DNA Structure In Cells Research Articles

Determination of Deleterious SNPs in NUDT15 Gene Related to Acute Lymphoblastic Leukemia by using Bioinformatics Tools

Aim: In acute lymphoblastic leukemia (ALL), thiopurine group drugs are the most basic drugs and are included in almost all treatment protocols, especially in maintenance treatment. The mechanism of action of thioguanine nucleotides is to enter the DNA structure in cells, disrupt DNA synthesis, and trigger programmed cell death. The impact of deleterious SNPs on nucleotide triphosphate diphosphatase protein regarding ALL is not yet fully understood. In this study, it was aimed to determine the possible deleterious impacts of missense variants in the NUDT15 gene on protein structure and stabilization that play a significant role in susceptibility to the disease, using modern bioinformatics software. Method: To access SNPs in the NUDT15, it was used National Center for Biotechnology Information (NCBI), Single Nucleotide Polymorphism Database (dbSNP). In bioinformatics tools used in this study included SIFT, PolyPhen-2, PROVEAN, SNAP2, and PANTHER, followed by I-Mutant, HOPE, and STRING. Results: The results of the analysis showed that in a total of 6663 SNPs in the NUDT15, 6 variants have been identified as ‘deleterious’. According to the I-Mutant software, 4 deleterious SNPs decreased protein stability while 2 deleterious SNPs increased protein stability. In the HOPE database analysis, E115G, E57G, F52L, and K33N mutant amino acids were found to be smaller and more hydrophobic than wild-type amino acids, while G53R and G145D mutant amino acids were found to be larger. Thus, all variations resulted in alterations in the net charge on the NUDT15 protein. Conclusion: Data on NUDT15 variants will contribute to the prediction of the patient’s response to thiopurine drugs in future studies, to a better understanding of the patient’s susceptibility to drug interactions, and ultimately to obtaining information about the prognosis.

Environment-Sensitive Nucleoside Probe Unravels the Complex Structural Dynamics of i-Motif DNAs.

Although evidence for the existence and biological role of i-motif (iM) DNA structures in cells is emerging, probing their structural polymorphism and identifying physiologically active conformations using currently available tools remain a major challenge. Here, we describe the development of an innovative device to investigate the conformation equilibrium of different iMs formed by C-rich telomeric repeat and oncogenic B-raf promoter sequences using a new conformation-sensitive dual-purpose nucleoside probe. The nucleoside is composed of a trifluoromethyl-benzofuran-2-yl moiety at the C5 position of 2'-deoxyuridine, which functions as a responsive fluorescent and 19F NMR probe. While the fluorescent component is useful in monitoring and estimating the folding process, the 19F label provides spectral signatures for various iMs, thereby enabling a systematic analysis of their complex population equilibrium under different conditions (e.g., pH, temperature, metal ions, and cell lysate). Distinct 19F signals exhibited by the iMs formed by the human telomeric repeat helped in calculating their relative population. A battery of fluorescence and 19F NMR studies using native and mutated B-raf oligonucleotides gave valuable insights into the iM structure landscape and its dependence on environmental conditions and also helped in predicting the structure of the major iM conformation. Overall, our findings indicate that the probe is highly suitable for studying complex nucleic acid systems.

Metal-Based G-Quadruplex Binders for Cancer Theranostics.

The ability of fluorescent small molecules, such as metal complexes, to selectively recognize G-quadruplex (G4) structures has opened a route to develop new probes for the visualization of these DNA structures in cells. The main goal of this review is to update the most recent research efforts towards the development of novel cancer theranostic agents using this type of metal-based probes that specifically recognize G4 structures. This encompassed a comprehensive overview of the most significant progress in the field, namely based on complexes with Cu, Pt, and Ru that are among the most studied metals to obtain this class of molecules. It is also discussed the potential interest of obtaining G4-binders with medical radiometals (e.g., 99mTc, 111In, 64Cu, 195mPt) suitable for diagnostic and/or therapeutic applications within nuclear medicine modalities, in order to enable their theranostic potential.

Helicases FANCJ, RTEL1 and BLM Act on Guanine Quadruplex DNA in Vivo.

Guanine quadruplex (G4) structures are among the most stable secondary DNA structures that can form in vitro, and evidence for their existence in vivo has been steadily accumulating. Originally described mainly for their deleterious effects on genome stability, more recent research has focused on (potential) functions of G4 structures in telomere maintenance, gene expression, and other cellular processes. The combined research on G4 structures has revealed that properly regulating G4 DNA structures in cells is important to prevent genome instability and disruption of normal cell function. In this short review we provide some background and historical context of our work resulting in the identification of FANCJ, RTEL1 and BLM as helicases that act on G4 structures in vivo. Taken together these studies highlight important roles of different G4 DNA structures and specific G4 helicases at selected genomic locations and telomeres in regulating gene expression and maintaining genome stability.

Furan induced ovarian damage in non-diabetic and diabetic rats and cellular protective role of lycopene.

In our work, furan, lycopene, and furan+lycopene treatments were applied to non-diabetic and diabetic female rats via gavage. Ovarian tissue alterations with histopathology, immunohistochemistry, malondialdehyde levels, oxidative stress parameters such as superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and harmful effect on ovarian tissue DNA were evaluated in all groups for 28days. Furan caused the changes histological, ovarian cell's DNA structure, malondialdehyde levels, antioxidant enzymes activities as in a statistically significant manner in each group. Useful effect of lycopene was determined both in non-diabetic and diabetic treatment groups against furan according to the used experimental parameters. Although some histopathological alterations were seen in diabetic and non-diabetic/diabetic plus furan-treated group's ovarians, lycopene restored these variations near to normal levels in furan+lycopene treated groups for in 28days. Additionally, the results of our immunohistochemical analysis and alterations of the oxidative stress parameters results also supported these findings. Our result confirms that lycopene has protective effect and significantly altered diabetes and furan-induced toxicity in the rat ovarian tissue.

DNA Sequence can Pin the Position of DNA Supercoils

While DNA obviously functions as the carrier of genetic information, it may also encode the three-dimensional spatial structure of DNA in cells. Here we study the relationship between structure and sequence in supercoiled DNA, which involves a reorganization of the DNA configuration via the formation of so-called plectonemes - coiled loop structures that locally form to minimize the torsional stress within the DNA. We use a novel high-throughput single-molecule assay called ISD (‘Intercalation-induced Supercoiling of DNA’, see Ganji et al, Nano Lett. 16, 4699, 2016), where we induce supercoiling of surface-attached DNA molecules by adding an intercalating dye. This assay allows us to visualize individual plectonemes and characterize their position-dependent frequency as well as dynamics such as nucleation, termination, and diffusion. From profiles of the average plectoneme density as a function of the position along the DNA molecule, we can study whether plectonemes exhibit any preference for certain DNA sequences. We indeed find that particular local DNA sequences pin plectonemes. Specifically, we identify DNA stretches with exclusively A or T bases (which we name ‘TorA tracts’) that strongly pin plectonemes. Interestingly, TorA tracts pin plectonemes just as well as poly-A tracts. This suggests that it is not a local intrinsic bending of the DNA (such as predicted for polyA tracts) that underlies the plectoneme pinning. Instead, we propose that local melting may lead to the formation of a kink at the tip of a plectoneme that provides a pinning site. Indeed we observed strong pinning at DNA mutation sites where 1 to 10 neighboring nucleotides are mispaired such that a ssDNA bubble is formed. Our data show that the DNA sequence does not merely encode for genetic information but also for structural information that governs the three-dimensional organization of supercoiled DNA.

G-quadruplex and G-rich sequence stimulate Pif1p-catalyzed downstream duplex DNA unwinding through reducing waiting time at ss/dsDNA junction

Alternative DNA structures that deviate from B-form double-stranded DNA such as G-quadruplex (G4) DNA can be formed by G-rich sequences that are widely distributed throughout the human genome. We have previously shown that Pif1p not only unfolds G4, but also unwinds the downstream duplex DNA in a G4-stimulated manner. In the present study, we further characterized the G4-stimulated duplex DNA unwinding phenomenon by means of single-molecule fluorescence resonance energy transfer. It was found that Pif1p did not unwind the partial duplex DNA immediately after unfolding the upstream G4 structure, but rather, it would dwell at the ss/dsDNA junction with a ‘waiting time’. Further studies revealed that the waiting time was in fact related to a protein dimerization process that was sensitive to ssDNA sequence and would become rapid if the sequence is G-rich. Furthermore, we identified that the G-rich sequence, as the G4 structure, equally stimulates duplex DNA unwinding. The present work sheds new light on the molecular mechanism by which G4-unwinding helicase Pif1p resolves physiological G4/duplex DNA structures in cells.

Detection of G-quadruplex DNA in mammalian cells

It has been proposed that guanine-rich DNA forms four-stranded structures in vivo called G-quadruplexes or G4 DNA. G4 DNA has been implicated in several biological processes, but tools to study G4 DNA structures in cells are limited. Here we report the development of novel murine monoclonal antibodies specific for different G4 DNA structures. We show that one of these antibodies designated 1H6 exhibits strong nuclear staining in most human and murine cells. Staining intensity increased on treatment of cells with agents that stabilize G4 DNA and, strikingly, cells deficient in FANCJ, a G4 DNA-specific helicase, showed stronger nuclear staining than controls. Our data strongly support the existence of G4 DNA structures in mammalian cells and indicate that the abundance of such structures is increased in the absence of FANCJ. We conclude that monoclonal antibody 1H6 is a valuable tool for further studies on the role of G4 DNA in cell and molecular biology.

Clear and present danger? The use of a yeast biosensor to monitor changes in the toxicity of industrial effluents subjected to oxidative colour removal treatments

Discharges of coloured effluents into surface waters provide conspicuous evidence of the impact of industry on the environment. The textile industry is an obvious candidate for sources of such discharges. Conventional treatment methods appear to alleviate this situation by removing colour, however the affect on toxicity is less obvious. The objective of this study was to examine the changes in effluent toxicity during the course of two alternative wastewater treatment methods, ozonation and electrochemical oxidation, using a novel toxicity biosensor, GreenScreen EM. The biosensor is capable of measuring both general acute toxicity (cytotoxicity), and more specifically genotoxicity, that is damage to a cell's DNA structure, replication or distribution, caused by substances that may be mutagenic and/or carcinogenic. The biosensor utilises a modified strain of the brewers yeast Saccharomyces cerevisiae, incorporating a gene encoding green fluorescent protein (GFP) linked to the inducible promoter of the DNA damage responsive RAD54 gene. Upon exposure to a genotoxin, the production of GFP is up-regulated in parallel with RAD54, and the resulting cellular fluorescence provides a measure of genotoxicity. Acute toxicity is simultaneously determined by monitoring relative total growth of the cell culture during incubation. The results presented in this paper show that a reduction in colouration does not necessarily correspond to a reduction in effluent toxicity.

17] Probing of DNA structure in cells with osmium tetroxide-2,2'-bipyridine

Publisher Summary This chapter discusses the probing of DNA structure in cells using osmium tetroxide-2,2´-bipyridine (Os-bipy). The hypersensitivity of local open DNA structures toward Os-bipy is exploited for analysis in bacterial cells. Cells are treated with a millimolar concentration of Os-bipy for a short time, the excess reagent is removed, plasmid DNA is isolated by the boiling method, and osmium binding sites are determined. Direct probing of DNA in cells with Os-bipy at neutral pH showed no triplex formation in the homopurine–homopyrimidine segment of the intracellular pEJ4 plasmid. If E. coli cells were preincubated and treated with Os-bipy at pH 4.5 or 5.0, a modification pattern characteristic of H triplexes was obtained. Shifting the intracellular superhelical density to more negative values resulted in a stronger site-specific modification. In another experiment, attempt was made to search for triplex DNA in cells with the Os-bipy probe. The presence of triplex H-DNA in vitro is manifested by a strong modification in the center of the (C-T) n sequence and a weaker modification at the triplex boundary. To probe the homopurine strand in the triplex structure in vitro , diethyl pyrocarbonate or glyoxal was applied. So far no reports on the application of these or other single-strand-selective probes reacting with purines to studies of DNA structure in cells have been published. Thus, information about triplex structure in cells obtained by direct chemical probing is limited to the pyrimidine strand.

Studies on the synthesis and structure of mitochondrial DNA in cells infected by rous sarcoma viruses and on the occurrence of intramitochondrial virus-like particles in certain RSV-induced tumor cells

1. The synthesis of mitochondrial DNA in CEF in vivo at 3,4 and 6 days after infection with RSV (Schmidt-Ruppin, subgroup A) was progressively stimulated 2 to 4-fold as compared with that in uninfected CEF cells grown in parallel. 2. The stimulation of mtDNA synthesis in vivo upon transformation was found to be temperature dependent when the thermosensitive mutant of RSV, T5, was used to infect the cells. 3. In contrast to mtDNA synthesis, nuclear DNA synthesis did not differ in transformed and uninfected cells, nor did it change significantly upon temperature shifts. 4. MtDNA (monomeric and catenated dimeric forms) in transformed and uninfected CEF replicate by displacement synthesis. Various replication intermediates are described. 5. The restriction endonuclease EcoRI cleaves closed circular mtDNA from CEF at one specific site. 6. Heteroduplex molecules formed between nicked circular and/or EcoRI cleaved mt DNA molecules from uninfected and transformed CEF revealed, with a few exceptions, no detectable base sequence heterogeneity in at least 98% of cases. 7. Intramitochondrial virus like particles (IMV) are described in hamster tumor cells. The evidence suggests both engulfment of cytoplasmic particles by mitochondria and the presence of intramitochondrial incomplete forms of particles. Bromodeoxyuridine was found to enhance the frequency of IMV.