A histone-like protein of Helicobacter pylori protects DNA from stress damage and aids host colonization

Genomic DNA in a bacterial cell is folded into a compact structure called a nucleoid, and nucleoid-associated proteins are responsible for proper assembly of active higher-order genome structures. The human gastric pathogen Helicobacter pylori express a nucleoid-associated protein encoded by the hup gene, which is the homolog to the Escherichia coli histone-like protein HU. An H. pylori hup mutant strain (X47 hup:cat) showed a defect in stationary phase survival. The X47 hup:cat mutant was more sensitive to the DNA damaging agent mitomycin C, and displayed a decreased frequency of DNA recombination, indicating Hup plays a significant role in facilitating DNA recombinational repair. The X47 hup:cat mutant was also sensitive to both oxidative and acid stress, conditions that H. pylori commonly encounters in the host. The hup mutant cells survived significantly (7-fold) less upon exposure to macrophages than the wild type strain. In a mouse infection model, the hup mutant strain displayed a greatly reduced ability to colonize host stomachs. The geometric means of colonization number for the wild type and hup mutant were 6×10(5) and 1.5×10(4)CFU/g stomachs, respectively. Complementation of the hup strain by chromosomal insertion of a functional hup gene restored oxidative stress resistance, DNA transformation frequency, and mouse colonization ability to the wild type level. We directly demonstrated that the purified His-tagged H. pylori Hup protein can protect (in vitro) an H. pylori-derived DNA fragment from oxidative damage.     

Cellular prion protein protects against reactive-oxygen-species-induced DNA damage

Although the cellular form of the prion protein (PrPC) is critical for the development of prion disease through its conformational conversion into the infectious form (PrPSc), the physiological role of PrPC is less clear. Using alkaline single-cell gel electrophoresis (the Comet assay), we show that expression of PrPC protects human neuroblastoma SH-SY5Y cells against DNA damage under basal conditions and following exposure to reactive oxygen species, either hydroxyl radicals following exposure to Cu2+ or Fe2+ or singlet oxygen following exposure to the photosensitizer methylene blue and white light. Cells expressing either PrPDeltaoct which lacks the octapeptide repeats or the prion-disease-associated mutants A116V or PG14 had increased levels of DNA damage compared to cells expressing PrPC. In PrPSc-infected mouse ScN2a cells there was a significant increase in DNA damage over noninfected N2a cells (median tail DNA 2.87 and 7.33%, respectively). Together, these data indicate that PrPC has a critical role to play in protecting cells against reactive-oxygen-species-mediated DNA damage; a function which requires the octapeptide repeats in the protein, is lost in disease-associated mutants of the protein or upon conversion to PrPSc, and thus provide further support for the neuroprotective role for PrPC.     

Prion protein protects against DNA damage induced by paraquat in cultured cells

Exposure of cells to paraquat leads to production of superoxide anion (O2*-). This reacts with hydrogen peroxide to give the hydroxyl radical (*OH), leading to lipid peroxidation and cell death. In this study, we investigated the effects of cellular prion protein (PrPC) overexpression on paraquat-induced toxicity by using an established model system, rabbit kidney epithelial A74 cells, which express a doxycycline-inducible murine PrPC gene. PrPC overexpression was found to significantly reduce paraquat-induced cell toxicity, DNA damage, and malondialdehyde acid levels. Superoxide dismutase (total SOD and CuZn-SOD) and glutathione peroxidase activities were higher in doxycycline-stimulated cells. Our findings clearly show that PrPC overexpression plays a protective role against paraquat toxicity, probably by virtue of its superoxide dismutase-like activity.     

N(6)-Furfuryladenine, kinetin, protects against Fenton reaction-mediated oxidative damage to DNA

N(6)-Furfuryladenine (kinetin) has been shown to have anti-ageing effects on several different systems including plants, human cells in culture, and fruitflies. Since most of the experimental data point toward kinetin acting as an antioxidant both in vitro and in vivo, and since much evidence supporting a causal role of oxidative damage in ageing is accumulating, we tested the antioxidant properties of kinetin directly. Using 8-oxo-2'deoxyguanosine (8-oxo-dG) in calf thymus DNA as a marker for oxidative damage, we demonstrate that kinetin significantly (P < 0.005) protects the DNA against oxidative damage mediated by the Fenton reaction. Kinetin inhibited 8-oxo-dG formation in a dose-dependent manner with a maximum of 50% protection observed at 100 microM kinetin.     

Nuclear ATR lysine-tyrosylation protects against heart failure by activating DNA damage response

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A putative Leishmania DNA polymerase theta protects the parasite against oxidative damage

Leishmania infantum is a protozoan parasite that is phagocytized by human macrophages. The host macrophages kill the parasite by generating oxidative compounds that induce DNA damage. We have identified, purified and biochemically characterized a DNA polymerase θ from L. infantum (LiPolθ), demonstrating that it is a DNA-dependent DNA polymerase involved in translesion synthesis of 8oxoG, abasic sites and thymine glycol lesions. Stably transfected L. infantum parasites expressing LiPolθ were significantly more resistant to oxidative and interstrand cross-linking agents, e.g. hydrogen peroxide, cisplatin and mitomycin C. Moreover, LiPolθ-overexpressing parasites showed an increased infectivity toward its natural macrophage host. Therefore, we propose that LiPolθ is a translesion synthesis polymerase involved in parasite DNA damage tolerance, to confer resistance against macrophage aggression.     

Overexpressed heat shock protein 70 protects cells against DNA damage caused by ultraviolet C in a dose-dependent manner

Heat shock protein 70 (Hsp70) comprises proteins that have been reported to protect cells, tissues, and organisms against damage from a wide variety of stressful stimuli; however, little is known about whether Hsp70 protects against DNA damage. In this study, we investigated the relationship between Hsp70 expression and the levels of ultraviolet C (UVC)-induced DNA damage in A549 cells with normal, inhibited, and overexpressed Hsp70 levels. Hsp70 expression was inhibited by treatment with quercetin or overexpressed by transfection of plasmids harboring the hsp70 gene. The level of DNA damage was assessed by the comet assay. The results showed that the levels of DNA damage (shown as the percentage of comet cells) in A549 cells increased in all cells after exposure to an incident dose of 0, 10, 20, 40, and 80 J/m2 whether Hsp70 was inhibited or overexpressed. This response was dose dependent: a protection against UVC-induced DNA damage in cells with overexpressed Hsp70 was observed at UVC dose 20 J/m2 with a maximum at 40 J/m2 when compared with cells with normal Hsp70 levels and in quercetin-treated cells. This differential protection disappeared at 80 J/m2. These results suggest that overexpressed Hsp70 might play a role in protecting A549 cells from DNA damage caused by UVC irradiation, with a threshold of protection from at UVC irradiation-induced DNA damage by Hsp70. The detailed mechanism how Hsp70 is involved in DNA damage and possible DNA repair warrants further investigation.     

Concentration-dependent organization of DNA by the dinoflagellate histone-like protein HCc3

The liquid crystalline chromosomes of dinoflagellates are the alternative to the nucleosome-based organization of chromosomes in the eukaryotes. These nucleosome-less chromosomes have to devise novel ways to maintain active parts of the genome. The dinoflagellate histone-like protein HCc3 has significant sequence identity with the bacterial DNA-binding protein HU. HCc3 also has a secondary structure resembling HU in silico. We have examined HCc3 in its recombinant form. Experiments on DNA-cellulose revealed its DNA-binding activity is on the C-terminal domain. The N-terminal domain is responsible for intermolecular oligomerization as demonstrated by cross-linking studies. However, HCc3 could not complement Escherichia coli HU-deficient mutants, suggesting functional differences. In ligation assays, HCc3-induced DNA concatenation but not ring closure as the DNA-bending HU does. The basic HCc3 was an efficient DNA condensing agent, but it did not behave like an ordinary polycationic compound. HCc3 also induced specific structures with DNA in a concentration-dependent manner, as demonstrated by atomic force microscopy (AFM). At moderate concentration of HCc3, DNA bridging and bundling were observed; at high concentrations, the complexes were even more condensed. These results are consistent with a biophysical role for HCc3 in maintaining extended DNA loops at the periphery of liquid crystalline chromosomes.     

DNA organization by the apicoplast-targeted bacterial histone-like protein of Plasmodium falciparum

Apicomplexans, including the pathogens Plasmodium and Toxoplasma, carry a nonphotosynthetic plastid of secondary endosymbiotic origin called the apicoplast. The P. falciparum apicoplast contains a 35 kb, circular DNA genome with limited coding capacity that lacks genes encoding proteins for DNA organization and replication. We report identification of a nuclear-encoded bacterial histone-like protein (PfHU) involved in DNA compaction in the apicoplast. PfHU is associated with apicoplast DNA and is expressed throughout the parasite's intra-erythocytic cycle. The protein binds DNA in a sequence nonspecific manner with a minimum binding site length of ~27 bp and a K_d of ~63 nM and displays a preference for supercoiled DNA. PfHU is capable of condensing Escherichia coli nucleoids in vivo indicating its role in DNA compaction. The unique 42 aa C-terminal extension of PfHU influences its DNA condensation properties. In contrast to bacterial HUs that bend DNA, PfHU promotes concatenation of linear DNA and inhibits DNA circularization. Atomic Force Microscopic study of PfHU–DNA complexes shows protein concentration-dependent DNA stiffening, intermolecular bundling and formation of DNA bridges followed by assembly of condensed DNA networks. Our results provide the first functional characterization of an apicomplexan HU protein and provide additional evidence for red algal ancestry of the apicoplast.     

DNA dynamic flexibility and protein recognition: differential stimulation by bacterial histone-like protein HU

The abundant E. coli "histone-like" protein HU is shown to be a differential effector of DNA recognition by three diverse control proteins. DNA recognition by lac repressor and catabolite activator protein is greatly stimulated, while specific aroH DNA recognition by trp repressor is inhibited. BaCl2, an agent previously shown to promote DNA bending, mimics the HU effect to give the same qualitative differential stimulation spectrum. The HU activation involves cooperativity, further suggesting that the various DNA bends and distortions induced during assembly of higher order HU:DNA structures are important for the HU stimulation. Thus, E. coli chromosomal DNA regulation is likely strongly influenced by HU protein that may promote a variety of alternative DNA structures that either facilitate or inhibit specific recognition by diverse control proteins.     

DNA polymerase lambda protects mouse fibroblasts against oxidative DNA damage and is recruited to sites of DNA damage/repair

DNA polymerase lambda (pol lambda) is a member of the X family of DNA polymerases that has been implicated in both base excision repair and non-homologous end joining through in vitro studies. However, to date, no phenotype has been associated with cells deficient in this DNA polymerase. Here we show that pol lambda null mouse fibroblasts are hypersensitive to oxidative DNA damaging agents, suggesting a role of pol lambda in protection of cells against the cytotoxic effects of oxidized DNA. Additionally, pol lambda co-immunoprecipitates with an oxidized base DNA glycosylase, single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1), and localizes to oxidative DNA lesions in situ. From these data, we conclude that pol lambda protects cells against oxidative stress and suggest that it participates in oxidative DNA damage base excision repair.     

Zinc protects against ultraviolet A1-induced DNA damage and apoptosis in cultured human fibroblasts

Ultraviolet Al (UVA1) radiation generates reactive oxygen species and the oxidative stress is known as a mediator of DNA damage and of apoptosis. We exposed cultured human cutaneous fibroblasts to UVA1 radiation (wavelengths in the 340–450-nm range with emission peak at 365 nm) and, using the alkaline unwinding method, we showed an immediate significant increase of DNA strand breaks in exposed cells. Apoptosis was determined by detecting cytoplasmic nucleosomes (enzyme-linked immunosorbent assay method) at different time points in fibroblasts exposed to different irradiation doses. In our conditions, UVA1 radiation induced an early (8 h) and a delayed (18 h) apoptosis. Delayed apoptosis increased in a UVA dosedependent manner. Zinc is an important metal for DNA protection and has been shown to have inhibitory effects on apoptosis. The addition of zinc (6.5 mg/L) as zinc chloride to the culture medium significantly decreased immediate DNA strand breaks in human skin fibroblasts. Moreover, zinc chloride significantly decreased UVA1-induced early and delayed apoptosis. Thus, these data show for the first time in normal cutaneous cultured cells that UVA1 radiation induces apoptosis. This apoptosis is biphasic and appears higher 18 h after the stress. Zinc supplementation can prevent both immediate DNA strand breakage and early and delayed apoptosis, suggesting that this metal could be of interest for skin cell protection against UVA1 irradiation.     

Oxidative damage of DNA induced by the reaction of methylglyoxal with lysine in the presence of ferritin

Methylglyoxal (MG) is an endogenous metabolite which is present in increased concentrations in diabetics and reacts with amino acids to form advanced glycation end products. In this study, we investigated whether ferritin enhances DNA cleavage by the reaction of MG with lysine. When plasmid DNA was incubated with MG and lysine in the presence of ferritin, DNA strand breakage was increased in a dose-dependent manner. The ferritin/MG/lysine system-mediated DNA cleavage was significantly inhibited by reactive oxygen species (ROS) scavengers. These results indicated that ROS might participate in the ferritin/MG/lysine system-mediated DNA cleavage. Incubation of ferritin with MG and lysine resulted in a time-dependent release of iron ions from the protein molecules. Our data suggest that DNA cleavage caused by the ferritin/MG/lysine system via the generation of ROS by the Fenton-like reaction of free iron ions released from oxidatively damaged ferritin. [BMB Reports 2013; 46(4): 225-229]     

Resveratrol protects against irradiation-induced hepatic and ileal damage via its anti-oxidative activity

The present study was undertaken to determine whether resveratrol (RVT) could ameliorate ionizing radiation-induced oxidative injury. After a 10-days pre-treatment with RVT (10 mg/kg/day p.o.), rats were exposed to whole-body IR (800 cGy) and the RVT treatment was continued for 10 more days after the irradiation. Irradiation caused a significant decrease in glutathione level, while malondialdehyde levels, myeloperoxidase activity and collagen content were increased in the liver and ileum tissues. Similarly, plasma lactate dehydrogenase and pro-inflammatory cytokine levels, 8-hydroxy-2′-deoxyguanosine and leukocyte apoptosis were elevated, while antioxidant-capacity was reduced in the irradiated rats as compared with the control group. Furthermore, Na⁺, K⁺-ATPase activity was inhibited and DNA fragmentation was increased in the ileal tissues. Resveratrol treatment reversed all these biochemical indices, as well as histopathological alterations induced by irradiation. In conclusion, supplementing cancer patients with adjuvant therapy of resveratrol may have some benefit for a more successful radiotherapy.     

Hypothermia postpones DNA damage repair in irradiated cells and protects against cell killing

Hibernation is an established strategy used by some homeothermic organisms to survive cold environments. In true hibernation, the core body temperature of an animal may drop to below 0°C and metabolic activity almost cease. The phenomenon of hibernation in humans is receiving renewed interest since several cases of victims exhibiting core body temperatures as low as 13.7°C have been revived with minimal lasting deficits. In addition, local cooling during radiotherapy has resulted in normal tissue protection. The experiments described in this paper were prompted by the results of a very limited pilot study, which showed a suppressed DNA repair response of mouse lymphocytes collected from animals subjected to 7-Gy total body irradiation under hypothermic (13°C) conditions, compared to normothermic controls. Here we report that human BJ-hTERT cells exhibited a pronounced radioprotective effect on clonogenic survival when cooled to 13°C during and 12h after irradiation. Mild hypothermia at 20 and 30°C also resulted in some radioprotection. The neutral comet assay revealed an apparent lack on double strand break (DSB) rejoining at 13°C. Extension of the mouse lymphocyte study to ex vivo-irradiated human lymphocytes confirmed lower levels of induced phosphorylated H2AX (γ-H2AX) and persistence of the lesions at hypothermia compared to the normal temperature. Parallel studies of radiation-induced oxidatively clustered DNA lesions (OCDLs) revealed partial repair at 13°C compared to the rapid repair at 37°C. For both γ-H2AX foci and OCDLs, the return of lymphocytes to 37°C resulted in the resumption of normal repair kinetics. These results, as well as observations made by others and reviewed in this study, have implications for understanding the radiobiology and protective mechanisms underlying hypothermia and potential opportunities for exploitation in terms of protecting normal tissues against radiation.     

Resveratrol ameliorates oxidative DNA damage and protects against acrylamide-induced oxidative stress in rats

Acrylamide (ACR), used in many fields from industrial manufacturing to laboratory personnel work is also formed during the heating process through interactions of amino acids. Therefore ACR poses a significant risk to human health. This study aimed to elucidate whether resveratrol (RVT) treatment could modulate ACR-induced oxidative DNA damage and oxidative changes in rat brain, lung, liver, kidney and testes tissues. Rats were divided into four groups as control (C); RVT (30 mg/kg i.p. dissolved in 0.9% NaCl), ACR (40 mg/kg i.p.) and RVT + ACR groups. After 10 days rats were decapitated and tissues were excised. 8-hydroxydeoxyguanosine (8-OHdG) is a biomarker of oxidative DNA damage. 8-OHdG content in the extracted DNA solution was determined by enzyme-linked immunosorbent assay method. Malondialdehyde (MDA), glutathione (GSH) levels and myeloperoxidase activity (MPO) were determined in tissues, while oxidant-induced tissue fibrosis was determined by collagen contents. Serum enzyme activities, cytokine levels, leukocyte apoptosis were assayed in plasma. As an indicator of oxidative DNA damage, 8-OHdG levels significantly increased in ACR group and this was reversed significantly by RVT treatment. In ACR group, GSH levels decreased significantly while the MDA levels, MPO activity and collagen content increased in the tissues suggesting oxidative organ damage. In RVT-treated ACR group, oxidant responses reversed significantly. Serum enzyme activities, cytokine levels and leukocyte late apoptosis which increased following ACR administration, decreased with RVT treatment. Therefore supplementing with RVT can be useful in individuals at risk of ACR toxicity.     

Growth arrest in G1 protects against oxygen-induced DNA damage and cell death

Although oxygen is required for normal aerobic respiration, hyperoxia (95% O(2)/5% CO(2)) damages DNA, inhibits proliferation in G1, S and G2 phases of the cell cycle, and induces necrosis. The current study examines whether growth arrest in G1 protects pulmonary epithelial cells from oxidative DNA damage and cell death. Mv1Lu pulmonary adenocarcinoma cells were chosen for studies because hyperoxia inhibits their proliferation in S and G2 phase, while they can be induced to arrest in G1 by altering culture conditions. Hyperoxia inhibited proliferation, increased intracellular redox, and rapidly reduced clonogenic survival. In contrast, Mv1Lu cells treated with transforming growth factor (TGF)-beta1, deprived of serum or grown to confluency, arrested and remained predominantly in G1 even during exposure. Growth arrest in G1 significantly enhanced clonogenic survival by 10-50-fold. Enhanced survival was not due to reduction in the intracellular redox-state of the cells, but instead was associated with reduced DNA strand breaks and p53 expression. Our findings suggest that the protective effects of G1 is mediated not simply by a reduction in intracellular ROS, but rather through an enhanced ability to limit or rapidly recognize and repair damaged DNA.     

Negative regulation of CHK2 activity by protein phosphatase 2A is modulated by DNA damage

Checkpoint kinase 2 (CHK2) is a major effector of the DNA damage response pathway and although its mechanism of activation has been well studied, the attenuation of its activity following DNA damage has not been explored. Here, we identify the B'α subunit of protein phosphatase 2A (PP2A) as a CHK2 binding partner and show that their interaction is modulated by DNA damage. B'α binds to the SQ/TQ repeat region of CHK2, which is a target of ATM phosphorylation. The induction of DNA double-strand breaks by gamma irradiation as well as treatment with doxorubicin causes dissociation of the B'α and CHK2 proteins. This dissociation correlates with an increase in the ATM-dependent phosphorylation of CHK2 at serines 33 and 35 in the SQ/TQ region. Indeed, mutating these sites to mimic phosphorylation increases the dissociation after irradiation. PP2A negatively regulates CHK2 phosphorylation at multiple sites, as well as its kinase activity. These data reveal a novel mechanism for PP2A to keep CHK2 inactive under normal conditions while also allowing for a rapid release from this regulation immediately following DNA damage. This is followed by a subsequent reconstitution of the PP2A/CHK2 complex in later time points after damage, which may help to attenuate the signal.     

RecA protein of Mycobacterium tuberculosis possesses pH-dependent homologous DNA pairing and strand exchange activities: implications for allele exchange in mycobacteria

To gain insights into inefficient allele exchange in mycobacteria, we compared homologous pairing and strand exchange reactions promoted by RecA protein of Mycobacterium tuberculosis to those of Escherichia coli RecA protein. The extent of single-stranded binding protein (SSB)-stimulated formation of joint molecules by MtRecA was similar to that of EcRecA over a wide range of pH values. In contrast, strand exchange promoted by MtRecA was inhibited around neutral pH due to the formation of DNA networks. At higher pH, MtRecA was able to overcome this constraint and, consequently, displayed optimal strand exchange activity. Order of addition experiments suggested that SSB, when added after MtRecA, was vital for strand exchange. Significantly, with shorter duplex DNA, MtRecA promoted efficient strand exchange without network formation in a pH-independent fashion. Increase in the length of duplex DNA led to incomplete strand exchange with concomitant rise in the formation of intermediates and networks in a pH-dependent manner. Treatment of purified networks with S1 nuclease liberated linear duplex DNA and products, consistent with a model in which the networks are formed by the invasion of hybrid DNA by the displaced linear single-stranded DNA. Titration of strand exchange reactions with ATP or salt distinguished a condition under which the formation of networks was blocked, but strand exchange was not significantly affected. We discuss how these results relate to inefficient allele exchange in mycobacteria.