Repair of UV-damaged DNA in mammalian skin followed by the immunohistochemical method

DNA repair in murine and guinea pig skin has been studied by the immunohistochemical method. For the detection of DNA photolesions in situ by the indirect immunofluorescence (IF) method two antisera directed against DNA-pyrimidine-dimers and DNA-psoralen-photoadducts have been applied. The IF assay enabled to detect the DNA photodamage induced by high UV-doses, exceeding more than fivefold minimal phototoxic response of the skin. It was found that IF staining gradually disappeared due to DNA repair, and at 48 h after UV-exposure both types of the DNA photolesions were no more detectable. Importantly, the IF method revealed that the persistence of DNA-pyrimidine-dimers could be traced for a longer time than that detectable by UV-endonuclease incision method.     

激光对DNA的作用

简述了激光对DNA的作用。介绍了与激光辐照导致DNA链断裂、光产物形成及其分子机理、激光损伤与突变的关系以及高能脉冲激光和双光子作用有关的一些研究结果。     

Effects of Niacin Restriction on Sirtuin and PARP Responses to Photodamage in Human Skin

Sirtuins (SIRTs) and poly(ADP-ribose) polymerases (PARPs), NAD(+)-dependent enzymes, link cellular energy status with responses to environmental stresses. Skin is frequently exposed to the DNA damaging effects of UV irradiation, a known etiology in skin cancer. Thus, understanding the defense mechanisms in response to UV, including the role of SIRTs and PARPs, may be important in developing skin cancer prevention strategies. Here, we report expression of the seven SIRT family members in human skin. SIRTs gene expressions are progressively upregulated in A431 epidermoid carcinoma cells (SIRTs1 and 3), actinic keratoses (SIRTs 2, 3, 5, 6, and 7) and squamous cell carcinoma (SIRTs 1-7). Photodamage induces dynamic changes in SIRT expression with upregulation of both SIRT1 and SIRT4 mRNAs. Specific losses of SIRT proteins occur early after photodamage followed by accumulation later, especially for SIRT4. Niacin restriction, which decreases NAD(+), the sirtuin substrate, results in an increase in acetylated proteins, upregulation of SIRTs 2 and 4, increased inherent DNA damage, alterations in SIRT responses to photodamage, abrogation of PARP activation following photodamage, and increased sensitivity to photodamage that is completely reversed by repleting niacin. These data support the hypothesis that SIRTs and PARPs play important roles in resistance to photodamage and identify specific SIRTs that respond to photodamage and may be targets for skin cancer prevention.     

Mechanistic aspects of Escherichia coli photodynamic inactivation by cationic tetra-meso(N-methylpyridyl)porphine.

The mechanistic aspects of Escherichia coli photodynamic inactivation (PDI) have been studied in bacteria expressing the reporter protein GFP, following transfection with wild type pGFP plasmid and treatment with the hydrophilic cationic sensitizer tetra-meso(N-methyl-4-pyridyl)porphine tetratosylate (TMPyP). Cell survival and morphology during PDI were correlated with plasmid-GFP degradation in comparison to DNA and RNA strand-breaks, while photobleaching of the GFP chromophore was used to monitor protein photodamage. Singlet oxygen generated upon TMPyP photoactivation interacted with target nucleic acid polymers in a drug-and light-dose dependent manner. The hierarchy and cascade of the photodamage was in the order: genomic-DNA > total RNA > plasmid-DNA, as revealed by specific extraction and agarose electrophoresis. The notable resistance of the plasmid DNA in comparison to genomic DNA has implications for PDI of antibiotic-resistant bacteria. Re-growth of the treated cells in fresh medium showed structural features of an SOS response. Under these conditions, DNA repair machinery was initiated by typical alignment of DNA-protein co-aggregates accompanied by lateral assembly of ribosomes, apart from damaged DNA-arrays, as depicted by electron microscopy. GFP-TMPyP interactions were demonstrated by double green and red fluorescence on electrophoresis plates analyzed by spectral imaging. Photobleaching measurements revealed specific GFP photodamage directly related to PDI of the E. coli. The kinetics of both the GFP photobleaching and the K(+) efflux, representing photodamage to cytosolic proteins and membrane damage, respectively, were found to be similar. The survival curves were correlated to chromosomal degradation and ultrastructural damage. We conclude that TMPyP-dependent PDI of E. coli is primarily dependent on genomic DNA photodamage rather than on protein or membrane malfunctions.     

Naphthalimide intercalators with chiral amino side chains: effects of chirality on DNA binding, photodamage and antitumor cytotoxicity

Several novel heterocyclic-fused naphthalimides intercalators with chiral amino side chains were investigated. Their side chains' chiral configuration determines DNA binding activities in the order: S-enantiomers > R-enantiomers. And their DNA photodamaging activities were in good agreement with their DNA binding constants, the S-enantiomers could photocleave circular supercoiled pBR322 DNA more efficiently than their R-enantiomers. S-enantiomer B(3) could photodamage DNA at 0.2 microM and cleave supercoiled plasmid DNA from form I to form II completely at 50 microM. Almost all of these intercalators showed effective cytoxicities against human lung cancer cells and murine leukemia cells. S-enantiomers showed different antitumor cytotoxicity by comparison with R-enantiomers. This work may provide additional information for the role of amino side chains on intercalators as antitumor agents.     

激光对DNA作用机理的AFM研究

激光作用质粒DNA和小牛胸腺DNA产生损伤效应,导致DNA结构变化,利用一种改进的试样制备过程和纳米显微镜－－原子力显微镜（AFM）能够获得可重现的激光作用质粒DNA和小牛胸腺DNA的AFM图像,显示它们的特殊的表达结构,讨论了激光辐照导致DNA链断裂的作用机理。     

激光作用质粒DNA和小牛胸腺DNA的AFM研究

激光作用质粒ＤＮＡ和小牛胸腺ＤＮＡ产生损伤效应,导致ＤＮＡ结构变化,利用一种改进的试样制备过程和纳米显微镜--原子力显微镜（ＡＦＭ）能够获得可重现的激光作用质粒ＤＮＡ和小牛胸腺ＤＮＡ的ＡＦＭ图象,显示它们的特殊的表面结构。     

Sequence-targeted photochemical modifications of nucleic acids by complementary oligonucleotides covalently linked to porphyrins

Porphyrins linked to oligonucleotides produce various types of photodamage on a complementary target DNA. The observed reactions include oxidation of guanine bases and cross-linking reactions of the oligonucleotide to its target sequence. Guanines located close to the porphyrin macrocycle were the most altered as compared to more remote guanines on the target sequence. No specific reaction was observed when the complexes were dissociated at temperatures above the melting temperature of the oligonucleotide-target hybrid. Both cross-linking and oxidation reactions accounted for ca. 60% modification of the target chains in the complex. Our results show that oligonucleotides covalently linked to porphyrins are efficient systems for inducing irreversible sequence-specific photodamage on a target DNA.     

Rethinking the role of many plant phenolics – protection from photodamage not herbivores?

Phenolics have been considered classic defence compounds for protecting plants from herbivores, ever since plant secondary metabolites were suggested to have evolved for that reason. The resource availability and carbon-nutrient balance hypotheses proposed that variation in phenolic levels between and within plant species reflects environmental availability of nutrients and light, and represents a trade-off in allocation by plants to growth and defence against herbivores. In contrast to these concepts, we suggest that (1) the main role of many plant phenolics may be to protect leaves from photodamage, not herbivores; (2) they can achieve this by acting as antioxidants; and (3) their levels may vary with environmental conditions in order to counteract this potential photodamage. We therefore suggest that patterns in phenolic levels, often used to support the concept of trade-off between growth and herbivore defence in relation to resource availability, may actually reflect different risks of photodamage. We suggest that the level of many phenolics is low under some environmental conditions, not because resources to produce them are limited, but simply because the risk of photodamage is low and they are not required. If our photodamage hypothesis is correct, a reassessment of the ecological and evolutionary role of many phenolics in plant defence theory is required.     

An anti-DNA antibody prefers damaged dsDNA over native

DNA–protein interactions, including DNA–antibody complexes, have both fundamental and practical significance. In particular, antibodies against double-stranded DNA play an important role in the pathogenesis of autoimmune diseases. Elucidation of structural mechanisms of an antigen recognition and interaction of anti-DNA antibodies provides a basis for understanding the role of DNA-containing immune complexes in human pathologies and for new treatments. Here we used Molecular Dynamic simulations of bimolecular complexes of a segment of dsDNA with a monoclonal anti-DNA antibody’s Fab-fragment to obtain detailed structural and physical characteristics of the dynamic intermolecular interactions. Using a computationally modified crystal structure of a Fab–DNA complex (PDB: 3VW3), we studied in silico equilibrium Molecular Dynamics of the Fab-fragment associated with two homologous dsDNA fragments, containing or not containing dimerized thymine, a product of DNA photodamage. The Fab-fragment interactions with the thymine dimer-containing DNA was thermodynamically more stable than with the native DNA. The amino acid residues constituting a paratope and the complementary nucleotide epitopes for both Fab–DNA constructs were identified. Stacking and electrostatic interactions were shown to play the main role in the antibody–dsDNA contacts, while hydrogen bonds were less significant. The aggregate of data show that the chemically modified dsDNA (containing a covalent thymine dimer) has a higher affinity toward the antibody and forms a stronger immune complex. These findings provide a mechanistic insight into formation and properties of the pathogenic anti-DNA antibodies in autoimmune diseases, such as systemic lupus erythematosus, associated with skin photosensibilization and DNA photodamage.     

Distinct mechanisms of guanine-specific DNA photodamage induced by nalidixic acid and fluoroquinolone antibacterials

Fluoroquinolone antibacterials, which have been used for the treatment of a variety of infectious diseases, are reported to be photocarcinogenic. We investigated the mechanisms of DNA damage by UVA radiation (365 nm) plus fluoroquinolone antibacterials using 32P-labeled DNA fragments obtained from the human c-Ha-ras-1 proto-oncogene and the p53 tumor suppressor gene. Photocarcinogenic nalidixic acid (NA), which is an old member of synthetic quinolone antibacterials, caused DNA damage specifically at 5'-GG-3' sequences, whereas lomefloxacin (LFLX) did not exhibit the site preference for consecutive guanines. LFLX-induced DNA photodamage was inhibited by sodium azide and enhanced in D2O, suggesting that singlet oxygen plays the key role in the DNA damage. LFLX plus UVA induced the formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) depending on LFLX concentrations, and 8-oxodG formation was enhanced in single-stranded DNA. In contrast, NA induced larger amounts of 8-oxodG in double-stranded DNA. ESR spin destruction method revealed that NA induced DNA photodamage through electron transfer but LFLX did not. These findings indicate that DNA damage induced by photoactivated LFLX and NA plays an important role in expression of their photocarcinogenicity.     

UV-induced pyrimidine dimers and trimethylpsoralen cross-links do not alter chromatin folding in vitro

We have examined the ability of intact and histone H1 depleted chromatin fibers to fold into higher ordered structures in vitro following DNA damage by two different agents: UV irradiation at 254 nm and trimethylpsoralen plus near-UV light. Both agents damage DNA specifically, yet cause different degrees of unwinding (and possibly bending) of the DNA helix. In addition, trimethylpsoralen forms interstrand DNA cross-links. The structural transitions of intact and histone H1 depleted chromatin fibers, induced by NaCl, were monitored by analytical ultracentrifugation, light scattering, and circular dichroism. Our results indicate that when chromatin fibers contain even large, nonphysiological amounts of DNA photodamage by either agent, the salt-induced folding of these fibers into higher ordered structures is unaffected. The compact 30-nm fiber must therefore be able to accommodate a large amount of DNA photodamage (greater than one UV-induced photoproduct or trimethylpsoralen interstrand cross-link per nucleosome) with little or no change in the overall size or compaction of this structure.     

Pterostilbene protects against UVB-induced photo-damage through a phosphatidylinositol-3-kinase-dependent Nrf2/ARE pathway in human keratinocytes

Objective: Ultraviolet B (UVB) irradiation is the initial etiological factor for various skin disorders, including erythema, sunburn, photoaging, and photocarcinogenesis. Pterostilbene (Pter) displayed remarkable antioxidant, anti-inflammatory, and anticarcinogenic activities. This study aimed to investigate the effective mechanism of Pter against UVB-induced photodamage in immortalized human keratinocytes.

Methods: Human keratinocytes were pretreated with Pter (5 and 10?μM) for 24?h prior to UVB irradiation (300?mJ/cm²). Harvested cells were analyzed by MTT, DCFH-DA, comet, western blotting, luciferase promoter, small interference RNA transfection, and quantitative real-time polymerase chain reaction assay.

Results: Pter significantly attenuated UVB-induced cell death and reactive oxygen species (ROS) generation, and effectively increased nuclear translocation of NF-E2-related factor-2 (Nrf2), expression of Nrf2-dependent antioxidant enzymes, and DNA repair activity. Moreover, the protective effects of Pter were abolished by small interference RNA-mediated Nrf2 silencing. Furthermore, Pter was also found to induce the phosphorylation of Nrf2 and the known phosphatidylinositol-3-kinase (PI3K) phosphorylated kinase, Akt. The specific inhibitor of PI3K, LY294002, successfully abrogated Pter-induced Nrf2 phosphorylation, activation of Nrf2-antioxidant response element pathway, ROS scavenging ability, and DNA repair activity.

Conclusion: The present study indicated that Pter effectively protected against UVB-induced photodamage by increasing endogenous defense mechanisms, scavenging UVB-induced ROS, and aiding in damaged DNA repair through a PI3K-dependent activation of Nrf2/ARE pathway.     

DNA intercalation and photosensitization by cationic meso substituted porphyrins.

Several cationic porphyrins are known to bind to DNA by intercalative and outside binding modes. This study identifies the cis and trans isomers of bis(N-methyl-4-phridiniumyl)diphenyl porphyrin as DNA intercalators based on evidence from a DNA topoisomerase I assay. Moreover, both isomers are shown to be potent photosensitizers of DNA, inducing multiple S1 nuclease sensitive breaks in the phosphodiester backbone. Porphyrin-induced photodamage in DNA was also shown to be quantitatively dependent upon ionic strength and to inhibit the action of restriction endonucleases. The results indicate that these porphyrins can be useful probes of DNA structure and have potential as DNA-targeted photosensitizers.     

Photofootprinting of DNA triplexes.

We have used a photofootprinting assay to study intermolecular and intramolecular DNA triplexes. The assay is based on the fact that the DNA duplex is protected against photodamage (specifically, against the formation of the (6-4) pyrimidine photoproducts) within a triplex structure. We have shown that this is the case for PyPuPu (YRR) as well as PyPuPy (YRY) triplexes. Using the photofootprinting assay, we have studied the triplex formation under a variety of experimentally defined conditions. At acid pH, d(C)n.d(G)n.d(C)n and d(CT)n.d(GA)n.d(CT)n triplexes are detected by this method. The d(CT)n.d(GA)n.d(CT)n triplexes are additionally stabilized by divalent cations and spermidine. PyPuPu triplexes are pH-independent and are stabilized by divalent cations, such as Mg++ and Zn++. The effect depends on the type of cation and on the DNA sequence. The d(CT)n.d(GA)n.d(GA)n triplex is stabilized by Zn++, but not by Mg++, whereas the d(C)n.d(G)n.d(G)n triplex is stabilized by Mg++. In H-DNA, virtually the entire pyrimidine chain is protected against photodimerization, whereas only half of the pyrimidine chain participating in a triplex is protected in the CGG intramolecular triplex.     

光合作用光能转换过程中与耗能代谢有关的光保护机制(综述)

对近年来光合电子传递过程中各种耗能代谢（包括光呼吸、Ｍｅｈｌｅｒ反应、循环电子传递、硝酸还原代谢等）的运转对光合机构的保护作用作一简要综述。     

Crude polysaccharide from an anti-UVB cell clone of <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> protect HaCaT cells against UVB-induced oxidative stress

Bupleurum scorzonerifolium Willd has been found to have a wide range of immunopharmacologic functions. We isolated an anti-UVB B. scorzonerifolium cell clone and found elevated level of polysaccharides. In this study, we investigated the ability of crude polysaccharide (CP) from the anti-UVB B. scorzonerifolium cell clone to inhibit UVB-induced photodamage using a human skin keratinocyte cell line, HaCaT. Cells were UVB irradiated and then incubated in presence of different concentrations of CP. MTT assay showed that the CP did not induce cytotoxic effect under 10 mg/mL and after UVB irradiation, CP can inhibit UVB-induced HaCaT cell death. Decreased reactive oxygen species and lipid peroxidation and increased superoxide dismutase activity showed that CP can act as a free radical scavenger. Furthermore, CP had a strong protective ability against UVB-induced DNA damage. These effects were compared to the crude polysaccharide (CP′) from normal B. scorzonerifolium callus at concentration of 20 mg/mL. The portion of crude polysaccharide (CP) from the anti-UVB B. scorzonerifolium cell clone was more than 2.5-fold higher than crude polysaccharide (CP′) from normal B. scorzonerifolium callus. Taken together, the protective mechanisms of crude polysaccharide from the anti-UVB B. scorzonerifolium cell clone against UVB-induced photodamage occur by the inhibition of UVB-induced reactive oxygen species production, lipid peroxidation and DNA damage.     

The mechanism of photoinhibition in vivo: Re-evaluation of the roles of catalase, α-tocopherol, non-photochemical quenching, and electron transport

Photoinhibition of photosystem II (PSII) occurs when the rate of light-induced inactivation (photodamage) of PSII exceeds the rate of repair of the photodamaged PSII. For the quantitative analysis of the mechanism of photoinhibition of PSII, it is essential to monitor the rate of photodamage and the rate of repair separately and, also, to examine the respective effects of various perturbations on the two processes. This strategy has allowed the re-evaluation of the results of previous studies of photoinhibition and has provided insight into the roles of factors and mechanisms that protect PSII from photoinhibition, such as catalases and peroxidases, which are efficient scavengers of H(2)O(2); α-tocopherol, which is an efficient scavenger of singlet oxygen; non-photochemical quenching, which dissipates excess light energy that has been absorbed by PSII; and the cyclic and non-cyclic transport of electrons. Early studies of photoinhibition suggested that all of these factors and mechanisms protect PSII against photodamage. However, re-evaluation by the strategy mentioned above has indicated that, rather than protecting PSII from photodamage, they stimulate protein synthesis, with resultant repair of PSII and mitigation of photoinhibition. This article is part of a Special Issue entitled: Photosynthesis Research for Sustainability: from Natural to Artificial.     

Protective action of copper (II) complex of a Schiff base against DNA damage induced by m-chloroperbenzoic acid using a novel DNA unwinding technique

DNA strand breaks can be detected with great sensitivity by exposing calf thymus DNA to alkaline solutions and monitoring the rate of strand unwinding. Fluorometric analysis of DNA unwinding (FADU) is a reliable method for detecting single-strand DNA breaks as an index of DNA damage induced by photosensitizer.m-Chloroperbenzoic acid (CPBA) was used as a photosensitizer in the photodamage of calf thymus DNA. When DNA is exposed to ionizing radiation, the radicals produced in the irradiated sample modify the base-pair regions of the double strands. The protective action of copper salt, Schiff base [ethylene diamine with ethyl acetate](L) and its Cu(II) complex (Cu(7) L Cl(14)) against DNA damage photoinduced by CPBA was studied using ethidium bromide as a fluorescent probe. Treatment of DNA with 5, 10, 50, 100, or 200 microM CPBA produced 75%, 48%, 38%, 32% and 30% double-stranded DNA remaining, respectively after 30 min of alkaline treatment at 15 degrees C. Treatment of calf thymus DNA irradiated with CPBA with a dose of 1 mM [Cu(7) L Cl(14)] produced 96% double-stranded remaining protection under the same conditions compared with irradiated DNA without addition of Cu(II) complex of Schiff base.     

Protein synthesis is the primary target of reactive oxygen species in the photoinhibition of photosystem II

Photoinhibition of photosystem II (PSII) occurs when the rate of photodamage to PSII exceeds the rate of the repair of photodamaged PSII. Recent examination of photoinhibition by separate determinations of photodamage and repair has revealed that the rate of photodamage to PSII is directly proportional to the intensity of incident light and that the repair of PSII is particularly sensitive to the inactivation by reactive oxygen species (ROS). The ROS-induced inactivation of repair is attributable to the suppression of the synthesis de novo of proteins, such as the D1 protein, that are required for the repair of PSII at the level of translational elongation. Furthermore, molecular analysis has revealed that the ROS-induced suppression of protein synthesis is associated with the specific inactivation of elongation factor G via the formation of an intramolecular disulfide bond. Impairment of various mechanisms that protect PSII against photoinhibition, including photorespiration, thermal dissipation of excitation energy, and the cyclic transport of electrons, decreases the rate of repair of PSII via the suppression of protein synthesis. In this review, we present a newly established model of the mechanism and the physiological significance of repair in the regulation of the photoinhibition of PSII.