DNA氧化性损伤与端粒缩短

末端复制问题（the end replication problem）不能完全解释端粒在某些细胞分裂过程中迅速缩短的现象.40％的高压氧下细胞传代次数降低,端粒缩短速率增大,细胞出现衰老特征,端粒DNA上单链断裂积累.推测端粒缩短的主要原因在于衰老过程中或氧胁迫下端粒DNA单链断裂增多,使端粒末端单链片段在DNA复制时丢失.端粒酶和活性氧对端粒长度的正负调控作用的准确机制还有待于更深入的研究.     

Isolated cell behavior drives the evolution of antibiotic resistance

Bacterial antibiotic resistance is typically quantified by the minimum inhibitory concentration (MIC), which is defined as the minimal concentration of antibiotic that inhibits bacterial growth starting from a standard cell density. However, when antibiotic resistance is mediated by degradation, the collective inactivation of antibiotic by the bacterial population can cause the measured MIC to depend strongly on the initial cell density. In cases where this inoculum effect is strong, the relationship between MIC and bacterial fitness in the antibiotic is not well defined. Here, we demonstrate that the resistance of a single, isolated cell—which we call the single‐cell MIC (scMIC)—provides a superior metric for quantifying antibiotic resistance. Unlike the MIC, we find that the scMIC predicts the direction of selection and also specifies the antibiotic concentration at which selection begins to favor new mutants. Understanding the cooperative nature of bacterial growth in antibiotics is therefore essential in predicting the evolution of antibiotic resistance.     

Homozygous mutation of MTPAP causes cellular radiosensitivity and persistent DNA double-strand breaks

The study of rare human syndromes characterized by radiosensitivity has been instrumental in identifying novel proteins and pathways involved in DNA damage responses to ionizing radiation. In the present study, a mutation in mitochondrial poly-A-polymerase (MTPAP), not previously recognized for its role in the DNA damage response, was identified by exome sequencing and subsequently associated with cellular radiosensitivity. Cell lines derived from two patients with the homozygous MTPAP missense mutation were radiosensitive, and this radiosensitivity could be abrogated by transfection of wild-type mtPAP cDNA into mtPAP-deficient cell lines. Further analysis of the cellular phenotype revealed delayed DNA repair, increased levels of DNA double-strand breaks, increased reactive oxygen species (ROS), and increased cell death after irradiation (IR). Pre-IR treatment of cells with the potent anti-oxidants, α-lipoic acid and n-acetylcysteine, was sufficient to abrogate the DNA repair and clonogenic survival defects. Our results firmly establish that mutation of the MTPAP gene results in a cellular phenotype of increased DNA damage, reduced repair kinetics, increased cell death by apoptosis, and reduced clonogenic survival after exposure to ionizing radiation, suggesting a pathogenesis that involves the disruption of ROS homeostasis.     

Metabolic constraints on the evolution of antibiotic resistance

Despite our continuous improvement in understanding antibiotic resistance, the interplay between natural selection of resistance mutations and the environment remains unclear. To investigate the role of bacterial metabolism in constraining the evolution of antibiotic resistance, we evolved Escherichia coli growing on glycolytic or gluconeogenic carbon sources to the selective pressure of three different antibiotics. Profiling more than 500 intracellular and extracellular putative metabolites in 190 evolved populations revealed that carbon and energy metabolism strongly constrained the evolutionary trajectories, both in terms of speed and mode of resistance acquisition. To interpret and explore the space of metabolome changes, we developed a novel constraint‐based modeling approach using the concept of shadow prices. This analysis, together with genome resequencing of resistant populations, identified condition‐dependent compensatory mechanisms of antibiotic resistance, such as the shift from respiratory to fermentative metabolism of glucose upon overexpression of efflux pumps. Moreover, metabolome‐based predictions revealed emerging weaknesses in resistant strains, such as the hypersensitivity to fosfomycin of ampicillin‐resistant strains. Overall, resolving metabolic adaptation throughout antibiotic‐driven evolutionary trajectories opens new perspectives in the fight against emerging antibiotic resistance.     

Inflammation-induced DNA damage and damage-induced inflammation: a vicious cycle

Inflammation is the ultimate response to the constant challenges of the immune system by microbes, irritants or injury. The inflammatory cascade initiates with the recognition of microorganism-derived pathogen associated molecular patterns (PAMPs) and host cell-derived damage associated molecular patterns (DAMPs) by the pattern recognition receptors (PRRs). DNA as a molecular PAMP or DAMP is sensed directly or via specific binding proteins to instigate pro-inflammatory response. Some of these DNA binding proteins also participate in canonical DNA repair pathways and recognise damaged DNA to initiate DNA damage response. In this review we aim to capture the essence of the complex interplay between DNA damage response and the pro-inflammatory signalling through representative examples.     

RAS/MAPK signaling functions in oxidative stress,DNA damage response and cancer progression

Mitogen-activated protein kinase (MAPK) signaling pathways organize a great constitution network that regulates several physiological processes, like cell growth, differentiation, and apoptotic cell death. Due to the crucial importance of this signaling pathway, dysregulation of the MAPK signaling cascades is involved in the pathogenesis of various human cancer types. Oxidative stress and DNA damage are two important factors which in common lead to carcinogenesis through dysregulation of this signaling pathway. Reactive oxygen species (ROS) are a common subproduct of oxidative energy metabolism and are considered to be a significant physiological modulator of several intracellular signaling pathways including the MAPK pathway. Studies demonstrated that the MAP kinases extracellular signal-regulated kinase (ERK) 1/2 and p38 were activated in response to oxidative stress. In addition, DNA damage is a partly common circumstance in cell life and may result in mutation, cancer, and even cell death. Recently, accumulating evidence illustrated that the MEK/ERK pathway is associated with the suitable performance of cellular DNA damage response (DDR), the main pathway of tumor suppression. During DDR, the MEK/ERK pathway is regularly activated, which contributes to the appropriate activation of DDR checkpoints to inhibit cell division. Therefore, the aim of this review is to comprehensively discuss the critical function of MAPK signaling in oxidative stress, DNA damage, and cancer progression.     

Aluminium-induced production of oxygen radicals, lipid peroxidation and DNA damage in seedlings of rice (Oryza sativa)

The effect of aluminium (Al) on seedlings of two rice cultivars, Pusa Basmati and Vikas was investigated after different hours of exposure to 80 mol/L of external Al supply. With increasing time of exposure, the growing seedlings readily absorbed Al and its localization was greater in roots than shoots. Prolonged exposure to Al intensified lipid peroxidation, changed the activities of SOD and peroxidase and caused DNA damage. However, differential responses were observed between the seedlings of two rice cultivars under Al stress. A close inverse relationship existed between decreased root growth and increased Al accumulation, lipid peroxidation, SOD, peroxidase activities and DNA damage. The results demonstrate that roots are the major sites of Al localization and accumulation of Al promoted oxygen free radicals mediated peroxidation of membranes as evidenced by increased MDA levels and the activities of SOD and peroxidase. Our results for the first time showed that Al can cause DNA damage in rice.     

Apelin-13 attenuates cisplatin-induced cardiotoxicity through inhibition of ROS-mediated DNA damage and regulation of MAPKs and AKT pathways

Platinum-based chemotherapy represents one of the most effective ways in combating human cancers. However, the cardiotoxicity subsequent severely limited its clinical application. Increased evidences indicate that oxidative stress plays a crucial role in the pathological process of platinum-induced cardiotoxicity. It is reported that apelin-13 a bioactive peptide has the scavenging capacity of free radical, and it has the potential to regulate the cardiovascular system. Hence, the potential of apelin-13 to antagonize cisplatin-induced cardiotoxicity was evaluated in H9c2 rat myocardial cells in vitro and in C57 mice in vivo. The results showed that cisplatin indeed caused DNA damage in H9c2 cells by promoting the accumulation of intracellular reactive oxygen species (ROS) and superoxide anion, which led to cell apoptosis and resulted in overt cardiotoxicity. However, apelin-13 pre-treatment effectively attenuated the cisplatin-induced ROS and superoxide anion generation, inhibited DNA damage, and suppressed the PARP cleavage and caspases activation. Further investigation revealed that apelin-13 blocked cisplatin-induced H9c2 cells apoptosis involving the regulation of MAPKs and PI3K/Akt signaling pathway. Importantly, apelin-13 co-treatment also significantly attenuated cisplatin-induced cardiotoxicity in vivo by inhibiting myocardial cells apoptosis and improving angiogenesis in mice heart. Taken together, our results suggest that the use of apelin-13 may be an effective strategy for antagonizing the cardiotoxicity-induced by platinum-based chemotherapy.     

Efflux-linked accelerated evolution of antibiotic resistance at a population edge

1. Download : Download high-res image (191KB)
2. Download : Download full-size image

     

Interaction of stannous chloride leads to alteration in DNA, triphosphate nucleotides and isolated bases

Stannous chloride (SnCl₂) is a reducing chemical agent used in several man-made products. SnCl₂ can generate reactive oxygen species (ROS); therefore, studies have been carried out in order to better understand its damaging action in biological systems. In this work, calf thymus DNA, triphosphate nucleotides and isolated bases were incubated with SnCl₂ and the results were analyzed through UV spectrophotometry. The presence of stannous ions altered the absorption spectra of all three isolates. The amount of stannous ions associated to DNA was measured by atomic absorption spectrophotometry. Data showed that more than 40% of the initial SnCl₂ concentration was present in the samples. Our results are in accordance with the damaging potential of this salt and present evidence that stannous ions can complex with DNA, inducing ROS in its vicinity, which may be responsible for the observed lesions. (Mol Cell Biochem xxx: 173–179, 2005)     

The mechanisms of oxidative DNA damage and apoptosis induced by norsalsolinol, an endogenous tetrahydroisoquinoline derivative associated with Parkinson's disease

Tetrahydroisoquinoline (TIQ) derivatives are putative neurotoxins that may contribute to the degeneration of dopaminergic neurons in Parkinson's disease. One TIQ, norsalsolinol (NorSAL), is present in dopamine-rich areas of human brain, including the substantia nigra. Here, we demonstrate that NorSAL reduces cell viability and induces apoptosis via cytochrome c release and caspase 3 activation in SH-SY5Y human neuroblastoma cells. Cytochrome c release, caspase 3 activation, and apoptosis induction were all inhibited by the antioxidant N -acetylcysteine. Thus, reactive oxygen species (ROS) contribute to apoptosis induced by NorSAL. Treatment with NorSAL also increased levels of oxidative damage to DNA, a stimulus for apoptosis, in SH-SY5Y. To clarify the mechanism of intracellular DNA damage, we examined the DNA damage caused by NorSAL using ³²P-5'-end-labeled isolated DNA fragments. NorSAL induced DNA damage in the presence of Cu(II). Catalase and bathocuproine, a Cu(I) chelator, inhibited this DNA damage, suggesting that ROS such as the Cu(I)-hydroperoxo complex derived from the reaction of H₂O₂ with Cu(I), promote DNA damage by NorSAL. In summary, NorSAL-generated ROS induced oxidative DNA damage, which led to caspase-dependent apoptosis in neuronal cells.     

Oxidative DNA Damage and Activation of c-Jun N-Terminal Kinase Pathway in Fibroblasts from Patients with Hereditary Spastic Paraplegia

1.Hereditary spastic paraplegia (HSP) is a genetically heterogeneous group of neurodegenerative disorders affecting 1 in 10,000 individuals. The present study was aimed to elucidate the role played by reactive oxygen species (ROS) in the pathogenesis of this disease. 2. To address this question we used 7-11 passaged fibroblasts from HSP patients to measure the extent of DNA damage induced by H2O2 treatment and to evaluate the JNK phosphorylation level after hydrogen peroxide and serum stimuli. 3. The present study demonstrates that HSP cells compared to controls are more sensitive to DNA damages induced by H2O2 treatment, and that JNK phosphorylation levels are increased in HSP fibroblasts compared to controls after hydrogen peroxide and serum stimuli. These results suggest a ROS-mediated pathogenetic mechanism for this disease.     

DNA repair and the evolution of transformation IV. DNA damage increases transformation

Natural genetic transformation in the bacterium Bacillus subtilis provides a model system to explore the evolutionary function of sexual recombination. In the present work, we study the response of transformation to UV irradiation using donor DNAs that differ in sequence homology to the recipient's chromosome and in the mechanism of transformation. The four donor DNAs used include homologous-chromosomal-DNA, two plasmids containing a fragment of B. subtilis trp⁺ operon DNA and a plasmid with no sequence homology to the recipient cell's DNA. Transformation frequencies for these DNA molecules increase with increasing levels of DNA damage (UV radiation) to recipient cells, only if their transformation requires homologous recombination (i.e. is recA⁺-dependent). Transformation with non-homologous DNA is independent of the recipient's recombination system and transformation frequencies for it do not respond to increases in UV radiation. The transformation frequency for a selectable marker increases in response to DNA damage more dramatically when the locus is present on small, plasmid-borne, homologous fragments than if it is carried on high molecular weight chromosomal fragments. We also study the kinetics of transformation for the different donor DNAs. Different kinetics are observed for homologous transformation depending on whether the homologous locus is carried on a plasmid or on chromosomal fragments. Chromosomal DNA- and non-homologous-plasmid-DNA-mediated transformation is complete (maximal) within several minutes, while transformation with a plasmid containing homologous DNA is still occurring after an hour. The results indicate that DNA damage directly increases rates of homologous recombination and transformation in B. subtilis. The relevance of these results and recent results of other labs to the evolution of transformation are discussed.     

Repair of Oxidative DNA Damage in Saccharomyces cerevisiae

Malfunction of enzymes that detoxify reactive oxygen species leads to oxidative attack on biomolecules including DNA and consequently activates various DNA repair pathways. The nature of DNA damage and the cell cycle stage at which DNA damage occurs determine the appropriate repair pathway to rectify the damage. Oxidized DNA bases are primarily repaired by base excision repair and nucleotide incision repair. Nucleotide excision repair acts on lesions that distort DNA helix, mismatch repair on mispaired bases, and homologous recombination and non-homologous end joining on double stranded breaks. Post-replication repair that overcomes replication blocks caused by DNA damage also plays a crucial role in protecting the cell from the deleterious effects of oxidative DNA damage. Mitochondrial DNA is also prone to oxidative damage and is efficiently repaired by the cellular DNA repair machinery. In this review, we discuss the DNA repair pathways in relation to the nature of oxidative DNA damage in Saccharomyces cerevisiae.     

Chemiluminescence assay for reactive oxygen species scavenging activities and inhibition on oxidative damage of DNA in Deinococcus radiodurans.

Free radical scavenging effects of the cellular protein extracts from two strains of Deinococcus radiodurans and Escherichia coli against O2-, H2O2 and *OH were investigated by chemiluminescence (CL) methods. The cellular protein extracts of D. radiodurans R1 and KD8301 showed higher scavenging effects on O2- than that of E. coli. D. radiodurans R1 and KD8301 also strongly scavenged H2O2 with an EC50 (50% effective concentration) of 0.12 and 0.2 mg/mL, respectively, compared to that of E. coli (EC50 = 3.56 mg/mL). The two strains of D. radiodurans were effective in scavenging *OH generated by the Fenton reaction, with EC50 of 0.059 and 0.1 mg/mL, respectively, compared to that of E. coli (EC50 > 1 mg/mL). Results from the chemiluminescence assay of *OH-induced DNA damage and the plasmid pUC18 DNA double-strand break (DSB) model in vitro showed that D. radiodurans had remarkably inhibitory effect on the *OH-induced oxidative damage of DNA. The scavenging effects of D. radiodurans on reactive oxygen species (ROS) played an important role in the response to oxidation stress and preventing against DNA oxidative damage, and may be attributed to intracellular scavenging proteins, including superoxide dismutase (SOD) and catalase.     

Oxidative and non-oxidative DNA damage and cardiovascular disease

Abstract

Evidence for the association of DNA damage with cardiovascular disease has been obtained from in vitro cell culture models, experimental cardiovascular disease and analysis of samples obtained from humans with disease. There is general acceptance that several factors associated with the risk of developing cardiovascular disease cause oxidative damage to DNA in cell culture models with both nuclear and mitochondrial DNA as targets. Moreover, evidence obtained over the past 10 years points to a possible mechanistic role for DNA damage in experimental atherosclerosis culminating in recent studies challenging the assumption that DNA damage is merely a biomarker of the disease process. This kind of mechanistic insight provides a renewed impetus for further studies in this area.     

The structure of the human AGT protein bound to DNA and its implications for damage detection

O6-Alklyguanine-DNA alkyltransferase (AGT) is an important DNA repair protein that protects cells from mutagenesis and toxicity arising from alkylating agents. We present an X-ray crystal structure of the wild-type human protein (hAGT) bound to double-stranded DNA with a chemically modified cytosine base. The protein binds at two different sites: one at the modified base, and the other across a sticky-ended DNA junction. The protein molecule that binds the modified cytosine base flips the base and recognizes it in its active site. The one that binds ends of neighboring DNA molecules partially flips an overhanging thymine base. This base is not inserted into the active-site pocket of the protein. These two different hAGT/DNA interactions observed in the structure suggest that hAGT may not detect DNA lesions by searching for the adduct itself, but rather for weakened and/or distorted base-pairs caused by base damage in the duplex DNA. We propose that hAGT imposes a strain on the DNA duplex and searches for DNA regions where the native structure is destabilized. The structure provides implications for pyrimidine recognition, improved inhibitor design, and a possible protein/protein interaction patch on hAGT.