DNA Protection Mechanisms Research Articles

DNA-induced structural changes in DNA-damage suppressor protein Dsup by NMR and circular dichroism

DNA damage suppressor protein (Dsup) from water bear Ramazzottius varieormatus can suppress X-ray-induced DNA damage and improve radio-tolerance in human cells [Hashimoto, et al. Nat Commun 7, 12808 (2016)]. The functional information about this unique protein is very limited and its structure is unknown. Sequence analysis suggests that Dsup is an intrinsically disordered protein with extended unstructured regions and a potential single-helix domain. We are studying Dsup using a variety of biophysical methods to understand the basis of its interaction with DNA and the mechanism of DNA protection.

A computational structural study on the DNA-protecting role of the tardigrade-unique Dsup protein

The remarkable ability of tardigrades to withstand a wide range of physical and chemical extremes has attracted a considerable interest in these small invertebrates, with a particular focus on the protective roles of proteins expressed during such conditions. The discovery that a tardigrade-unique protein named Dsup (damage suppressor) protects DNA from damage produced by radiation and radicals, has raised expectations concerning its potential applications in biotechnology and medicine. We present in this paper what might be dubbed a “computational experiment” on the Dsup-DNA system. By means of molecular modelling, calculations of electrostatic potentials and electric fields, and all-atom molecular dynamics simulations, we obtained a dynamic picture of the Dsup-DNA interaction. Our results suggest that the protein is intrinsically disordered, which enables Dsup to adjust its structure to fit DNA shape. Strong electrostatic attractions and high protein flexibility drive the formation of a molecular aggregate in which Dsup shields DNA. While the precise mechanism of DNA protection conferred by Dsup remains to be elucidated, our study provides some molecular clues of their association that could be of interest for further investigation in this line.

Marine macroalgae as a dietary source of genoprotection in gilthead seabream (Sparus aurata) against endogenous and exogenous challenges

DNA integrity and stability are essential to organisms' health and survival. However, it has been neglected in what concerns to fish farming, disregarding the potential impact of endogenous/ exogenous factors. As marine macroalgae constitute a source of natural compounds with a large spectrum of biological activities, this study, situated in the interface of nutritional-genetic research and development of algae practical applications, aimed to evaluate the genoprotective properties of a macroalgae-enriched diet (total percentage of 5%, incorporating equal percentages of Ulva rigida, Gracilaria gracilis and Fucus vesiculosus) in gilthead seabream (Sparus aurata). Protection was assessed in relation to a basal genome integrity and against an exogenous genotoxic challenge (cyclophosphamide; CP). Fish were reared for 30 days with the supplemented diet, being then injected with CP and sampled at days 3 and 10 post-injection (p.i.). To evaluate whether the favorable effects remain after the end of supplementation, a fish subgroup previously fed with algae-enriched diet was submitted to a diet reversion at day 3 p.i., being thereafter fed with the standard diet. Genetic damage was evaluated through the erythrocytic nuclear abnormalities (ENA) and comet assays and complemented by the assessment of the antioxidant system. Results pointed out that algae-enriched feed exhibits anti-genotoxic properties, mostly expressed in relation to the exogenous pressure, manifest in relation to DNA strand breaks and chromosomal lesions, also reducing oxidative DNA damage. Nonetheless, blood antioxidants were only punctually altered by the supplemented diet (e.g. catalase and glutathione-S-transferase). Analyzing the effect persistence, it was perceived that 7 days without algae uptake was enough to partially reduce the protection efficacy. Overall, these findings are promising towards the benefits of macroalgae inclusion in fish diet, and thus, to invigorate mariculture activity and the commercial use of algae, also providing new insights on the DNA protection mechanisms.

DNA Damage and Repair in Human Reproductive Cells.

The fundamental underlying paradigm of sexual reproduction is the production of male and female gametes of sufficient genetic difference and quality that, following syngamy, they result in embryos with genomic potential to allow for future adaptive change and the ability to respond to selective pressure. The fusion of dissimilar gametes resulting in the formation of a normal and viable embryo is known as anisogamy, and is concomitant with precise structural, physiological, and molecular control of gamete function for species survival. However, along the reproductive life cycle of all organisms, both male and female gametes can be exposed to an array of “stressors” that may adversely affect the composition and biological integrity of their proteins, lipids and nucleic acids, that may consequently compromise their capacity to produce normal embryos. The aim of this review is to highlight gamete genome organization, differences in the chronology of gamete production between the male and female, the inherent DNA protective mechanisms in these reproductive cells, the aetiology of DNA damage in germ cells, and the remarkable DNA repair mechanisms, pre- and post-syngamy, that function to maintain genome integrity.

A novel bacterial class V dye-decolourizing peroxidase from the extremophile Deinococcus radiodurans: cloning, expression optimization, purification, crystallization, initial characterization and X-ray diffraction analysis.

Deinococcus radiodurans is a bacterium with extreme resistance to desiccation and radiation. The resistance mechanism is unknown, but an efficient reactive oxygen species (ROS) scavenging system and DNA-repair and DNA-protection mechanisms are believed to play important roles. Here, the cloning and small- and medium-scale expression tests of a novel dye-decolourizing peroxidase from D. radiodurans (DrDyP) using three different Escherichia coli strains and three different temperatures in order to identify the optimum conditions for the expression of recombinant DrDyP are presented. The best expression conditions were used for large-scale expression and yielded ∼10 mg recombinant DrDyP per litre of culture after purification. Initial characterization experiments demonstrated unusual features with regard to the haem spin state, which motivated the crystallization experiment. The obtained crystals were used for data collection and diffracted to 2.2 Å resolution. The crystals belonged to the trigonal space group P31 or P32, with unit-cell parameters a=b=64.13, c=111.32 Å, and are predicted to contain one DrDyP molecule per asymmetric unit. Structure determination by molecular replacement using previously determined structures of dye-decolourizing peroxidases with ∼30% sequence identity at ∼2 Å resolution as templates are ongoing.

Tritiated water exposure disrupts myofibril structure and induces mis-regulation of eye opacity and DNA repair genes in zebrafish early life stages

Tritium (3H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). The present study aimed to identify early biomarkers of HTO contamination through the use of an aquatic model, the zebrafish (Danio rerio). We used the zebrafish embryo-larvae model to investigate the modes of action of HTO exposure at dose rates of 0.4 and 4 mGy/h, dose rates expected to induce deleterious effects on fish. Zebrafish were exposed to HTO from 3 hpf (hours post fertilization) to 96 hpf. The transcriptomic effects were investigated 24 h and 96 h after the beginning of the contamination, using mRNAseq. Results suggested an impact of HTO contamination, regardless of the dose rate, on genes involved in muscle contraction (tnnt2d, tnni2a.4, slc6a1a or atp2a1l) and eye opacity (crygm2d9, crygmxl1, mipb or lim2.3) after 24 h of contamination. Interestingly, an opposite differential expression was highlighted in genes playing a role in muscle contraction and eye opacity in 24 hpf embryos when comparing dose rates, suggesting an onset of DNA protective mechanisms. The expression of h2afx and ddb2 involved in DNA repair was enhanced in response to HTO exposure. The entrainment of circadian clock and the response to H2O2 signalling pathways were enriched at 96 hpf at 0.4 mGy/h and in both stages after 4 mGy/h. Genes involved in ROS scavenging were differentially expressed only after 24 h of exposure for the lowest dose rate, suggesting the onset of early protective mechanisms against oxidative stress. Effects highlighted on muscle at the molecular scale were confirmed at a higher biological scale, as electron microscopy observations revealed sarcomere impairments in 96 hpf larvae for both dose rates. Together with other studies, the present work provides useful data to better understand modes of action of tritium on zebrafish embryos-larvae.

The Role of the Mitochondria in the Evolution of Stem Cells, Including MUSE Stem Cells and Their Biology.

From the transition of single-cell organisms to multicellularity of metazoans, evolutionary pressures selected new genes and phenotypes to cope with the oxygenation of the Earth's environment, especially via the symbiotic acquisition of the mitochondrial organelle. There were many new genes and phenotypes that appeared, namely, stem cells, low-oxygen-micro-environments to house these genes ("niches"), new epigenetic mechanisms to regulate , selectively, the gene repertoire to control proliferation, differentiation, apoptosis, senescence and DNA protection mechanisms, including antioxidant genes and DNA repair. This transition required a critical regulation of the metabolism of glucose to produce energy for both the stem cell quiescent state and the energy-requiring differentiated state. While the totipotent-, embryonic-, pluripotent-, and a few adult organ-specific stem cells were recognized, only relatively recently, because of the isolation of somatic cell nuclear transfer (SCNT) stem cells and "induced pluripotent stem" cells, challenges to the origin of these "iPS" cells have been made. The isolation and characterization of human MUSE stem cells and more adult organ-specific adult stem cells have indicated that these MUSE cells have many shared characteristics of the "iPS" cells, yet they do not form teratomas but can give rise to the trigeminal cell layers. While the MUSE cells are a subset of human fibroblastic cells, they have not been characterized, yet, for the mitochondrial metabolic genes, either in the stem cell state or during their differentiation processes. A description of other human adult stem cells will be made to set future studies of how the MUSE stem cells compare to all other stem cells.

Genotoxicity inhibition by Syzygium cumini (L.) seed fraction and rutin: understanding the underlying mechanism of DNA protection.

Considering the ethnopharmacological importance of Syzygium cumini's seed and the lack of information on the antimutagenic and DNA-protecting mechanisms, a fraction-based study was conducted. Four different (hexane, chloroform, ethyl acetate, and aqueous) fractions were obtained from the sequential extraction of the methanolic extract of the seed. The most active antioxidant fraction (ethyl acetate) contained significant amount of phenolics and flavonoids. LC-qTOF-MS analysis of the ethyl acetate fraction revealed the presence of rutin, myricetin, naringin, cuscohygrin, and epoxycarryophyllone as constituent phytocompounds. The ethyl acetate fraction (100 μg ml-1) and a selected compound (rutin, 40 μg ml-1) showed remarkable decrease in the revertants frequency range from 74-77% and 66-84%, respectively, against both the mutagens (sodium azide (NaN3) and methyl methane sulfonate (MMS)) in the Salmonella typhimurium tester strains. All the statistical analyses were at a significance level of 0.05 between the different treatment groups. Moreover, the underlying mechanism of antimutagenicity using different treatment regime for rutin was explored. MMS-mediated DNA fragmentation and oxidation in lymphocytes were also shown to be decreased significantly when treated with the ethyl acetate fraction and rutin. Oxidative damage to pBR322 plasmid DNA was also reduced when incubated with different concentration of the ethyl acetate fraction and rutin. Biophysical (UV, fluorescence, ITC, etc.) and computational methods were employed to obtain a closer look at the DNA-rutin interaction. The data obtained clearly revealed that the ethyl acetate fraction exhibited promising antimutagenic and DNA-protective activity and its flavonoid constituents, including rutin, contribute significantly to the observed activity.

PpGpp couples transcription to DNA repair in E. coli.

The small molecule alarmone (p)ppGpp mediates bacterial adaptation to nutrient deprivation by altering the initiation properties of RNA polymerase (RNAP). ppGpp is generated in Escherichia coli by two related enzymes, RelA and SpoT. We show that ppGpp is robustly, but transiently, induced in response to DNA damage and is required for efficient nucleotide excision DNA repair (NER). This explains why relA-spoT-deficient cells are sensitive to diverse genotoxic agents and ultraviolet radiation, whereas ppGpp induction renders them more resistant to such challenges. The mechanism of DNA protection by ppGpp involves promotion of UvrD-mediated RNAP backtracking. By rendering RNAP backtracking-prone, ppGpp couples transcription to DNA repair and prompts transitions between repair and recovery states.

Parkinson’s Disease—Apoptosis and Dopamine Oxidation

Tyrosine hydroxylase, monoamine oxidase and aldehyde dehydrogenase all form oxygen radicals as part of their mechanisms of action. These oxygen radicals damage dopaminergic neurons in the substantianigra of the midbrain and cause them to die by a process of necrosis or apoptosis. Oxygen radicals quickly abstract hydrogen from DNA forming DNA radicals and causing DNA fragmentation, activation of DNA protective mechanisms, NAD depletion and cell death. Tyrosine hydroxylase is present in all dopaminergic neurons, is involved in the synthesis of dopamine and forms oxygen radicals in a redox mechanism involving its cofactor, tetrahydrobiopterin. Levodopa is used therapeutically in Parkinson’s disease patients since it is a precursor for dopamine, an inhibitor of tyrosine hydroxylase, and prolongs pa-tient’s lives. Monoamine oxidase converts dopamine into 3,4-dihydroxyphenylacetaldehyde and forms oxygen radi-cals.Aldehyde dehydrogenase oxidizes the aldehyde and forms oxygen radicals and 3,4-dihydroxyphenylacetic acid. The treatment of Parkinson’s disease should involveinhibitors of oxygen radical formation in dopaminergic neurons and neuroprotective agents that stimulate DNA repair and prevent cell death.

Amifostine protection against induced DNA damage in γ‐irradiated Escherichia coli cells depend on recN DNA repair gene product activity

Amifostine is the most effective radioprotector known and the only one accepted for clinical use in cancer radiotherapy. In this work, the antigenotoxic effect of amifostine against gamma-rays was studied in Escherichia coli cells deficient in DNA damage repair activities. Assays of irradiated cells treated with amifostine showed that the drug reduced the genotoxicity induced by radiation in E. coli wild-type genotypes and in uvr, recF, recB, recB-recC-recF mutant strains, but not in recN defective cells. Thus, the mechanism of DNA protection by amifostine against gamma-radiation-induced genotoxicity appears to involve participation of the RecN protein that facilitates repair of DNA double-strand breaks. The results are discussed in relation to amifostine's chemopreventive potential.

Adaptive mutation: shall we survive bacterial genetic skills?

The origin and dynamics of genetic variations is one of the key questions in the modem science that has still not come out with a final answer. Emerging concepts regarding genetic variation have always produced a great controversy because they hold a key to unlock a great mystery of evolution. With such a powerful motivation scientist working in the molecular biology, genetics and biochemistry gathered a vast amount of experimental data showing us that a genome is a dynamic, hierarchically organized and complex integrated system for storing and processing information. Dynamic balance between stability and mutability of DNA nucleotide sequences is essential for a proper functioning of the organism. Beside many DNA repairing proteins and DNA protective mechanisms organisms possess also biochemical systems capable of changing DNA information. One of the most controversial and at the same time the most informative one is a phenomenon called adaptive mutation. We shall review findings concerning the phenomenon of adaptive mutation in prokaryotes and point out an urgent need for the upgrade of the awkward neo-darvinistic view on the origin .of the genetic variation.

DNA protection mechanisms are not involved in the radioresistance of the hyperthermophilic archaea Pyrococcus abyssi and P. furiosus.

Hyperthermophilic archaea of the genus Pyrococcus are resistant to gamma radiation, suggesting that efficient mechanisms for DNA repair exist in these organisms. To determine whether protective mechanisms might also be implicated in this radioresistance, we have estimated the linear density of DNA double-stranded breaks caused by gamma irradiation in the genomic DNA of two Pyrococcus species, using Escherichia coli and the radioresistant bacterium Deinococcus radiodurans as controls. The linear density of double-stranded breaks was essentially the same in all four microorganisms when irradiation was carried under similar anaerobic conditions, indicating that no specific DNA protection mechanisms exist in Pyrococcus species. Using one- and two-dimensional gel electrophoresis we compared the protein patterns from Pyrococcus abyssi and P. furiosus cells that had or had not been exposed to gamma rays. We did not detect any significant protein induction following DNA damage in either species.

Parkinson's disease--redox mechanisms.

Parkinson's disease occurs in 1% of people over the age of 65 when about 60% of the dopaminergic neurons in the substantia nigra of the midbrain are lost. Dopaminergic neurons appear to die by a process of apoptosis that is induced by oxidative stress. Oxygen radicals abstract hydrogen from DNA forming DNA radicals that lead to DNA fragmentation, activation of DNA protective mechanisms, NAD depletion and apoptosis. Oxygen radicals can be formed in dopaminergic neurons by redox cycling of MPP+, the active metabolite of MPTP. This redox cycling mechanism involves the reduction of MPP+ by a number of enzymes, especially flavin containing enzymes, some of which are found in mitochondria. Tyrosine hydroxylase is present in all dopaminergic neurons and is responsible for the synthesis of dopamine. However, tyrosine hydroxylase can form oxygen radicals in a redox mechanism involving its cofactor, tetrahydrobiopterin. Dopamine may be oxidized by monoamine oxidase to form oxygen radicals and 3,4-dihydroxyphenylacetaldehyde. This aldehyde may be oxidized by aldehyde dehydrogenase with the formation of oxygen radicals and 3,4-dihydroxyphenylacetic acid. The redox mechanisms of oxygen radical formation by MPTP, tyrosine hydroxylase, monoamine oxidase and aldehyde dehydrogenase will be discussed. Possible clinical applications of these mechanisms will be briefly presented.