8-oxodG for which the syn-conformation represents the energetic minimum. Kinetic primer extension studies using S. cerevisiae Pol eta reveal that cFaPydG is replicated in an error-free fashion. dC is inserted 2-3 orders of magnitude more efficiently than dT or dA, showing that FaPydG is a lesion which retains the coding potential of dG. This is also in contrast to 8-oxodG, for which base pairing with dC and dA was established.     

2',3'-二脱氧-2',3'-二去氢鸟嘌呤核苷构象稳定性的理论研究

采用密度泛函方法在B3LYP/6-31＋G**水平上研究了2',3'-二脱氧-2',3'-二去氢鸟嘌呤核苷分子(D4G)的构象. 分别研究在气相中的孤立分子和一水合物异构体的相对稳定性和异构体之间的相互转变过程, 分析了水分子的参与对D4G异构体的相对稳定性和几何结构参数以及自然电荷的影响. 结果表明, 孤立的D4G分子在气相中存在8种稳定构象, 其中构象d4g-2是所有构象中最稳定的, 气相中D4G主要以d4g-2存在. 气相中各构象的相对稳定性为: d4g-2＞d4g-1＞d4g-5＞d4g-3＞d4g-6＞d4g-4＞d4g-8＞d4g-7. 计算得到的各构象键长和键角数据与实验值接近. 一个水分子的加入对D4G分子的构型参数有所影响, 基本不改变D4G分子各构象的稳定性顺序, 但构象转变的能垒有所提高. 氢键在分子构象中发挥了重要作用.     

2',3'-二脱氧-2',3'-二去氢鸟嘌呤核苷构象稳定性的理论研究

采用密度泛函方法在B3LYP/6-31＋G**水平上研究了2',3'-二脱氧-2',3'-二去氢鸟嘌呤核苷分子(D4G)的构象. 分别研究在气相中的孤立分子和一水合物异构体的相对稳定性和异构体之间的相互转变过程, 分析了水分子的参与对D4G异构体的相对稳定性和几何结构参数以及自然电荷的影响. 结果表明, 孤立的D4G分子在气相中存在8种稳定构象, 其中构象d4g-2是所有构象中最稳定的, 气相中D4G主要以d4g-2存在. 气相中各构象的相对稳定性为: d4g-2＞d4g-1＞d4g-5＞d4g-3＞d4g-6＞d4g-4＞d4g-8＞d4g-7. 计算得到的各构象键长和键角数据与实验值接近. 一个水分子的加入对D4G分子的构型参数有所影响, 基本不改变D4G分子各构象的稳定性顺序, 但构象转变的能垒有所提高. 氢键在分子构象中发挥了重要作用.     

Site-specific probing of oxidative reactivity and telomerase function using 7,8-dihydro-8-oxoguanine in telomeric DNA

Telomeres at the ends of human chromosomes contain the repeating sequence 5'-d[(TTAGGG)(n)]-3'. Oxidative damage of guanine in DNAs that contain telomeric and nontelomeric sequence generates 7,8-dihydro-8-oxoguanine (8OG) preferentially in the telomeric segment, because GGG sequences are more reactive in duplex DNA. We have developed a general strategy for probing site-specific oxidation reactivity in diverse biological structures through substitution of minimally modified building blocks that are more reactive than the parent residue, but preserve the parent structure. In this study, 8OG was substituted for guanine at G(8), G(9), G(14), or G(15) in the human telomeric oligonucleotide 5'-d[AGGGTTAG(8)G(9)GTT AG(14)G(15)GTTAGGGTGT]-3'. Replacement of G by 8OG in telomeric DNA can affect the formation of intramolecular G quadruplexes, depending on the position of substitution. When 8OG was incorporated in the 5'-position of a GGG triplet, G quadruplex formation was observed; however, substitution of 8OG in the middle of a GGG triplet produced multiple structures. A clear correspondence between structure and reactivity was observed when oligonucleotides containing 8OG in the 5'-position of a GGG triplet were prepared in the quadruplex or duplex forms and interrogated by mediated electrocatalytic oxidation with Os(bpy)(3)(2+) (bpy = 2,2'-bipyridine). The rate constant for one-electron oxidation of a single 8OG in the 5'-position of a GGG triplet was (6.2 +/- 1.7) x 10(4) M(-1) s(-1) in the G quadruplex form. The rate constant was 2-fold lower for the same telomeric sequence in the duplex form ((3.0 +/- 1.3) x 10(4) M(-1) s(-1)). The position of 8OG in the GGG triplet affects telomerase activity and synthesis of telomeric repeat products. Telomerase activity was decreased significantly when 8OG was substituted in the 5'-position of the GGG triplet, but not when 8OG was substituted in the middle of the triplet. Thus, biological oxidation of G to 8OG in telomeres has the potential to modulate telomerase activity. Further, small molecules that inhibit telomerase by stabilizing telomeric G quadruplexes may not be as effective under oxidative stress.     

In Vitro Tests for a Rapid Evaluation of Antidiabetic Potential of Plant Species Containing Caffeic Acid Derivatives: A Validation by Two Well-Known Antidiabetic Plants,Ocimum gratissimum L. Leaf and Musanga cecropioides R. Br. ex Tedlie (Mu) Stem Bark

Plant bioactive extracts represent a major resource for identifying drugs and adjuvant therapy for type 2 diabetes. To promote early screening of plants’ antidiabetic potential, we designed a four in vitro tests strategy to anticipate in vivo bioactivity. Two antidiabetic plants were studied: Ocimum gratissimum L. (Oc) leaf extract and Musanga cecropoides R. Br. ex Tedlie (Mu) stem bark extract. Chemical compositions were analyzed by LCMS and HPLC. Antidiabetic properties were measured based on (1) INS-1 cells for insulin secretion, (2) L6 myoblast cells for insulin sensitization (Glut-4 translocation), (3) L6 myoblast cells for protection against hydrogen peroxide (H₂O₂) oxidative stress (cell mortality), and (4) liver microsomial fraction for glucose-6-phosphastase activity (G6P). Oc extract increased insulin secretion and insulin sensitivity, whereas it decreased oxidative stress-induced cell mortality and G6P activity. Mu extract decreased insulin secretion and had no effect on insulin sensitivity or G6P activity, but it increased oxidative stress-induced cell mortality. Results were compared with NCRAE, an antidiabetic plant extract used as reference, previously characterized and reported with increased insulin secretion and insulin sensitivity, protection against oxidative stress, and decreased G6P activity. The proposed set of four in vitro tests combined with chemical analysis provided insight into the interest in rapid early screening of plant extract antidiabetic potential to anticipate pharmaco-toxicological in vivo effects.     

A DFT study of hydrogen bond interactions between oxidative 2′-deoxyadenosine nucleotides and RNA nucleotides

In this study, the interactions between oxidative 2′-deoxyadenosine nucleotides (2OHA, 8OHA, 8OXOA, fapyA) and canonical ribonucleotides (A, C, G, U) were investigated at B3LYP level with 6-31G(d) basis set. The binding energies calculated were corrected for the basis set superposition error at the same level. The result shows that syn 8OXOA:G complex is the most stable among all the complexes. According to energetic analysis, the species and position of substitution of 2′-deoxyadenosine nucleotide significantly influence the stability of conformers. The intermolecular and intramolecular hydrogen bonds (HBs) were characterized based on atoms in molecules theory (AIM) and natural bond orbital (NBO) analysis, indicating that the type and geometry of HB significantly influence the stability of monomer and complex. Furthermore, in most cases, the intramolecular HBs in monomer and complex exhibit similar properties because they own nearly the same geometry and parameters obtained from AIM and NBO analysis.     

Oxidation of guanine bases mediated by a manganese-porphyrin-oligonucleotide conjugate onto a G-rich DNA target: 8-oxo-7,8-dihydroguanine is not the major lesion

The oxidative alkali-labile G lesions mediated by manganese porphyrin oligonucleotide conjugate on a DNA target could not be attibuted to the formation of 8-oxo-7,8-dihydroguanine since they were not substrate of the Fpg protein. In order to identify the nature of these lesions, an analysis of the oxidized derivatives of 2′-deoxyguanosine, imidazolone and oxazolone, generated by photooxidation, was efficiently performed by using the positive electrospray ionisation-mass spectrometry method.     

Biological consequences associated with DNA oxidation mediated by singlet oxygen.

Singlet oxygen is a major oxidative species that can be generated by numerous biological processes such as photosensitization. This oxidant can react with deoxyguanosine and with guanine in deoxyribonucleic acid (DNA) leading to the induction of at least four different reaction products such as 4,8-dihydro-4-hydroxy-8-oxodeoxyguanosine and 7,8-dihydro-8-oxodeoxyguanosine. The induction of true single-stranded breaks in the oxidated DNA is still a matter of controversy and is not yet clearly established. This paper focuses mainly on several biological consequences which can be associated with the induction of DNA lesions by singlet oxygen. Oxidated DNA loses its transformation efficiency probably because unrepaired lesions can partially inhibit DNA replication. Mutagenesis is one of the main effects induced by guanine oxidation products. Molecular analysis of mutated genes reveals that G to T transversions are the most frequent mutations; these are probably introduced in DNA by misincorporation of deoxyadenosine monophosphate (dAMP) opposite to the lesion. Efficient repair of these oxidated guanine residues can take place via specific glycosylase, endonuclease or the SOS network. However, the data concerning the toxicity of singlet oxygen for eukaryotic cells are not frequent enough in the literature to draw a clear picture of the effects of this activated species in several biologically revelant phenomena.     

Biliverdin reductase A in the prevention of cellular senescence against oxidative stress

Biliverdin reductase A (BLVRA), an enzyme that converts biliverdin to bilirubin, has recently emerged as a key regulator of the cellular redox cycle. However, the role of BLVRA in the aging process remains unclear. To study the role of BLVRA in the aging process, we compared the stress responses of young and senescent human diploid fibroblasts (HDFs) to the reactive oxygen species (ROS) inducer, hydrogen peroxide (H2O2). H2O2 markedly induced BLVRA activity in young HDFs, but not in senescent HDFs. Additionally, depletion of BLVRA reduced the H2O2-dependent induction of heme oxygenase-1 (HO-1) in young HDFs, but not in senescent cells, suggesting an aging-dependent differential modulation of responses to oxidative stress. The role of BLVRA in the regulation of cellular senescence was confirmed when lentiviral RNAi- transfected stable primary HDFs with reduced BLVRA expression showed upregulation of the CDK inhibitor family members p16, p53, and p21, followed by cell cycle arrest in G0-G1 phase with high expression of senescence-associated β-galactosidase. Taken together, these data support the notion that BLVRA contributes significantly to modulation of the aging process by adjusting the cellular oxidative status.     

Insights into intrastrand cross-link lesions of DNA from QM/MM molecular dynamics simulations

DNA damages induced by oxidative intrastrand cross-links have been the subject of intense research during the past decade. Yet, the currently available experimental protocols used to isolate such lesions only allow to get structural information about linked dinucleotides. The detailed structure of the damaged DNA macromolecule has remained elusive. In this study we generated in silico the most frequent oxidative intrastrand cross-link adduct, G[8,5-Me]T, embedded in a solvated DNA dodecamer by means of quantum mechanics/molecular mechanics (QM/MM) Car-Parrinello simulations. The free energy of activation required to bring the reactant close together and to form the C-C covalent-bond is estimated to be ~10 kcal/mol. We observe that the G[8,5-Me]T tandem lesion is accommodated with almost no perturbation of the Watson-Crick hydrogen-bond network and induces bend and unwinding angles of ~20° and 8°, respectively. This rather small structural distortion of the DNA macromolecule compared to other well characterized intrastrand cross-links, such as cyclobutane pyrimidines dimers or cisplatin-DNA complex adduct, is a probable rationale for the known lack of efficient repair of oxidative damages.     

TDAG51 deficiency promotes oxidative stress-induced apoptosis through the generation of reactive oxygen species in mouse embryonic fibroblasts

Apoptosis has an important role in maintaining tissue homeostasis in cellular stress responses such as inflammation, endoplasmic reticulum stress, and oxidative stress. T-cell death-associated gene 51 (TDAG51) is a member of the pleckstrin homology-like domain family and was first identified as a pro-apoptotic gene in T-cell receptor-mediated cell death. However, its pro-apoptotic function remains controversial. In this study, we investigated the role of TDAG51 in oxidative stress-induced apoptotic cell death in mouse embryonic fibroblasts (MEFs). TDAG51 expression was highly increased by oxidative stress responses. In response to oxidative stress, the production of intracellular reactive oxygen species was significantly enhanced in TDAG51-deficient MEFs, resulting in the activation of caspase-3. Thus, TDAG51 deficiency promotes apoptotic cell death in MEFs, and these results indicate that TDAG51 has a protective role in oxidative stress-induced cell death in MEFs.     

MD2 blockade prevents modified LDL-induced retinal injury in diabetes by suppressing NADPH oxidase-4 interaction with Toll-like receptor-4

Modified LDL-induced inflammation and oxidative stress are involved in the pathogenesis of diabetic retinopathy. Recent studies have also shown that modified LDL activates Toll-like receptor 4 (TLR4) to mediate retinal injury. However, the mechanism by which modified LDL activates TLR4 and the potential role of the TLR4 coreceptor myeloid differentiation protein 2 (MD2) are not known. In this study, we inhibited MD2 with the chalcone derivatives L2H17 and L6H21 and showed that MD2 blockade protected retinal Müller cells against highly oxidized glycated-LDL (HOG-LDL)-induced oxidative stress, inflammation, and apoptosis. MD2 inhibition reduced oxidative stress by suppressing NADPH oxidase-4 (NOX4). Importantly, HOG-LDL activated TLR4 and increased the interaction between NOX4 and TLR4. MD2 was required for the activation of these pathways, as inhibiting MD2 prevented the association of NOX4 with TLR4 and reduced NOX4-mediated reactive oxygen species production and TLR4-mediated inflammatory factor production. Furthermore, treatment of diabetic mice with L2H17 significantly reduced LDL extravasation in the retina and prevented retinal dysfunction and apoptosis by suppressing the TLR4/MD2 pathway. Our findings provide evidence that MD2 plays a critical role in mediating modified LDL-induced cell injury in the retina and suggest that targeting MD2 may be a potential therapeutic strategy.Subject terms: Obesity, Type 2 diabetes     

基于金属的神经退行性疾病治疗策略*

本文综述了金属离子在神经退行性疾病中的重要作用以及针对该类疾病金属治疗药物的研究进展。以老年痴呆症和帕金森氏症为代表,结合本课题组的初步研究结果,讨论了金属离子在蛋白质聚集与氧化应激反应中的重要作用,暗示金属螯合策略应成为治疗该类疾病的首选策略,并介绍了数种已用于或即将用于临床实验的金属螯合制剂;还介绍了烷基化神经退行性疾病相关蛋白的金属结合位点,可以显著抑制该蛋白质聚集体的形成和活性氧的产生,这可能是继螯合策略后一种更有发展潜力的神经退行性疾病治疗方法。     

Oxidation Chemistry of DNA and p53 Tumor Suppressor Gene

The chemistry of DNA and its repair selectivity control the influence of genomic oxidative stress on the development of serious disorders such as cancer and heart diseases. DNA is oxidized by endogenous reactive oxygen species (ROS) in vivo or in vitro as a result of high energy radiation, non-radiative metabolic processes, and other consequences of oxidative stress. Some oxidations of DNA and tumor suppressor gene p53 are thought to be mutagenic when not repaired. For example, site-specific oxidations of p53 tumor suppressor gene may lead to cancer-related mutations at the oxidation site codon. This review summarizes the research on the primary products of the most easily oxidized nucleobase guanine (G) when different oxidation methods are used. Guanine is by far the most oxidized DNA base. The primary initial oxidation product of guanine for most, but not all, pathways is 8-oxoguanine (8-oxoG). With an oxidation potential much lower than G, 8-oxoG is readily susceptible to further oxidation, and the products often depend on the oxidants. Specific products may control the types of subsequent mutations, but mediated by gene repair success. Site-specific oxidations of p53 tumor suppressor gene have been reported at known mutation hot spots, and the codon sites also depend on the type of oxidants. Modern methodologies using LC–MS/MS for codon specific detection and identification of oxidation sites are summarized. Future work aimed at understanding DNA oxidation in nucleosomes and interactions between DNA damage and repair is needed to provide a better picture of how cancer-related mutations arise.     

Facile Synthesis of Dendritic Hydroxyl‐terminated Cyanuric Chloride Derivatives and Their Properties

Novel dendritic compounds G1.5(OH)₄ and G1.5(OH)₈ with peripheral hydroxyl were synthesized under mild conditions from cyanuric chloride, ethylene diamine, ethanolamine and diethanolamine. The products could be separated and purified through dispersion and precipitation in organic solvents with good yields (over 93.0%) and high purity (above 98.0%, HPLC). The structures of the products were characterized by IR, MS, ¹ H NMR and elementary analysis. G1.5(OH)₄ and G1.5(OH)₈ could be dissolved in polar solvents such as methanol, water and dimethylsulfoxide. TGA analysis showed that G1.5(OH)₄ and G1.5(OH)₈ had good thermo stability. The aqueous solutions of G1.5(OH)₄ and G1.5(OH)₈ exhibited low surface tension.