Formation Of Linear DNA Research Articles

Prevalence and Genomic Sequence Analysis of Domestic Cat Hepadnavirus in the United States.

Hepadnaviruses are partially double-stranded DNA viruses that infect a variety of species. The prototypical virus in this family is the human hepatitis B virus, which chronically infects approximately 400 million people worldwide and is a risk factor for progressive liver disease and liver cancer. The first hepadnavirus isolated from carnivores was a domestic cat hepadnavirus (DCH), initially identified in Australia and subsequently detected in cats in Europe and Asia. As with all characterized hepadnaviruses so far, DCH infection has been associated with hepatic disease in its host. Prevalence of this infection in the United States has not been explored broadly. Thus, we utilized conventional and quantitative PCR to screen several populations of domestic cats to estimate DCH prevalence in the United States. We detected DCH DNA in 1 out of 496 animals (0.2%) in the U.S. cohort. In contrast, we detected circulating DCH DNA in 7 positive animals from a cohort of 67 domestic cats from Australia (10.4%), consistent with previous studies. The complete consensus genome of the U.S. DCH isolate was sequenced by Sanger sequencing with overlapping PCR products. An in-frame deletion of 157 bp was identified in the N-terminus of the core open reading frame. The deletion begins at the direct repeat 1 sequence (i.e., the 5′ end of the expected double-stranded linear DNA form), consistent with covalently closed circular DNA resultant from illegitimate recombination described in other hepadnaviruses. Comparative genome sequence analysis indicated that the closest described relatives of the U.S. DCH isolate are those previously isolated in Italy. Motif analysis supports DCH using NTCP as an entry receptor, similar to human HBV. Our work indicates that chronic DCH prevalence in the U.S. is likely low compared to other countries.

Study of In vitro antioxidant and DNA damage protection activity of a novel luteolin derivative isolated from Terminalia chebula

ABSTRACTThe present study aimed to investigate the efficacy of flavonoids isolated from the fruit of Terminalia chebula against the hydrogen peroxide-induced DNA damage. Five fractions were screened and were compared for their in vitro antioxidant activity by DPPH method and DNA protection activity by DNA nicking assay. All the extracts showed excellent antioxidant activity with IC50 values ranging from 11.23 ± 0.18 to 36.70 ± 0.12 µg/ml. The band intensity of supercoiled, circular and linear DNA was measured in DNA nicking assay and it is very clear that the addition of extract significantly inhibited the formation of circular and linear forms of DNA. Methanol–Water 1:1 fraction which showed the highest activity in antioxidant and DNA protection was subjected to structural characterization using different spectroscopic techniques. The structure of the compound was identified as a derivative of luteolin. The result presented in this work strongly supports the hypothesis that DNA protection activities were positively associated with the antioxidant property.

Fluorescence and Absorption Titrations of Bio-relevant Imidazole Based Organometallic Pd(II) Complexes with DNA: Synthesis, Characterization, DNA Interaction, Antimicrobial, Cytotoxic and Molecular Docking Studies

Organometallic Pd(II) complexes(4a–4f) of type Na[Pd(3n)Cl2]; where 3n = imidazole ligands (3a–3f), were synthesized. The complexes have been characterized by elemental analysis, thermogravimetric analysis, conductance measurement, mass spectrometry, UV–visible absorption spectroscopy, IR, 1H NMR, 13C APT NMR and Energy-dispersive X-ray spectroscopy. The compounds were screened for various biological activities such as nucleolytic, DNA binding, bacteriostatic (MIC) and cytotoxicity. DNA binding aptitude has been studied by UV–visible absorption titration, fluorescence spectroscopy, molecular modelling and viscosity measurement, which suggests Pd(II) complexes as DNA intercalating agents. Gibb’s free energy, enthalpy and entropy change parameters were evaluated for DNA–complex interaction by UV–visible absorption titration at different temperature, in which negative value of all the thermodynamic parameters suggests that the ligand–DNA complex is stabilized by hydrogen bonding and van der Waals forces. The gel electrophoresis of pUC19 DNA in presence of Pd(II) complexes showed the conversion of supercoiled DNA into open circular and linear form of DNA. Compounds were screened against different pathogens for evaluating their bacteriostatic activity in terms of MIC. In vitro cytotoxicity on Artemia cysts and in vivo cytotoxicity on S. pombe cells propose potent cytotoxic nature of Pd(II) complexes.

Oxidative Assets Toward Biomolecules and Cytotoxicity of New Oxindolimine-Copper(II) and Zinc(II) Complexes

A new oxindolimine ligand derived from isatin (1H-indole-2,3-dione) and 2-aminomethylbenzimidazole was synthesized, leading to two novel complexes after metalation with copper(II) perchlorate or zinc(II) chloride, [Cu(isambz)2](ClO4)2 (complex 1) and [Zn(isambz)Cl2] (complex 2). This new ligand was designed as a more lipophilic compound, in a series of oxindolimine–metal complexes with antitumor properties, having DNA, mitochondria, and some proteins, such as CDK1 kinase and topoisomerase IB, as key targets. The new complexes had their reactivity to human serum albumin (HSA) and DNA, and their cytotoxicity toward tumor cells investigated. The binding to CT-DNA was monitored by circular dichroism (CD) spectroscopy and fluorescence measurements using ethidium bromide in a competitive assay. Consequent DNA cleavage was verified by gel electrophoresis with complex 1, in nmolar concentrations, with formation of linear DNA (form III) after 60 min incubation at 37 °C, in the presence of hydrogen peroxide, which acts as a reducing agent. Formation of reactive oxygen species (ROS) was observed, monitored by spin trapping EPR. Interaction with HSA lead to α-helix structure disturbance, and formation of a stable radical species (HSA–Tyr·) and carbonyl groups in the protein. Despite showing oxidative ability to damage vital biomolecules such as HSA and DNA, these new complexes showed moderate cytotoxicity against hepatocellular carcinoma (HepG2) and neuroblastoma (SHSY5Y) cells, similarly to previous compounds in this series. These results confirm DNA as an important target for these compounds, and additionally indicate that oxidative damage is not the leading mechanism responsible for their cytotoxicity. Additionally, this work emphasizes the importance of ligand characteristics and of speciation in activity of metal complexes.

Critical low-energy Ar-ion beam conditions to induce direct DNA double strand break

Abstract Ultra-low-energy ion interaction with naked DNA has been an interesting research topic as it can reveal fundamentals involved in charged particle irradiation effect on life as well as biological evolution. This study was a detailed investigation on ultra-low-energy argon ion bombardment effects on naked DNA. Argon ion beam was decelerated by a deceleration lens to ultra low energy ranging from about 1 keV down to a few tens of eV to bombard naked DNA plasmid pGFP to fluences of 1, 2 and 4 × 1015 ions/cm2. After ion bombardment, gel electrophoresis analyzed changes in the DNA topological form. The result showed that the DNA form changed, including from original supercoiled to damaged relaxed and linear forms, certainly depending on the ion energy and fluence. The DNA form changes as functions of the ion energy and fluence were quantified. It was found that the critical Ar-ion beam conditions were the energy 750 eV to be the lowest with the fluence 2 × 1015 ions/cm2 which could induce the linear DNA form, indicating direct double strand breaks, a more severe damage of DNA which might lead to mutation.

Detection of DNA double-strand breaks in boron neutron capture reaction

We evaluated DNA double-strand breaks (DSBs) induced by boron neutron capture reaction (BNCR) using plasmid DNA, boron solution, and gel electrophoresis. The amount of the linear form of DNA produced by DSBs increased with the neutron-beam irradiation dose. The amount of the open-circular form of DNA produced by single-strand breaks (SSBs) increased with the neutron-beam irradiation dose and the 10B concentration. The model facilitated quantification of BNCR-induced DSBs and SSBs, irrespective of the DNA repair mechanism.

A strategy for enhanced circular DNA construction efficiency based on DNA cyclization after microbial transformation.

BackgroundWith the rapid development of synthetic biology, the demand for assembling multiple DNA (genes) fragments into a large circular DNA structure in one step has dramatically increased. However, for constructions of most circular DNA, there are two contradictions in the ligation/assembly and transformation steps. The ligation/assembly consists of two different reactions: 1) the ligation/assembly between any two pieces of a linear form DNA; 2) the cyclization (or self-ligation) of a single linear form DNA. The first contradiction is that the bimolecular ligation/assembly requires a higher DNA concentration while the cyclization favors a lower one; the second contradiction is that a successful transformation of a ligation/assembly product requires a relatively high DNA concentration again. This study is the first attempt to use linear plasmid and Cyclization After Transformation (CAT) strategy to neutralize those contradictions systematically.ResultsThe linear assembly combined with CAT method was demonstrated to increase the overall construction efficiency by 3–4 times for both the traditional ligation and for the new in vitro recombination-based assembly methods including recombinant DNA, Golden Gate, SLIC (Sequence and Ligation Independent Cloning) and Gibson Isothermal Assembly. Finally, the linear assembly combined with CAT method was successfully applied to assemble a pathway of 7 gene fragments responsible for synthesizing precorrin 3A which is an important intermediate in VB12 production.ConclusionThe linear assembly combined with CAT strategy method can be regarded as a general strategy to enhance the efficiency of most existing circular DNA construction technologies and could be used in construction of a metabolic pathway consisting of multiple genes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-015-0204-x) contains supplementary material, which is available to authorized users.

Abstract 830: Disulfiram is a potent inhibitor of topoisomerase II

Abstract Disulfiram is used in the treatment of alcoholism. It has also shown promise as an anticancer drug. The disulfide bond in disulfiram is metabolized in vivo to diethyldithiocarbamate (DDTC) which forms an extremely strong complex with copper(II) through which it may exert its cytotoxicity. Both disulfiram and diethyldithiocarbamate inhibited K562 cell growth in the low nanomolar range. The addition of the copper(II) chelators Trien and ammonium tetrathiomolybdate to the cell culture medium significantly reduced the cytotoxicity of disulfiram and diethyldithiocarbamate towards K562 cells, which suggests that their cytotoxicity is mediated by uptake of trace amounts of copper from the culture medium. Disulfiram, but not DDTC, strongly inhibited the decatenation activity of topoisomerase IIα in the low micromolar concentration range. Disulfiram, but not DDTC, also was shown to be a topoisomerase IIα poison as evidenced by its ability to induce formation of linear DNA. However, the K/VP.5 cell line, a K562-derived etoposide-resistant cell line with reduced levels of topoisomerase II, was not cross-resistant to either disulfiram or DDTC. K562 cells depleted in GSH levels by treatment with buthionine sulfoximine did not show increased sensitivity to disulfiram or DDTC. Disulfiram and DDTC also did not increase dichlorofluorescein fluorescence in K562 cells. These latter two results suggest that copper-mediated oxidative stress may not be a significant factor in their cytotoxicity. In summary, these results showed that while disulfiram and DDTC inhibited topoisomerase IIα, this inhibition may be only partly responsible for their cytotoxicity. Support: CIHR, a Canada Research Chair in Drug Development to B.B.H., and a National Institutes of Health grant CA090787 to J.C.Y. Citation Format: Xing Wu, Jack C. Yalowich, Daywin Patel, Brian B. Hasinoff. Disulfiram is a potent inhibitor of topoisomerase II. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 830. doi:10.1158/1538-7445.AM2014-830

Sequestering HMGB1 via DNA-conjugated beads ameliorates murine colitis.

Inflammatory bowel disease (IBD) is chronic inflammation of the gastrointestinal tract that affects millions of people worldwide. Although the etiology of IBD is not clear, it is known that products from stressed cells and enteric microbes promote intestinal inflammation. High mobility group box 1 (HMGB1), originally identified as a nuclear DNA binding protein, is a cytokine-like protein mediator implicated in infection, sterile injury, autoimmune disease, and IBD. Elevated levels of HMGB1 have been detected in inflamed human intestinal tissues and in feces of IBD patients and mouse models of colitis. Neutralizing HMGB1 activity by administration of anti-HMGB1 antibodies or HMGB1-specific antagonist improves clinical outcomes in animal models of colitis. Since HMGB1 binds to DNA with high affinity, here we developed a novel strategy to sequester HMGB1 using DNA immobilized on sepharose beads. Screening of DNA-bead constructs revealed that B2 beads, one linear form of DNA conjugated beads, bind HMGB1 with high affinity, capture HMGB1 ex vivo from endotoxin-stimulated RAW 264.7 cell supernatant and from feces of mice with colitis. Oral administration of B2 DNA beads significantly improved body weight, reduced colon injury, and suppressed colonic and circulating cytokine levels in mice with spontaneous colitis (IL-10 knockout) and with dextran sulfate sodium-induced colitis. Thus, DNA beads reduce inflammation by sequestering HMGB1 and may have therapeutic potential for the treatment of IBD.

Order effect of repair processes to clustered DNA damage

In a living cell, a multiply damaged site in DNA is thought to be processed by several different pathways simultaneously or sequentially. Under this situation, the cellular response to the lesion cluster might depend on the order of repair processes because the configuration of the lesions will be modified by the reaction of initial repair protein, affecting the DNA-binding or lesion-excision activities of latter repair proteins. For example, a cluster comprised an AP site or SSB and base lesions is formed after one of the lesions in a base lesion cluster is excised by a glycosylase. It has been predicted that the enzymatic activity of a protein binding to a lesion could be affected by a second lesion located nearby. In the present study, we investigate whether initial base excision repair process affects secondary process on the clustered DNA damage site produced by high linear energy transfer irradiation.Plasmid DNA (pUC18) used as model DNA were irradiated in a solution (10 mM Tris and 1 mM EDTA) with C6+ ions (60 keV/µm) obtained from HIMAC (NIRS, Chiba, Japan) according to our previous method [ 1], or He2+ ions from TIARA (JAEA, Takasaki, Japan) in a fully hydrated film [ 2]. We used two base excision repair enzymes to examine the order effect of BER processes, Nth and Fpg, which convert pyrimidine and purine lesions to an SSB, respectively. After exposure, the sample was incubated with Nth (or Fpg) at 37°C for 30 min. Then, we added another enzyme Fpg (or Nth) and incubate for another 30 min. We also simultaneously treated the DNA sample with both enzymes for 1 h. After the incubation, the samples were analysed by agarose (1%) gel electrophoresis to determine the yield of conformational changes of the plasmid from closed circular form to open or linear form. These changes show productions of an enzymatically induced SSB (the former) or DSB (the latter).A typical example of the dependence of the loss of closed circular form DNA on radiation dose is shown in Fig. 1a for carbon ion irradiation. The reciprocal of the slope of each graph shows yield of prompt SSBs [n(SSB)prompt] for the sample without any enzymatic treatment, or [n(SSB)prompt] + enzymatically induced SSBs [n(SSB)Nth(or Fpg, or Nth+Fpg)]. The SSB yield for the sample treated with Nth first and then Fpg is very slightly less than that of other treatments (<10%). The dose responses of the linear form DNA production are shown in Fig. 1b. There were no significant differences among the enzymatic treatments. These results indicate that the configuration change of the cluster by the first enzymatic treatment does not significantly influence the activity of secondary enzyme. The most of base lesion clusters produced in the track of carbon ions might be converted to strand breaks, some of which form double-strand breaks (DSBs), by glycosylase activity in living cells. Fig. 1.Dependence of (a) the loss of closed-circular DNA and (b) the number of linear form DNA DSB determined from the fraction of the linear form of DNA, on carbon ion dose. The arrows indicate the order of the enzymatic treatments.

Zinc(ii) complexes with the quinolone antibacterial drug flumequine: structure, DNA- and albumin-binding

The interaction of Zn(II) with the quinolone antibacterial drug flumequine (Hflmq) in the presence or absence of an N,N′-donor heterocyclic ligand, 2,2′-bipyridine (bipy), is being investigated. Interaction of equimolar quantities of ZnCl2 with flumequine and 2,2′-bipyridine results in the formation of a structurally characterized [Zn(flmq)(bipy)Cl] (2) complex, while excess of flumequine leads to a structurally characterized [Zn(flmq)2(bipy)] (3) compound. The reaction of ZnCl2 with flumequine in the absence of 2,2′-bipyridine leads to formation of complex [Zn(flmq)2(H2O)2] (1). In all these complexes, the deprotonated bidentate flumequinato ligands are coordinated to zinc ions through pyridone and carboxylato oxygens. The complexes exhibit good binding propensity to human or bovine serum albumin protein having relatively high binding constant values. UV study of interaction of the complexes with calf-thymus DNA (CT DNA) has shown that they bind to CT DNA and [Zn(flmq)(bipy)Cl] exhibits the highest binding constant. A competitive study with ethidium bromide (EB) has shown that the complexes can displace DNA-bound EB, indicating that they bind to DNA in strong competition with EB. The complexes bind to CT DNA in an intercalative binding mode which has also been verified by DNA solution viscosity measurements. DNA electrophoretic mobility experiments showed that all complexes bind to pDNA possibly in an intercalative manner resulting in catenanes formation as well as in double-stranded cleavage reflecting (or ending) in the formation of linear DNA.

First- and second-generation quinolone antibacterial drugs interacting with zinc(II): Structure and biological perspectives

Interaction of equimolar quantities of ZnCl2 with the quinolone antibacterial drugs flumequine (Hflmq), oxolinic acid (Hoxo) or enrofloxacin (Herx) and the N,N′-donor heterocyclic ligands 1,10-phenanthroline (phen) or 2,2′-bipyridine (bipy) results in the formation of 1:1 drug to metal complexes with the general formula [Zn(quinolone)(N,N′-donor)Cl], while excess of the quinolone leads to 1:2 metal to drug [Zn(quinolone)2(N,N′-donor)] complexes. In all complexes, the deprotonated bidentate quinolonato ligands are coordinated to zinc ion through the pyridone oxygen and a carboxylato oxygen. The crystal structures of [Zn(oxo)(phen)Cl], [Zn(flmq)(phen)Cl] and [Zn(flmq)2(phen)] have been determined by X-ray crystallography. All complexes exhibit good binding propensity to human or bovine serum albumin protein showing relatively high binding constant values. Interaction of the complexes with calf-thymus (CT) DNA, studied by UV spectroscopy, has shown that they bind to CT DNA, while [Zn(flmq)(phen)Cl] and [Zn(flmq)2(phen)] complexes exhibit the highest binding constants to CT DNA. Competitive study with ethidium bromide (EB) has shown that all complexes can displace the DNA-bound EB indicating that they bind to DNA in strong competition with EB. Intercalative binding mode is proposed for the interaction of the complexes with CT DNA and has also been verified by DNA solution viscosity measurements. DNA electrophoretic mobility experiments suggest that all complexes bind to linearized pDNA and supercoiled pDNA by intercalative manner resulting in catenanes formation as well as in double-stranded cleavage reflecting (or ending) in the formation of linear DNA. The complexes exhibit significant antimicrobial activity tested on five different microorganisms.

DNA cleavage behavior of a new p-xylyl spaced bisCu(BPA)Cl2 complex: the steric effect of a bulky p-xylyl-derived spacer

The different DNA cleavage behaviors of 1,3- and 1,4-xylylene spaced bisCu(BPA)Cl2 complexes (BPA, bis(pyridyl-2-methyl)amine) display the positioning effect of xylylene spacers, implying the essential roles of spacers in the related cleavage (Y. Zhao et al., Chem.–Eur. J., 2006, 12, 6621–6629). Herein, a p-xylyl derivative (2,1,3-benzoxadiazole-4,7-dimethylene (BDDM)) was utilized as a bulky spacer to construct a new p-xylyl spaced bisCu(BPA)Cl2 complex 1 to explore the spacer's steric effect on the related DNA cleavage. Agarose gel electrophoresis demonstrated that complex 1 is able to convert the supercoiled plasmid pUC19 DNA into the nicked DNA and linear DNA in the presence of excessive reductant. This DNA cleavage behavior is different from that of simple p-xylylene spaced Cu2(pTPXA)Cl4, which mediates only the nicked DNA and random DNA fragments. The DNA binding ability of complex 1 is higher than that of Cu2(pTPXA)Cl4, while its DNA nicking ability is lower than that of Cu(BPA)Cl2, as well as the dinuclear Cu2(pTPXA)Cl4, which displays a high DNA nicking efficiency due to the internuclear synergy in activating O2 to damage DNA. The linear DNA formation induced by complex 1 is realized via a non-random double-stranded scission process. The temporal profiles of the nicked DNA and linear DNA in the presence of 8 μM complex 1 imply that the two sequential neighboring breaks in the complementary strands lead to the formation of linear DNA. With the DNA cleavage behavior in the presence of different reactive oxygen species (ROS) quenchers, the DNA-bound hydroxyl radical was proposed as the main ROS involved in the cleavage. Structural optimization of this complex disclosed its larger intramolecular Cu–Cu distance than Cu2(pTPXA)Cl4, which may disfavor the internuclear synergic O2 activation. It was also proposed that the bulky BDDM spacer favors the two spaced copper centers function solely to damage in situ the two adjacent deoxyriboses from complementary strands to form the linear DNA, and the specific DNA binding mode of complex 1 due to the BDDM spacer disfavors the DNA-bound ROS to abstract a proton from deoxyribose and results in a lower DNA cleavage efficiency than mononuclear Cu(BPA)Cl2. The non-random linear DNA mediating ability and the decreased DNA cleavage efficiency caused by the bulky internuclear spacer shown in the current study provides a new alternative to regulate the DNA cleavage behavior of polycopper artificial nucleases via spacer modification.

Vacuum effect on DNA lesion and genetic mutation of cells

DNA topological forms can be changed by environmental factors thus to potentially cause genetic mutation. Vacuum of low pressure is considered to be such a factor. An investigation was carried out to check topological form changes of extracellular plasmid DNA due to lesion in DNA under the vacuum condition. Pumping the experimental stage of biological samples to vacuum may result in three effects on the environment of the sample, namely, low pressure, low temperature and low humidity, all of which may impact DNA. In the experiment, the DNA topological form change and related lesion after plasmid DNA samples were exposed to vacuum with varied time was analyzed with gel electrophoresis and fluorometric assay. The electrophoresis results were quantified to obtain percentages of the supercoiled and relaxed forms but no linear form of DNA. The fluorometer measured concentrations of single strand and double strand DNAs. The results showed that the single strand break was the dominant lesion in DNA. The DNA form change and the lesion were found to depend mainly on the pressure change but not much on the pressure itself. The vacuum-exposed DNA was subsequently transformed into bacteria Escherichia coli ( E. coli) for checking mutation occurrence. No observable mutation of the DNA-transformed bacteria was found. This study concluded that certain light lesion in DNA dominated by the single strand break could be induced by vacuum exposure but with negligible risk of genetic mutation.

An Extracellular S1-Type Nuclease of Marine Fungus Penicillium melinii

An extracellular nuclease was purified 165-fold with a specific activity of 41,250 U/mg poly(U) by chromatography with modified chitosan from the culture of marine fungus Penicillium melinii isolated from colonial ascidium collected near Shikotan Island, Sea of Okhotsk, at a depth of 123 m. The purified nuclease is a monomer with the molecular weight of 35 kDa. The enzyme exhibits maximum activity at pH 3.7 for DNA and RNA. The enzyme is stable until 75°C and in the pH range of 2.5-8.0. The enzyme endonucleolytically degrades ssDNA and RNA by 3'-5' mode to produce 5'-oligonucleotides and 5'-mononucleotides; however, it preferentially degrades poly(U). The enzyme can digest dsDNA in the presence of pregnancy-specific beta-1-glycoprotein-1. The nuclease acts on closed circular double-stranded DNA to produce opened circular DNA and then the linear form DNA by single-strand scission. DNA sequence encoding the marine fungus P. melinii endonuclease revealed homology to S1-type nucleases. The tight correlation found between the extracellular endonuclease activity and the rate of H³-thymidine uptake by actively growing P. melinii cells suggests that this nuclease is required for fulfilling the nucleotide pool of precursors of DNA biosynthesis during the transformation of hyphae into the aerial mycelium and conidia in stressful environmental conditions.

Abstract 2530: The anticancer kinase inhibitor dovitinib (TKI258/CHIR258) may have multiple modes of action because it also binds DNA and inhibits topoisomerase I and topoisomerase IIα

Abstract Dovitinib (TKI258/CHIR258) is a kinase inhibitor in phase II development for the treatment of renal cell carcinoma, advanced breast cancer, and relapsed multiple myeloma. Dovitinib is thought to exert anticancer effects through its binding to and kinase inhibition of fibroblast growth factor receptor 3 (FGFR3). It also strongly inhibits a number of other kinases that are members of the RTK superfamily, including the vascular endothelial growth factor receptor (VEGFR); fibroblast growth factor receptor 1 (FGFR1); platelet-derived growth factor receptor type 3; FMS-like tyrosine kinase 3 (FLT3); stem cell factor receptor (c-KIT) and colony-stimulating factor receptor 1 (CSF1R) all of which may also contribute to its anticancer activity. In terms of its chemical structure dovitinib is a benzimidazole-quinolinone compound. Thus, structurally it resembles the bisbenzimidazole minor groove binding dye Hoechst 33258. Molecular modeling studies of the docking of dovitinib into an X-ray structure of a Hoechst 33258-DNA complex showed that dovitinib could be reasonably accommodated in the minor groove. Dovitinib was shown to bind to DNA by its ability to increase the DNA melting temperature. Changes in the visible absorbance and fluorescence spectrum of dovitinib that occurred upon addition of DNA were also consistent with DNA binding. Because DNA binders are often topoisomerase I and topoisomerase II inhibitors, the ability of dovitinib to inhibit these DNA processing enzymes was also investigated. Dovitinib inhibited both the decatenation activity and the ATPase activity of topoisomerase IIα in the low micromolar concentration range. Dovitinib was also able to stabilize the topoisomerase IIα-cleavage complex and act as a topoisomerase IIα poison with activity comparable to etoposide as measured by its ability to induce formation of linear DNA. Dovitinib was also examined for its ability to increase phosphorylation of H2AX in K562 cells to determine if it could act as a cellular topoisomerase II poison and induce double strand DNA breaks. Dovitinib was also able to inhibit the topoisomerase I-catalyzed relaxation of DNA. In conclusion, the anticancer activity of dovitinib may result not only from its ability to inhibit multiple kinases, but also from its ability to target topoisomerase I and topoisomerase IIα. Support: CIHR and a Canada Research Chair in Drug Development to BBH and an NIH grant (CA090787) to JCY. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2530. doi:10.1158/1538-7445.AM2011-2530

Implications of double-stranded DNA structure for development, cancer and evolution

Genomes consist of DNA and the genetic information is encoded in a linear form of DNA. According to the central dogma of molecular biology, the genetic information is transcribed into mRNA, and mRNA translated into a polypeptide. Gene expression should be precisely regulated in order to create progeny. Unlike RNA, DNA has double-stranded structure. Is there any specific biological reason why DNA has evolved to possess double-stranded structure? In this presentation, biological implications of the double-stranded structure of the DNA molecule will be reviewed. In eukaryotes, it has been reported that cells might have the machinery that distinguishes one DNA-strand from the other, and that the strand-recognition mechanism might control development, cancer and evolution. Three prominent models concerning biological implications of replication of double-stranded DNA will be discussed: 1) Klar’s “somatic strand-specific imprinting and selective chromatid segregation model” for differential gene regulation, 2) Cairns’ “immortal strand inheritance model” for cancer prohibition, and 3) the “disparity mutagenesis model” for the acceleration of evolution proposed by the present author.

Evaluation of antioxidant and DNA protection activities in the extracts of the Terminalia catappa leaves

Terminalia catappa (Combretaceae) is used commonly as a folk medicine in Taiwan, and has been claimed to have therapeutic effects in hepatitis and liver related diseases [1, 2]. The leaves, bark and fruit of the Terminalia catappa have also been commonly used as a folk medicine for antidiarrheic, antipyretic and haemostatic purposes in India, Philippines, Malaysia and Indonesia [3]. The extracts from the Terminalia catappa leaves were used in this study. Antioxidant activities were measured by both 1, 1-diphenyl-2-picrylhydrazyl radical (DPPH·) scavenging and 2, 2-azino-bis (3-ethylbenz- thiazoline-6-sulfonic acid) (ABTS·+) decolourisation methods. To further evaluate the effect of Terminalia catappa extracts on UV induced DNA damages, the DNA protection assay was employed. Terminalia catappa leaf extracts had effective DPPH bleaching activity and ABTS·+ radical scavenging activity in a concentration dependent manner. Furthermore, the Terminalia catappa leaf extracts to inhibit the oxidative DNA damages were assessed by measuring the conversion of supercoiled pUC119 plasmid DNA to the linear forms. UV irradiation of DNA with hydrogen peroxide resulted in the formation of linear forms of DNA, indicating double-strand DNA breaks. Addition of Terminalia catappa plant extracts to DNA resulted in a partial inhibition of the conversion of supercoiled DNA to linear forms, indicating that the Terminalia catappa plant extract was able to protect plasmid DNA against hydroxyl radical induced oxidative damage. The inhibition of hydroxyl radical induced DNA strand breaks by Terminalia catappa plant extracts exhibited a concentration dependent relationship.

Preparation of DNA dry powder for non-viral gene delivery by spray-freeze drying: effect of protective agents (polyethyleneimine and sugars) on the stability of DNA.

This study investigates the feasibility of using the process of spray-freeze drying (SFD) to produce DNA dry powders for non-viral gene delivery. The effect of protective agents was assessed on the stability of DNA dry powders after SFD. The process of SFD had adverse effects on the tertiary structure of DNA with the protective agents of sucrose, trehalose and mannitol. With the protection of these sugars, a band corresponding to the linear form of DNA was observed during gel electrophoresis between the supercoiled form (SC) and the open circular (OC) form. On the contrary, excess cationic condensing polyethyleneimine (PEI), in conjunction with the above sugars, had the ability to provide protection for DNA from degradation after SFD. This is indicated by the reservation in SC and OC forms of DNA during agarose gel electrophoresis. The electrostatic forces between PEI polymer and DNA are critical for providing protection against various stresses generated by the process of SFD. Furthermore, on rehydration, the particle size and zeta potential of PEI/DNA complexes at weight ratios 3:1 of SFD dry powders were well maintained. Also, no transfection activity loss of PEI/DNA complexes at weight ratios 3:1 on NIH/3T3 cells was observed for reconstituted powders as compared with untreated control solutions. These results give a better understanding of preparing stable DNA dry powders by the process of SFD.

Revealing Programmable Ion-Exchange in a G-quadruplex using the Nanopore Detector

Guanine-rich DNA and RNA can form high order G-quadruplexes through metal ion-coordinated guanine-guanine base-pairs. G-quadruplexes in genome actively participate in gene regulation, and in vitro designed G-quadruplexes are potent pharmaceuticals, biosensors and building bricks of nanostructures. We have electrically visualized the trapping of a single G-quadruplex in the nanocavity enclosed by the alpha-hemolysin nanopore. The characteristic conductance blocks allowed us to discriminate between a folded G-quadruplex that is trapped in the nanocavity and an unfolded linear-form DNA that simply translocates through the nanopore [J.Phys.Chem.B 112, 8354-8360 (2008)]. This ability has enabled the study on the ion-selective folding/unfolding of a single G-quadruplex [Nucl.Acids.Res 37, 972-982 (2009)]. In this report, we uncover another important G-quadruplex process, ion-exchange, by examining the G-quadruplex formed by the thrombin-binding aptamer (TBA) in various designated ion mixtures. In the mixture of Na+ and K+, the G-quadruplex residing time in nanopore was prolonged and the occurrence of unfolded linear TBA translocation was reduced as the K+ concentration gradually increases from 0 mM to 500 mM, convincing that K+ is highly sensitively binding with G-quadruplex, and the continuous K+ exchange in G-quadruplex elongates the lifetime of G-quadruplex. In contrast, in the mixture of Li+ and Na+, the G-quadrpulex stays shorter in nanocavity and the occurrence of linear TBA was reduced to the similar level with that by pure Na+, indicating that the Na+ is highly preferred, compared to Li+, to intrude into G-quaruplex after Li+ leaves, and unfolds G-quadruplex into linear TBA. This research is to support the understanding of molecular kinetics tuned by environmental factors, and the result may apply for ion-regulating programmable biosensors and novel nanobiotechnology.