Cold atmospheric pressure plasma jet interactions with plasmid DNA

The effect of a cold (<40 °C) radio frequency-driven atmospheric pressure plasma jet on plasmid DNA has been investigated. Gel electrophoresis was used to analyze the DNA forms post-treatment. The experimental data are fitted to a rate equation model that allows for quantitative determination of the rates of single and double strand break formation. The formation of double strand breaks correlates well with the atomic oxygen density. Taken with other measurements, this indicates that neutral components in the jet are effective in inducing double strand breaks.

DNA-dependent Protein Kinase-mediated Phosphorylation of Protein Kinase B Requires a Specific Recognition Sequence in the C-terminal Hydrophobic Motif

DNA-dependent protein kinase (DNA-PK) has been implicated in a variety of nuclear processes including DNA double strand break repair, V(D)J recombination, and transcription. A recent study showed that DNA-PK is responsible for Ser-473 phosphorylation in the hydrophobic motif of protein kinase B (PKB/Akt) in genotoxic-stressed cells, suggesting a novel role for DNA-PK in cell signaling. Here, we report that DNA-PK activity toward PKB peptides is impaired in DNA-PK knock-out mouse embryonic fibroblast cells when compared with wild type. In addition, human glioblastoma cells expressing a mutant form of DNA-PK (M059J) displayed a lower DNA-PK activity when compared with glioblastoma cells expressing wild-type DNA- PK (M059K) when PKB peptide substrates were tested. DNA- PK preferentially phosphorylated PKB on Ser-473 when compared with its known in vitro substrate, p53. A consensus hydrophobic amino acid surrounding the Ser-473 phospho-acceptor site in PKB containing amino acids Phe at position +1 and +4 and Tyr at position -1 are critical for DNA- PK activity. Thus, these data define the specificity of DNA- PK action as a Ser-473 kinase for PKB in DNA repair signaling.

In vitro isoflavone supplementation reduces hydrogen peroxide-induced DNA damage in sperm

Isoflavones are plant compounds, proposed to have health benefits in a variety of human diseases, including coronary heart disease and endocrine-responsive cancers. Their physiological effects include possible antioxidant activity, therefore suggesting a role for isoflavones in the prevention of male infertility. The aim of this study was to test the antioxidant effects of the isoflavones genistein and equol on sperm DNA integrity, assessed in vitro after hydrogen peroxide-mediated damage, using the cornet assay. Pre-treatment with genistein or equol at doses of 0.01-100 mumol/l significantly protected sperm DNA against oxidative damage. Both ascorbic acid (10-600 mumol/l) and alpha-tocopherol (1-100 mumol/l) also protected. Compared with ascorbic acid and alpha-tocopherol, added at physiological concentrations, genistein was the most potent antioxidant, followed by equol, ascorbic acid, and alpha-tocopherol. Genistein and equol added in combination were more protective than when added singly. Based on these preliminary data, which are similar to those observed previously in lymphocytes, these compounds may have a role to play in antioxidant protection against male infertility.

Cells deficient in the base excision repair protein, DNA polymerase beta, are hypersensitive to oxaliplatin chemotherapy

A significant proportion of human cancers overexpress DNA polymerase beta (Pol beta), the major DNA polymerase involved in base excision repair. The underlying mechanism and biological consequences of overexpression of this protein are unknown. We examined whether Pol beta, expressed at levels found in tumor cells, is involved in the repair of DNA damage induced by oxaliplatin treatment and whether the expression status of this protein alters the sensitivity of cells to oxaliplatin. DNA damage induced by oxaliplatin treatment of HCT116 and HT29 colon cancer cells was observed to be associated with the stabilization of Pol beta protein on chromatin. In comparison with HCT116 colon cancer cells, isogenic oxaliplatin-resistant (HCT-OR) cells were found to have higher constitutive levels of Pol beta protein, faster in vitro repair of a DNA substrate containing a single nucleotide gap and faster repair of 1,2-GG oxaliplatin adduct levels in cells. In HCT-OR cells, small interfering RNA knockdown of Pol beta delayed the repair of oxaliplatin-induced DNA damage. In a different model system, Pol beta-deficient fibroblasts were less able to repair 1,2-GG oxaliplatin adducts and were hypersensitive to oxaliplatin treatment compared with isogenic Pol beta-expressing cells. Consistent with previous studies, Pol beta-deficient mouse fibroblasts were not hypersensitive to cisplatin treatment. These data provide the first link between oxaliplatin sensitivity and DNA repair involving Pol beta. They demonstrate that Pol beta modulates the sensitivity of cells to oxaliplatin treatment. Oncogene (2010) 29, 463-468; doi:10.1038/onc.2009.327; published online 19 October 2009

Spatio-temporal analysis of DNA damage repair using the X-ray microbeam

Cellular response to radiation damage is made by a complex network of pathways and feedback loops whose spatiotemporal organization is still unclear despite its decisive role in determining the fate of the damaged cell. The single-cell approach and the high spatial resolution offered by microbeams provide the perfect tool to study and quantify the dynamic processes associated with the induction and repair of DNA damage. The soft X-ray microbeam has been used to follow the development of radiation induced foci in live cells by monitoring their size and intensity as a function of dose and time using yellow fluorescent protein (YFP) tagging techniques. Preliminary data indicate a delayed and linear rising of the intensity signal indicating a slow kinetic for the accumulation of DNA repair protein 53BP1. A slow and limited foci diffusion has also been observed. Further investigations are required to assess whatever such diffusion is consistent with a random walk pattern or if it is the result of a more structured lesion processing phenomenon. In conclusion, our data indicates that the use of microbeams coupled to live cell microscopy represent a sophisticated approach for visualizing and quantifying the dynamics changes of DNA proteins at the damaged sites.

Resonance Raman probing of the interaction between dipyridophenazine complexes of Ru(II) and DNA

Resonance Raman (RR) spectroscopy has been used to probe the interaction between dipyridophenazine (dppz) complexes of ruthenium(II), [Ru(L)(2)(dppz)](2+) (L = 1,10-phenanthroline (1) and 2,2-bipyridyl (2)), and calf-thymus DNA. Ground electronic state RR spectra at selected probe wavelengths reveal enhancement patterns which reflect perturbation of the dppz-centered electronic transitions in the UV-vis spectra in the presence of DNA. Comparison of the RR spectra recorded of the short-lived MLCT excited states of both complexes in aqueous solution with those of the longer-lived states of the complexes in the DNA environment reveals changes to excited state modes, suggesting perturbation of electronic transitions of the dppz ligand in the excited state as a result of intercalation. The most prominent feature, at 1526 cm(-1), appears in the spectra of both 1 and 2 and is a convenient marker band for intercalation. For 1, the excited state studies have been extended to the A and A enantiomers. The marker band appears at the same frequency for both but with different relative intensities. This is interpreted as reflecting the distinctive response of the enantiomers to the chiral environment of the DNA binding sites. The results, together with some analogous data for other potentially intercalating complexes, are considered in relation to the more general application of time-resolved RR spectroscopy for investigation of intercalative interactions of photoexcited metal complexes with DNA.

Regulatory regime change in Turkish banks:Reactive or proactive?

This paper examines the positive contributions made toward restructuring the regulatory framework of Turkey's banking and financial sectors prior to and post the 2000–2001 financial crisis. Drawing on a framework initially developed by Onis and Senses, 2007 and Onis and Senses, 2009 and further referred to by Onis, 2009 and Onis, 2010 it argues that financial reforms undertaken by the Turkish government would not have been successful without the strong support of domestic coalitions. While the external pressures put on the Turkish government from the International Monetary Fund, The World Bank and the European Union for financial reforms were necessary to kick start the reforms as a reactive process, these pressures on their own may have served only the interests of financial business elites at the expense of the broader stakeholders. Empirical data for the study was collected from documentary analysis of key financial institutions and interviews with twenty major Turkish regulatory agents and other stakeholders. The paper then discusses how the perceptions of these stakeholders are embodied into, and have influenced, regulatory regime change in Turkey from a reactive state to a more proactive one.

DNA damage induction in dry and hydrated DNA by synchrotron radiation

Radiation biophysics has sought to understand at a molecular level, the mechanisms through which ionizing radiations damage DNA, and other molecules within living cells. The complexity of lesions produced in the DNA by ionizing radiations is thought to depend on the amount of energy deposited at the site of each lesion. To study the relationship between the energy deposited and the damage produced, we have developed novel techniques for irradiating dry prasmid DNA, partially re-hydrated DNA and DNA in solution using monochromatic vacuum-UV synchrotron radiation. We have used photons in the energy range 7-150 eV, corresponding to the range of energies typically involved in the efficient production of DNA single-strand (SSB), and double-strand breaks (DSB) by ionizing radiation. The data show that both types of breaks are produced at all energies investigated (with, or without water present). Also, the energy dependence for DSB induction follows a similar trend to SSB induction but at a 20-30-fold reduced incidence, suggesting a common precursor for both types of damage. Preliminary studies where DNA has been irradiated in solution indicate a change in the shape of the dose-effect curve (from linear, to linear-quadratic for double-strand break induction) and a large increase in sensitivity due to the presence of water.

Measurement of DNA damage by electrons with energies between 25 and 4000 eV.

All ionizing radiations deposit energy stochastically along their tracks. The resulting distribution of energies deposited in a small target such as the DNA helix leads to a corresponding spectrum in the severity of damage produced. So far, most information about the probable spectra of DNA lesion complexity has come from Monte Carlo studies which endeavour to model the relationship between the energy deposited in DNA and the damage induced. The aim of this paper is to establish methods of determining this relationship by irradiating pBR322 plasmid DNA using low energy electrons with energies comparable with the minimum energy thought to produce critical damage. The technique of agarose gel electrophoresis has been used to ascertain the fraction of DNA single- and double-strand breaks induced by monoenergetic electrons with energies as low as 25 eV. Our data show that the threshold electron energy for induction of single-strand breaks is

A study of DNA fragmentation patterns in cells irradiated with charged particles: evidence for non-random distributions

Many studies have shown that the effectiveness of radiations of varying LET is similar when yields of dsb have been measured, despite large differences in biological response. Recent evidence has suggested however, that current techniques underestimate the yields of dsb. By monitoring the fragmentation of DNA over a wide range of fragment sizes ( 6 Mbp) by pulsed field electrophoresis, RBE values greater than 1.0 for radiations of around 100 keV/mm have been determined. The data provide evidence for the production of correlated breaks produced within cells as particle tracks traverse the nucleus. The highly ordered structure of DNA within mammalian cells may lead to clustering of breaks over distances related to the repeating unit structures of the chromatin. As well as these regionally damaged sites, a major contributor to radiation effectiveness will be the localised clustering of damage in the 1 - 20 bp region. A major effort is required to elucidate the relative importance of these levels of clustering and their importance in biological response.

Critical energies for ssb and dsb induction in plasmid DNA by vacuum-UV photons: an arrangement for irradiating dry or hydrated DNA with monochromatic photons

Purpose: Theoretical modelling techniques are often used to simulate the action of ionizing radiations on cells at the nanometre level, Using monoenergetic vacuum-UV (VUV) radiation to irradiate DNA either dry or humidified, the action spectra for the induction of DNA damage by low energy photons and the role of water and can be studied. These data provide inputs for the theoretical models.

Evidence for complexity at the nanometer scale of radiation-induced DNA DSBs as a determinant of rejoining kinetics

The rejoining kinetics of double-stranded DNA fragments, along with measurements of residual damage after postirradiation incubation, are often used as indicators of the biological relevance of the damage induced by ionizing radiation of different qualities. Although it is widely accepted that high-LET radiation-induced double-strand breaks (DSBs) tend to rejoin with kinetics slower than low-LET radiation-induced DSBs, possibly due to the complexity of the DSB itself, the nature of a slowly rejoining DSB-containing DNA lesion remains unknown. Using an approach that combines pulsed-field gel electrophoresis (PFGE) of fragmented DNA from human skin fibroblasts and a recently developed Monte Carlo simulation of radiation-induced DNA breakage and rejoining kinetics, we have tested the role of DSB-containing DNA lesions in the 8-kbp-5.7-Mbp fragment size range in determining the DSB rejoining kinetics. It is found that with low-LET X rays or high LET alpha particles, DSB rejoining kinetics data obtained with PFGE can be computer-simulated assuming that DSB rejoining kinetics does not depend on spacing of breaks along the chromosomes. After analysis of DNA fragmentation profiles, the rejoining kinetics of X-ray-induced DSBs could be fitted by two components: a fast component with a half-life of 0.9 +/- 0.5 h and a slow component with a half-life of 16 +/- 9 h. For a particles, a fast component with a half-life of 0.7 +/- 0.4 h and a slow component with a half-life of 12 5 h along with a residual fraction of unrepaired breaks accounting for 8% of the initial damage were observed. In summary, it is shown that genomic proximity of breaks along a chromosome does not determine the rejoining kinetics, so the slowly rejoining breaks induced with higher frequencies after exposure to high-LET radiation (0.37 +/- 0.12) relative to low-LET radiation (0.22 +/- 0.07) can be explained on the basis of lesion complexity at the nanometer scale, known as locally multiply damaged sites. (c) 2005 by Radiation Research Society.

Label-free detection of nanomolar unmodified single- and double-stranded dna by using surface-enhanced raman spectroscopy on Ag and Au colloids

Unlabelled single- and double-stranded DNA (ssDNA and dsDNA, respectively) has been detected at concentrations =10-9?M by surface-enhanced Raman spectroscopy. Under appropriate conditions the sequences spontaneously adsorbed to the surface of both Ag and Au colloids through their nucleobases; this allowed highly reproducible spectra with good signal-to-noise ratios to be recorded on completely unmodified samples. This eliminated the need to promote absorption by introducing external linkers, such as thiols. The spectra of model ssDNA sequences contained bands of all the bases present and showed systematic changes when the overall base composition was altered. Initial tests also showed that small but reproducible changes could be detected between oligonucleotides with the same bases arranged in a different order. The spectra of five ssDNA sequences that correspond to different strains of the Escherichia coli bacterium were found to be sufficiently composition-dependent so that they could be differentiated without the need for any advanced multivariate data analysis techniques.

Molecular dynamics studies of the STAT3 homodimer:DNA complex: relationships between STAT3 mutations and protein-DNA recognition.

Signal Transducers and Activators of Transcription (STAT) proteins are a group of latent cytoplasmic transcription factors involved in cytokine signaling. STAT3 is a member of the STAT family and is expressed at elevated levels in a large number of diverse human cancers and is now a validated target for anticancer drug discovery.. Understanding the dynamics of the STAT3 dimer interface, accounting for both protein-DNA and protein-protein interactions, with respect to the dynamics of the latent unphosphorylated STAT3 monomer, is important for designing potential small-molecule inhibitors of the activated dimer. Molecular dynamics (MD) simulations have been used to study the activated STAT3 homodimer:DNA complex and the latent unphosphorylated STAT3 monomer in an explicit water environment. Analysis of the data obtained from MD simulations over a 50 ns time frame has suggested how the transcription factor interacts with DNA, the nature of the conformational changes, and ways in which function may be affected. Examination of the dimer interface, focusing on the protein-DNA interactions, including involvement of water molecules, has revealed the key residues contributing to the recognition events involved in STAT3 protein-DNA interactions. This has shown that the majority of mutations in the DNA-binding domain are found at the protein-DNA interface. These mutations have been mapped in detail and related to specific protein-DNA contacts. Their structural stability is described, together with an analysis of the model as a starting-point for the discovery of novel small-molecule STAT3 inhibitors.

The effect of sperm DNA fragmentation on miscarriage rates: a systematic review and meta-analysis

STUDY QUESTION Is there an association between high levels of sperm DNA damage and miscarriage?SUMMARY ANSWERMiscarriage rates are positively correlated with sperm DNA damage levels.WHAT IS KNOWN ALREADYMost ejaculates contain a subpopulation of sperm with DNA damage, also referred to as DNA fragmentation, in the form of double or single-strand breaks which have been induced in the DNA prior to or following ejaculation. This DNA damage may be particularly elevated in some subfertile men, hence several studies have examined the link between sperm DNA damage levels and conception and miscarriage rates.STUDY DESIGN, SIZE, DURATIONA systematic review and meta-analysis of studies which examined the effect of sperm DNA damage on miscarriage rates was performed. Searches were conducted on MEDLINE, EMBASE and the Cochrane Library without any language restrictions from database inception to January 2012.PARTICIPANTS/MATERIALS, SETTING, METHODSWe used the terms 'DNA damage' or 'DNA fragmentation' combined with 'miscarriage', 'abortion' or 'pregnancy' to generate a set of relevant citations. Data extraction was performed by two reviewers. Study quality was assessed using the Newcastle-Ottawa Scale. Meta-analysis of relative risks of miscarriage was performed with a random effects model. Subgroup analyses were performed by the type of DNA damage test, whether the sperm examined were prepared or from raw semen and for pregnancies resulting from IVF or ICSI treatment.MAIN RESULTS AND THE ROLE OF CHANCEWe identified 16 cohort studies (2969 couples), 14 of which were prospective. Eight studies used acridine orange-based assays, six the TUNEL assay and two the COMET assay. Meta-analysis showed a significant increase in miscarriage in patients with high DNA damage compared with those with low DNA damage [risk ratio (RR) = 2.16 (1.54, 3.03), P <0.00001)]. A subgroup analysis showed that the miscarriage association is strongest for the TUNEL assay (RR = 3.94 (2.45, 6.32), P <0.00001).LIMITATIONS, REASONS FOR CAUTIONThere is some variation in study characteristics, including the use of different assays and different thresholds for DNA damage and the definition of pregnancy loss.WIDER IMPLICATIONS OF THE FINDINGSThe use of methods which select sperm without DNA damage for use in assisted conception treatment may reduce the risk of miscarriage. This finding indicates that assays detecting DNA damage could be considered in those suffering from recurrent pregnancy loss. Further research is necessary to study the mechanisms of DNA damage and the potential therapeutic effects of antioxidant therapy.STUDY FUNDING/COMPETING INTEREST(S)None.