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2008-01-01
The energies of homo-guanine pairs (5primeG/G-3prime) in stacked conformations found in crystallographic B-DNA and A-DNA were estimated by means of DF-MP2/aDZ method with inclusion of the correction for basis superposition error. The significant heterogeneity was noticed related to the structural properties, the intermolecular interaction energies, the values of the ionization potential and the localization of the HOMO densities. The direct consequence of polymorphism of DNA is the variation of only two structural parameters defining guanine-guanine stacking. These two distinguishing variables are slide and roll. The rest of base step parameters exhibit similar variability for both A-DNA and B-DNA helices. Besides, the polymorphism of nucleic acid directly influences the stabilization of t...
A preliminary neutron crystallographic study of an A-DNA crystal
2009-01-01
The LADI-III diffractometer at the Institut Laue-Langevin has been used to carry out a preliminary neutron crystallographic study of the self-complementary DNA oligonucleotide d(AGGGGCCCCT)2 in the A conformation. The results demonstrate the viability of a full neutron crystallographic analysis with the aim of providing enhanced information on the ion-water networks that are known to be important in stabilizing A-DNA. This is the first account of a single-crystal neutron diffraction study of A-DNA. The study was carried out with the smallest crystal used to date for a neutron crystallographic study of a biological macromolecule.
Molecular simulations of ultra-low-energy nitrogen ion bombardment of A-DNA in vacuum
2010-01-01
For investigating mechanisms involved in low-energy ion beam induced mutation, besides experiments using low-energy and low-fluence ions to bombard naked DNA, molecular simulations were carried out as an effort towards the insight in molecular interactions between ions and DNA. In the current study, Monte Carlo (MC) and molecular dynamics (MD) simulations were applied. The results of MC simulations provide some clues about the interaction energies and sites of preference of N-ion bombardment on an A-DNA short duplex strand. MD simulations of a single N-ion moving towards the same DNA strand with different linear velocities corresponding to bombardment energies of 0.1, 1, 10 and 100eV revealed information about changes in bond lengths and visibly distorted structures of bombarded nucleotide...
2009-01-01
The energies of intra- and inter-strand stacking interactions in model d(GpC) and d(CpG) two-base-pair steps were estimated by MP2/aug-cc-pVDZ single point calculations corrected for basis superposition errors. The stacked two-nucleobase pairs were constructed using experimental values of base pair and base step parameters taken from Nucleic Acid Database ( External Reference Not Shown ). Three distinct polymorphic forms were analysed, namely A-, B- and Z-DNA. The applied methodology enables statistical analysis of structural and energetic diversities. The structural relationships between polymorphic forms are quite complex and depend on the sequence of pairs. The variability of parameters such as shift and tilt is almost the same irrespective of the polymorphic form and sequence of steps ...
2007-01-01
The capillary flow hydrothermal reactor system for the UV-vis spectrophotometric detection system (CHUS) has been applied to inspect the behavior of DNA at temperatures up to 300degreeC. Double-stranded DNA (dsDNA) was monitored with and without ethidium bromide in the presence of MgCl2. The ratio (A+DNA/A-DNA) of the absorbance of EB with DNA (A+DNA) to that without DNA (A-DNA) increased steeply over 1 at temperatures around 100degreeC, which was not observed in the absence of MgCl2. This was found to be due to the decrease of solubility of DNA in hot water, where dsDNA is converted to single-stranded DNA and become insoluble. At temperatures over 175degreeC, DNA becomes soluble again because of the degradation of long insoluble DNA polymers to short soluble DNA oligomers. This study poin...
Parallel RNA-strand recognition by 2prime-amino-b-l-LNA
2009-01-01
A short synthetic route to the first b-l-ribo configured locked nucleic acid (LNA), that is, 2prime-amino-b-l-LNA thymine phosphoramidite 6, has been developed from bicyclic nucleoside 1. Incorporation of 2prime-amino-b-l-LNA thymine monomers into a-DNA strands results in probes forming stable duplexes with complementary RNA in parallel orientation.
Intrinsic Low Temperature Paramagnetism in B-DNA
2005-04-19
We present experimental study of magnetization in $\lambda$-DNA in conjunction with structural measurements. The results show the surprising interplay between the molecular structures and their magnetic property. In the B-DNA state, $\lambda$-DNA exhibits paramagnetic behaviour below 20 K that is non-linear in applied magnetic field whereas in the A-DNA state, remains diamagnetic down to 2 K. We propose orbital paramagnetism as the origin of the observed phenomena and discuss its relation to the existence of long range coherent transport in B-DNA at low temperature.
Inevitability of spiral-shape in DNA
2008-01-01
Cyto-fluid dynamic theory, which clarifies the inevitability of the size ratio of purine and pyrimidine in the base-pairs, is extended to reveal the inevitability of the spiral-shape in DNA, i.e., the double-strand of bases-pairs. First, we will define the cluster consisting of a nitrogenous base and water molecules as continuum flexible column. Then, the differential equation describing deformation motions of the columns having spiral-shapes is derived based on the momentum conservation law. The stability theory, which is based on the concept of the quasistability weaker than the neutral stability, clarifies the inevitability of the spiral shape of B-DNA. It is stressed that the other mode corresponding to A-DNA or C-DNA is also predicted by the present approach.
Excised damaged base determines the turnover of human N-methylpurine-DNA glycosylase
2009-01-01
N-Methylpurine-DNA glycosylase (MPG) initiates base excision repair in DNA by removing a wide variety of alkylated, deaminated, and lipid peroxidation-induced purine adducts. In this study, we tested the role of excised base on MPG enzymatic activity. After the reaction, MPG produced two products: free damaged base and AP-site containing DNA. Our results showed that MPG excises 1,N6-ethenoadenine (?A) from ?A-containing oligonucleotide (?A-DNA) at a similar or slightly increased efficiency than it does hypoxanthine (Hx) from Hx-containing oligonucleotide (Hx-DNA) under similar conditions. Real-time binding experiments by surface plasmon resonance (SPR) spectroscopy suggested that both the substrate DNAs have a similar equilibrium binding constant (KD) towards MPG, but under single-turnover...
Adaptation of the base-paired double-helix molecular architecture to extreme pressure
2007-01-01
The behaviour of the d(GGTATACC) oligonucleotide has been investigated by X-ray crystallography at 295 K in the range from ambient pressure to 2 GPa (∼20 000 atm). Four 3D-structures of the A-DNA form (at ambient pressure, 0.55, 1.09 and 1.39 GPa) were refined at 1.60 or 1.65 Å resolution. In addition to the diffraction pattern of the A-form, the broad meridional streaks previously explained by occluded B-DNA octamers within the channels of the crystalline A-form matrix were observed up to at least 2 GPa. This work highlights an important property of nucleic acids, their capability to withstand very high pressures, while keeping in such conditions a nearly invariant geometry of base pairs that store and carry genetic i...
Single crystal X-ray diffraction studies of DNA and DNA-drug complexes
The structure of the brominated oligonucleotide d(ACGTACG(5-BrU)) sub 2 was solved using the multiwavelength anomalous diffraction (MAD) technique. The space group was P4 sub 3 2 sub 1 2, with unit cell a=b=43.60A, c=26.27A. This structure was an A-DNA, isomorphous with many other previously solved octomers. Single crystal X-ray diffraction data were collected from crystals of the intercalation complexes N-[2-(dimethylamino)ethyl] acridine-4-carboxamide (DACA), d(CGTACG) sub 2 and N-[2-(dimethylamino)ethyl] 9-aminoacridine-4-carboxamide (9- aminoDACA) and some of their derivatives. An attempt was made to solve the structure of the DACA derivative N-[2-(dimethylamino)butyl]-acridine-4-carboxamide (DACA4) by molecular replacement, using the crystal structure of the daunomycin d(CGTACG) sub 2 complex as a search model. Attempts were made to position the molecule in the unit cell based on an SIR map, knowledge of the symmetry and unit cell dimensions. The structure of the 9-amino-5-bromo DACA - d(CGT(5-BrU)CG) sub 2 complex was solved using the MAD technique. The crystal was in space group P6 sub 4 with unit cell dimensions a=b=30.14A c=39.57A. The complex consisted of two drug molecules intercalated between the CpG steps and a further disordered drug molecule at the end of the DNA stack allowing the complexes to pack with a continuous PI - stack. The intercalated drug molecules were bound with the long axis of the acridine chromophore parallel to the long axis of the base pairs and the side-chain protruding into the major groove. The tertiary amine of the drug side-chain was oriented towards the adjacent guanine group and was shown to be capable of interacting with both N7 and 06 of the guanine. The model from the 9-amino-5-bromoDACA d(CGT(5-BrU)CG) sub 2 complex was used for further attempts to solve the structure of the DACA4 d(CGTACG) sub 2 complex by the same methods as before. The self-rotation function for these data indicated possible NCS in the xy plane of
http://tux.lib.deakin.edu.au./adt-VDU/public/adt-VDU20051110.135641
DNA repair mechanisms constitute an essential cellular response to DNA damage arising either from metabolic processes or from environmental sources such as ultraviolet radiation. Repair of these lesions may be via direct reversal, or by processes such as nucleotide excision repair (NER), a coordinated pathway in which lesions and the surrounding nucleotides are excised and replaced via DNA resynthesis. The importance of repair is illustrated by human disease states such as xeroderma pigmentosum and Cockayne's syndrome which result from defects in the NER system arising from mutations in XP- genes or XP- and CS- genes respectively Little detail is known of DNA damage repair processes in plants, despite the economic and ecological importance of these organisms. This study aimed to expand our knowledge of the process of NER in plants, largely via a polymerase chain reaction (PCR)-based approach involving amplification, cloning and characterisation of plant genomic DNA and cDNA. Homologues of the NER components XPF/RAD1 and XPD/RAD3 were isolated as both genomic and complete cDNA sequences from the model dicotyledonous plant Arabidopsis thaliana. The sequence of the 3'-untranslated region of atXPD was also determined. Comparison of genomic and cDNA sequences allowed a detailed analysis of gene structures, including details of intron/exon processing. Variable transcript processing to produce three distinct transcripts was found in the case of atXPF. In an attempt to validate the proposed homologous function of these cDNAs, assays to test complementation of resistance to ultraviolet radiation in the relevant yeast mutants were performed. Despite extensive amino acid sequence conservation, neither plant cDNA was able to restore UV-resistance. As the yeast RAD3 gene product is also involved in vivo in transcription, and so is required for viability, the atXPD cDNA was tested in a complementation assay for this function in an appropriate yeast mutant. The plant cDNA was found to substantially increase the viability of the yeast mutant. The structural and functional significance of these results is discussed comparatively with reference to yeast, human and other known homologues. Other putative NER homologues were identified in A. thaliana database sequences, including those of ERCC1/RAD10 and XPG/ERCC5/RAD2, and are now the subjects of ongoing investigations. This study also describes preliminary investigations of putative REVS and RAD30 translesion synthesis genes from A. thaliana. Publisher: Deakin University. School of Biological and Chemical Sciences Language: en Rights: http://www.deakin.edu.au/disclaimer.html); Copyright Edward J Vonarx
http://research.usc.edu.au/vital/access/manager/Repository/usc:1552
Three highly repeated DNA sequences have been used to determine relationships of species within the Macropodidae (kangaroos and wallabies). Two highly repeated DNA sequences were isolated as buoyant density satellites in the red-necked wallaby and in the wallaroo-euro group. The third probe was a cloned representative of one class of highly repeated species from the red kangaroo. Radioactively labelled probes of these three repeated sequences were used to determine the incidence and distribution of each in number of macropodid species. The results were consistent with a monophyletic origin of the macropodid species and showed in particular, that the red-necked wallaby is closely related to the red kangaroo and to the euro-wallaroo group. In addition, the data indicated that the tammar and the agile wallaby were closely related. The results also favour the current taxonomic status of the eastern and western grey kangaroos as closely related but separate species. Publisher: CSIRO Publishing Relation: Australian Journal of Biological Sciences: Vol. 34, No. 3, pp325-340; Other identifier: URN:ISSN: 0004-9417 Language: eng
http://hdl.handle.net/2440/51067
DNA profiling has ignited public interest and consequently their expectations for the capabilities of forensic criminal and science investigations. The prospect of characterising the genetic makeup of individuals or trace samples from a wide variety of depositional and post-mortem circumstances raises the question of how reliable the methods are given the potential for prolonged exposure to variation in environmental factors, i.e. temperature, pH, UV irradiation and humidity, that are known to induce damage to DNA. Thus, it is crucial to verify the validity of the DNA profiling for characterising the genetic makeup of post-mortem tissues. This project aimed to assess the reliability of sequence and microsatellite based genotyping of tissues (muscle, hair and bone) sampled from carcasses over a two year post-mortem period. This assessment investigated the impact of environment induced DNA degradation in the local geographic region that is typical of the circumstances that confront forensic practitioners in southern Australia and to utilise rigorous controls by studying animals whose time of death and burial was known and for which we had pre-decay tissue samples available. A ‘body farm’ with 12 pig carcasses on the northern Adelaide plains, ~60km north of Adelaide, which has a typical southern Australian Mediterranean climate, i.e. cold wet winters and hot dry summers. Pigs (Sus scrofa) were used as an experimental analogue for human subjects because of the logistical and ethical reasons. The pig carcasses were allocated among three treatments: four were left on the surface, four were buried at 1m depth, and four were buried at 2 m depth. These ‘burial’ conditions mimic a range of conditions encountered typically in forensic and archaeological studies. Cortical bone samples were taken from each pig carcass at one week, one month, three months, six months, one year and two years post-mortem and muscle and hair over the same sampling period for as long as those tissue types were present. A set of PCR primers to amplify two (short and a long) fragments from the hypervariable part of the mitochondrial control region (HVRI) that is used in forensic and evolutionary studies of humans and many other mammal species were developed. Also a panel of four pig microsatellite loci with fluorescent labels to facilitate automated multiplex genotyping. These loci matched as closely as possible the core motifs and allele lengths typical of the commercially available microsatellite marker kits used in Australian forensic science labs so that our experiments were as good a model as possible of the human forensic DNA technology. In this study it was possible to retrieve samples from muscle tissue up to 90 days, hair up to one year and bone at two years post-mortem. The analyses showed that the long and short HVRI region PCR fragments were only amplifiable up to 30 days from muscle tissue and that these fragments were amplifiable up to one year from hair. In contrast, in cortical bone both PCR fragments were amplifiable up to two years. The long fragment disappeared in muscle tissue completely after 30 days and in hair after six months. However, the long fragment was present in cortical bone even at two years. Overall, there was a general trend of loss of concentration of both the long and short fragments over time. Comparisons of the HVRI nucleotide sequences among tissues sampled from individual animals showed substitution changes in muscles as early as 30 days (3 out of 6 individuals) and hair at six months (1 out of 6 individuals). In contrast, in cortical bone substitutions first appeared at 365 days (1 out of 6 individuals). The most common substitution observed in all tissues types was the C-T transition, with A-G transversions observed in two episodes and C-A transversion observed in one episode. Analyses of microsatellite genotypes in muscle tissues showed high allele peaks on chromatograms up to day seven samples. However, by three months PCR was not successful from muscle tissue. While, bone tissue had lower allele peak heights compared to the muscle tissues, alleles were detectable up to six months. Allele drop out occurred for one animal (at 2 meters) in muscle tissue at the dinucleotide locus and for another animal (kept on surface) also in muscle tissue at a tetranucleotide locus. Stuttering was observed for a single animal at dinucleotide locus in muscle tissue (buried sample 2 meter depth). No stuttering or allele drop outs were seen in the bone tissue. Overall the four loci completely disappeared after 30 days in muscle tissue and after 180 days in bone tissue. In summary, analyses showed that post-mortem DNA degradation was present in all the three tissue types (muscle, hair and bone). The types of damage identified were DNA fragmentation, nucleotide substitutions and DNA loss, which resulted in a diminished frequency of successful PCR for mitochondrial and nuclear markers over time and stuttering and allele drop out in microsatellite genotyping. In addition, two nucleotide substitutions were concentrated in ‘hotspots’ that correlate with sites of elevated mutation rate in vivo. Also the frequency of successful PCR of longer nuclear and mitochondrial PCR products declined markedly more quickly than for shorter products. These changes were first observed at much shorter post mortem intervals in muscle and much longer post mortem intervals in hair and bone tissue. When considering the carcass deposition treatments, tissues that were retrieved from buried carcases showed higher levels of DNA degradation compared to tissues retrieved from carcases left on the surface. Overall, muscle tissue is a good source for DNA analysis in immediate post mortem samples, whereas hair and bone tissue are good source for DNA analysis from older samples. When comparing the microsatellite genotyping and mtDNA analyses, mtDNA is a reliable source for DNA analysis from tissue recovered from bodies that had decayed for longer post-mortem durations such as months to years, whereas microsatellite genotyping gives reliable results for tissue from shorter post mortem intervals (hours to few days). Therefore it is recommended that when analysing mtDNA sequences, cloning and sequencing PCR products can help to identify the base pair substitutions especially for tissue retrieved from longer post mortem intervals. In addition, increasing the template DNA concentrations and "neutralising" co-extracted DNA inhibitors should be considered when dealing with tissue from longer post mortem intervals. Finally, the more stringent protocols used in ancient DNA studies should be considered when dealing with tissue with much longer post mortem intervals in forensic settings.Thesis (Ph.D.) -- University of Adelaide, School of Medical Sciences, 2009 Contributor: Donnellan, Stephen; Henneberg, Maciej; School of Medical Sciences : Anatomical Sciences Other identifier: 20090917173815
http://hdl.handle.net/2440/2353
© World Scienti c Publishing CompanyThis paper examines two methods for finding whether long-range correlations exist in DNA: a fractal measure and a mutual information technique. We evaluate the performance and implications of these methods in detail. In particular we explore their use comparing DNA sequences from a variety of sources. Using software for performing in silico mutations, we also consider evolutionary events leading to long range correlations and analyse these correlations using the techniques presented. Comparisons are made between these virtual sequences, randomly generated sequences, and real sequences. We also explore correlations in chromosomes from different species.M. J. Berryman, A. Allison and D. Abbott Publisher: World Scientific Publishing Contributor: School of Electrical and Electronic Engineering Other identifier: Fluctuation and Noise Letters, 2004; 4 (2):L237-L246; 0219-4775; 0020040853; 10.1142/S0219477504001574 Language: en_US Source: http://ejournals.wspc.com.sg/journals/fnl/04/0402/S0219477504001574.html
http://handle.uws.edu.au:8081/1959.7/24805
The extent of genetic structuring of a population results from a balance of forces producing local genetic differentiation, and counter-forces producing genetic homogeneity. An understanding of these forces is essential when investigating evolutionary processes in a species. It has been predicted that, when populations experience severe reductions in size, they lose genetic variability. Small population size may occur as a consequence of founder events, such as with the introduction and spread of a pest species. The predictions as described were examined by studying the molecular evolution and population genetics of European rabbit (Oryctolagus cuniculus (L)) populations in Australia, using variation in mitochondrial DNA control region sequences. The results suggest that there is a higher rate of gene flow within populations in semi arid/arid areas, which is attributable to the extreme environmental heterogeneity found in these areas and may be best explained by a process whereby local populations becoming extinct and are recolonised by individuals drawn from other populations.Master of Science (Hons) Publisher: Language: eng
http://handle.unsw.edu.au/1959.4/43601
Human cytomegalovirus (HCMV) is a ubiquitous virus that causes significant morbidity and mortality in immunocompromised individuals. Although there are prophylactic treatments available, all current antiviral drugs ultimately target the DNA polymerase, resulting in the increasing emergence of antiviral resistant strains in the clinical setting. There is a fundamental need for understanding the role of other essential genes in DNA replication as a foundation for developing new antiviral treatments that are safe and which utilize a mechanism of action different to existing therapies. In this study we looked at six HCMV replication genes encoding for the DNA polymerase accessory protein (UL44), single stranded DNA binding protein (UL57), primase (UL70), helicase (UL105), primase-helicase associated protein (UL102), and the putative initiator protein (UL84) in order to increase our understanding of their role in DNA replication. The aim of this project was to identify variation within these genes as well as to predict putative domains and motifs in order to ultimately express and study the functional properties of the HCMV primase (UL70) through the use of recombinant mutants. Sequencing of these genes revealed a high degree of conservation between the isolates with amino acid sequence identity of >97% for all genes. Using ScanProsite software from the Expert Protein Analysis System (ExPASy) proteomics server, we have mapped putative motifs throughout these HCMV replication genes. In particular, highly conserved putative Nlinked glycosylation sites were identified in UL105 that were also conserved across 33 homologues as well as several unique motifs including casein kinase II phosphorylation sites (CKII) in UL105 and UL84, a microbodies signal motif in UL57 and an integrin binding site in the UL102 helicase-primase associated protein. Our investigations have also elucidated motif-rich regions of the UL44 DNA polymerase accessory protein, mapped functionally important domains of the UL105 helicase and identified cysteine motifs that have implications for folding of the UL70 primase. Taken together, these findings provide insights to regions of these HCMV replication proteins that are important for post-translation modification, activation and overall function, and this information can be utilized to target further research into these proteins and advance the development of novel antiviral agents that target these processes. Publisher: Publisher:University of New South Wales. Medical Sciences Language: EN Rights: http://unsworks.unsw.edu.au/copyright
http://handle.unsw.edu.au/1959.4/41275
Since its first use in criminal investigations in 1987, DNA profiling has become the new gold standard for investigations and prosecutions. Academics, politicians and law enforcement officials have presented DNA evidence as a ??scientific hero?? that is capable of solving crimes and preventing miscarriages of justice. However, in spite of this reputation, few studies have explored the impact of this technology on criminal justice practitioners, or on the process of investigating and processing criminal offences. This dissertation provides a comparative study of the use of DNA profiling in two jurisdictions: New South Wales (NSW) in Australia and the Thames Valley in the United Kingdom (UK). Interviews canvassed the perspectives and experiences of police officers, scene of crime officers (SOCOs), forensic scientists, criminal lawyers, and judicial officers from these areas. These interviews were analysed in conjunction with appeal judgments and police statistics to reveal how DNA evidence has been used in the NSW and Thames Valley. The research presented in this dissertation indicates that DNA profiling is having a number of far-reaching effects on both criminal justice systems and is seen as a reliable forensic tool by criminal justice practitioners. Practitioners routinely use DNA evidence throughout the various stages of the criminal justice process and are actively changing their practices to utilise the technology more effectively. One of the main impacts of the introduction of DNA evidence into criminal investigations has been the need to provide substantial resources and infrastructure for the collection, analysis, and storage of samples. Both jurisdictions encountered a number of problems because they provided insufficient resources to effectively use DNA profiling. This study also offers insight into how criminal justice practitioners perceive the dangers of using DNA evidence and how miscarriages of justice can occur. Finally, through an analysis of the combined experiences of criminal justice practitioners, this dissertation challenges the widespread acceptance and routine use of forensic DNA profiling. It further suggests that it is now time to re-consider current practices in relation to how resources are devoted to the technology, and how criminal justice practitioners are using the technology. Publisher: Publisher:University of New South Wales. Social Sciences & International Studies Language: EN Rights: http://unsworks.unsw.edu.au/copyright
http://handle.unsw.edu.au/1959.4/22815
For the successful detection of selected DNA sequences or mutated genes associated with human disease, there are several challenges that the current research aims to overcome - higher sensitivity, greater selectivity and rapid assaying time. An electrochemical device using redox-active intercalators to transduce DNA hybridization via long-range electron transfer is presented in this thesis which aims to address the above challenges. The DNA recognition interface is composed of thiolated single-stranded DNA (ss-DNA) and a diluent component both of which are self-assembled onto a gold electrode. This project seeks to advance fundamental insight into issues that impact the structure and behavior of surface-immobilized DNA towards hybridization with target complementary ss-DNA. After the optimal conditions have been identified for the construction of a reproducible DNA recognition layer, a stepwise detection scheme using an anionic intercalator, as the redox molecule is introduced for the DNA transduction. The stepwise detection relies on the absence of any electrochemistry prior to DNA hybridization. Upon hybridization, the perfectly stacked DNA is capable of mediating the electrochemical oxidation and reduction of intercalated species and hence voltammetric peaks become evident. Although excellent selectivity towards single-base mismatch detection is achieved, this detection scheme has a high detection limit and slow assaying time. However, this is overcome by a novel in situ approach where the electrochemistry is performed in the presence of both complementary target DNA and intercalator. The effect of different DNA recognition interfaces on hybridization is also investigated using electrochemical and gravimetric techniques where the hybridization efficiency, kinetics and affinity constant of hybridization were assessed. These measurements showed that the length of the diluent layer has a large impact on the time taken to form a perfect duplex but no impact on the initial recognition of the target DNA by the immobilized probe DNA. Fundamental aspects of the DNA technology towards assaying small molecules which have binding affinity to DNA are also investigated. The probe ss-DNA sensing interface was found to be highly sensitive towards detection of Cd2+. The long-range electron transfer approach was also utilized in gaining more insight knowledge of the interaction of cisplatin, an anti-cancer drug with the DNA. Publisher: Awarded by:University of New South Wales. School of Chemistry Language: EN Rights: Copyright Elicia Leh See Wong; http://unsworks.unsw.edu.au/copyright
http://espace.library.uq.edu.au/view/UQ:192795
EDD, the human orthologue of Drosophila melanogaster “hyperplastic discs,” is overexpressed or mutated in a number of common human cancers. Although EDD has been implicated in DNA damage signaling, a definitive role has yet to be demonstrated. Here we report a novel interaction between EDD and the DNA damage checkpoint kinase CHK2. EDD and CHK2 associate through a phospho-dependent interaction involving the CHK2 Forkhead-associated domain and a region of EDD spanning a number of putative Forkhead-associated domain-binding threonines. Using RNA interference, we demonstrate a critical role for EDD upstream of CHK2 in the DNA damage signaling pathway. EDD is necessary for the efficient activating phosphorylation of CHK2 in response to DNA damage following exposure to ionizing radiation or the radiomimetic, phleomycin. Cells depleted of EDD display impaired CHK2 kinase activity and an inability to respond to DNA damage. These results identify EDD as a novel mediator in DNA damage signal transduction via CHK2 and emphasize the potential importance of EDD in cancer. Coverage: 2006-10-01T00:00:00Z
http://eprints.infodiv.unimelb.edu.au/00001883/01/Wong_2006.pdf
The Centromere is a vital chromosomal structure that ensures faithful segregation of replicated chromosomes to their respective daughter cells. With such an important structure, one would expect the underlying centromeric DNA sequence would be highly conserved across all species. It turns out that the underlying centromeric DNA sequences between species ranging from the yeast, fly, mouse to humans are in fact highly diverged suggesting a DNA sequence independent or an epigenetic mechanism of centromere formation.Neocentromeres are centromeres that form de-novo at genomic locations that are devoid of highly repetitive a-satellite DNA sequences of which normal centromeres are usually comprised from. To date, the 10q25 neocentromere is the most well-characterised, fully functional human centromere that has been used previously to characterise the extent of a number of centromeric protein binding domains and characterise the properties of the underlying DNA sequence. Along with other factors, the existence of neocentromeres has given rise to a hypothesis where centromeres are defined by epigenetic or DNA sequence independent mechanisms.The putative 10q25 neocentromere domain was recently redefined by high resolution mapping of Centromeric protein A (CENP-A) binding through a chromatin immunoprecipitation and array (CIA) analysis. The underlying DNA sequence was investigated to determine and confirm that the formation of the 10q25 neocentromere was through an epigenetic mechanism. Through a high-density restriction fragment length polymorphism (RFLP) analysis using overlapping PCR amplified DNA derived from genomic DNA representing the 10q25 region before and after neocentromere activation. No sequence polymorphisms, large insertions or deletions were detected and confirmed the epigenetic hypothesis of centromere formation.DNA methylation is one of many epigenetic factors that are important for cellular differentiation, gene regulation and genomic imprinting. As the mechanisms and functions of DNA methylation have been well characterised, its role at the 10q25 neocentromere was investigated to try and identify the candidate epigenetic mechanism involved in the formation of centromeres. DNA methylation across the neocentromere was assessed using sodium bisulfite PCR and sequencing of selected CpG islands located across the 10q25 neocentromere. Overall, the methylation level of the selected CpG islands demonstrated no difference in DNA methylation before and after neocentromere activation. However, significant hypomethylation upon neocentromere formation was detected close to the protein-binding domain boundaries mapped previously suggesting that this may have a role in demarcating protein binding domains at the neocentromere.Further analysis of DNA methylation investigated non-CpG island methylation at sites defined as CpG islets and CpG orphans. Interestingly, the DNA methylation level measured at selected CpG islets and CpG orphans across the 10q25 neocentromere were not completely hypermethylated as previously thought, but demonstrated variable methylation that became fully hypermethylated upon neocentromere activation in most sites investigated. These results suggested that a role for DNA methylation existed at the 10q25 neocentromere and that it occurred at sites devoid of CpG islands.This study has found that DNA methylation at non-CpG island sites was variable contrary to popular belief and, was linked with neocentromere formation through the observation of increased DNA methylation at the 10q25 neocentromere. Inhibition of DNA methylation demonstrated increased neocentromere instability and a decrease in methylation of these CpG islets and CpG orphans confirming the importance of DNA methylation at neocentromeres. This study has characterised a new class of sequences that are involved in the maintenance of chromatin structure through DNA methylation at the 10q25 neocentromere. Language: eng Rights: validuser; Terms and Conditions: Copyright in works deposited in the University of Melbourne Eprints Repository (UMER) is retained by the copyright owner. The work may not be altered without permission from the copyright owner. Readers may only, download, print, and save electronic copies of whole works for their own personal non-commercial use. Any use that exceeds these limits requires permission from the copyright owner. Attribution is essential when quoting or paraphrasing from these works.; Restricted Access – University of Melbourne Staff and Students Only; Login required please enter your University of Melbourne email username and password in the login boxes at the top righthand of this repository page to access this item.
Very low-energy and low-fluence ion beam bombardment of naked plasmid DNA
2009-01-01
Ion beam bombardment of biological organisms has been recently applied to mutation breeding of both agricultural and horticultural plants. In order to explore relevant mechanisms, this study employed low-energy ion beams to bombard naked plasmid DNA. The study aimed at simulation of the final stage of the process of the ion beam bombardment of real cells to check whether and how very low-energy and low-fluence of ions can induce mutation. Argon and nitrogen ions at 5 keV and 2.5 keV respectively bombarded naked plasmid DNA pGFP to very low-fluences, an order of 1013 ions/cm2. Subsequently, DNA states were analyzed using electrophoresis. Results provided evidences that the very low-energy and low-fluence ion bombardment indeed altered the DNA structure from supercoil to short linear fragments through multiple double strand breaks and thus induced ... >>
Typing some of lactic acid bacteria in Syria using PCR and FT-IR techniques
2010-01-01
Lactic Acid Bacteria (LAB) are considered to be the most useful microorganisms. They are beneficial in flavoring foods, inhibiting pathogenic as well as spoilage bacteria in food products. The isolates of LAB were obtained from traditional Syrian dairy products (white cheese and curdled yogurt) obtained from different regions in Syria. The isolates were subjected to phenotypic characterization analyses. The PCR technique of bacterial DNA was evaluated as an advanced tool for the identification of LAB. It was found that strains: E. faecium, E. faecalis and S. thermophilus dominate in white cheese and in yogurt. Our results demonstrated that we could identify LAB using Fourier transform infrared spectroscopy (FT-IR) patterns. (author)
2010-03-01
Full Text Available.At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, σS, involves the small regulatory RNA (sRNA) DsrA and the RNA chaperone Hfq. The Hfq-mediated DsrA-rpoS interaction relieves an intramolecular secondary structure that impedes ribosome access to the rpoS ribosome binding site. In addition, DsrA/rpoS duplex formation creates an RNase III cleavage site within the duplex. Previous biochemical studies suggested that DsrA and Hfq associate with the 30S ribosomal subunit protein S1, which implied a role for the ribosome in sRNA-mediated post-transcriptional regulation. Here, we show by ribosome profiling that Hfq partitions with the cytoplasmic fraction rather than with 30S subunits. Besides, by employing immunological techniques, no evidence for a physical interaction between Hfq and S1 was obtained. Similarly, in vitro studies did not reveal a direct interaction between DsrA and S1. By employing a ribosome binding deficient rpoS mRNA, and by using the RNase III clevage in the DsrA/rpoS duplex as a diagnostic marker, we provide in vivo evidence that the Hfq-mediated DsrA/rpoS interaction, and consequently the structural changes in rpoS mRNA precede ribosome binding. These data suggest a simple mechanistic model in which translational activation by DsrA provides a translationally competent rpoS mRNA to which 30S subunits can readily bind.
2010-03-01
At low temperature, translational activation of rpoS mRNA, encoding the stationary phase sigma-factor, σS, involves the small regulatory RNA (sRNA) DsrA and the RNA...Full Text Available
The protective mechanisms induced by a fish rhabdovirus DNA vaccine depend on temperature
2009-01-01
DNA vaccines encoding the viral glycoproteins of viral haemorrhagic septicaemia virus (VHSV) and infectious haematopoietic necrosis Virus (IHNV) have proved highly efficient in rainbow trout (Oncorhynchus mykiss) under experimental conditions. Non-specific as well as specific immune mechanisms seem to be activated. Temperature is an important external parameter affecting the immune response in fish. The present study aimed at determining the effectiveness of a DNA vaccine against VHS at different temperatures. Rainbow trout fingerlings acclimated at 5 degrees C, 10 degrees C or 15 degrees C, were given an intramuscular injection of 1 mu g purified plasmid DNA and challenged with virulent VHSV 8 or 36-40 days later. The vaccine protected the fish well at all three temperatures, but the involvement of innate and adaptive mechanisms differed: at low temperature. non-specific protection lasted longer and at 36 dpv fish kept at 5 degrees C had no detectable response of neutralizing antibodies while 67% of the fish kept at 15 degrees C had seroconverted. Induction of Mx as measured in liver samples was delayed at 5 degrees C with no detectable response 7 dpv whereas fish maintained at 10 C had significantly elevated levels of Mx3-transcripts at that time point. Immunohistochemical studies of the injection site of vaccinated fish also showed a clear effect of temperature: in fish maintained at 15 degrees C the vhsG-protein appeared earlier on the surface of transfected myocytes and the inflammatory response clearing away these myocytes arose earlier Compared to fish kept at the lower temperatures of 5 and 10 degrees C.
The DNA damage response at eroded telomeres and tethering to the nuclear pore complex
2009-01-01
The ends of linear eukaryotic chromosomes are protected by telomeres, which serve to ensure proper chromosome replication and to prevent spurious recombination at chromosome ends. In this study, we show by single cell analysis that in the absence of telomerase, a single short telomere is sufficient to induce the recruitment of checkpoint and recombination proteins. Notably, a DNA damage response at eroded telomeres starts many generations before senescence and is characterized by the recruitment of Cdc13 (cell division cycle 13), replication protein A, DNA damage checkpoint proteins and the DNA repair protein Rad52 into a single focus. Moreover, we show that eroded telomeres, although remaining at the nuclear periphery, move to the nuclear pore complex. Our results link the DNA damage response at eroded telomeres to changes in subnuclear localization and suggest the existence of collapsed replication forks at eroded telomeres.
The CCAAT-binding complex coordinates the oxidative stress response in eukaryotes
2010-03-01
The heterotrimeric CCAAT-binding complex is evolutionary conserved in eukaryotic organisms. The corresponding Aspergillus nidulans CCAAT- binding factor (AnCF) consists of the subunits...Full Text Available
The CCAAT-binding complex coordinates the oxidative stress response in eukaryotes
2010-03-01
Full Text Available.The heterotrimeric CCAAT-binding complex is evolutionary conserved in eukaryotic organisms. The corresponding Aspergillus nidulans CCAAT- binding factor (AnCF) consists of the subunits HapB, HapC and HapE. All of the three subunits are necessary for DNA binding. Here, we demonstrate that AnCF senses the redox status of the cell via oxidative modification of thiol groups within the histone fold motif of HapC. Mutational and in vitro interaction analyses revealed that two of these cysteine residues are indispensable for stable HapC/HapE subcomplex formation and high-affinity DNA binding of AnCF. Oxidized HapC is unable to participate in AnCF assembly and localizes in the cytoplasm, but can be recycled by the thioredoxin system in vitro and in vivo. Furthermore, deletion of the hapC gene led to an impaired oxidative stress response. Therefore, the central transcription factor AnCF is regulated at the post-transcriptional level by the redox status of the cell serving for a coordinated activation and deactivation of antioxidative defense mechanisms including the specific transcriptional activator NapA, production of enzymes such as catalase, thioredoxin or peroxiredoxin, and maintenance of a distinct glutathione homeostasis. The underlying fine-tuned mechanism very likely represents a general feature of the CCAAT-binding complexes in eukaryotes.
Systems Biology Modeling of the Radiation Sensitivity Network: A Biomarker Discovery Platform
2009-01-01
Purpose: The discovery of effective biomarkers is a fundamental goal of molecular medicine. Developing a systems-biology understanding of radiosensitivity can enhance our ability of identifying radiation-specific biomarkers. Methods and Materials: Radiosensitivity, as represented by the survival fraction at 2 Gy was modeled in 48 human cancer cell lines. We applied a linear regression algorithm that integrates gene expression with biological variables, including ras status (mut/wt), tissue of origin and p53 status (mut/wt). Results: The biomarker discovery platform is a network representation of the top 500 genes identified by linear regression analysis. This network was reduced to a 10-hub network that includes c-Jun, HDAC1, RELA (p65 subunit of NFKB), PKC-beta, SUMO-1, c-Abl, STAT1, AR, CDK1, and IRF1. Nine targets associated with radiosensitization drugs ... >>
2005-08-22
The overall goal of this project was to elucidate the structure/function relationships between oxidized DNA bases and the DNA repair enzymes that recognize and remove them. The NMR solution structure of formamidopyrimidine DNA glycosylase (Fpg) that recognizes oxidized DNA purines was to be determined. Furthermore, the solution structures of DNA molecules containing specific lesions recognized by Fpg was to be determined in sequence contexts that either facilitate or hinder this recognition. These objectives were in keeping with the long-term goals of the Principal Investigator's laboratory, that is, to understand the basic mechanisms that underpin base excision repair processing of oxidative DNA lesions and to elucidate the interactions of unrepaired lesions with DNA polymerases. The results of these two DNA transactions can ultimately determine the fate of the cell. These objectives were also in keeping with the goals of our collaborator, Dr. Michael Kennedy, who is studying the repair and recognition of damaged DNA. Overall the goals of this project were congruent with those of the Department of Energy's Health Effects and Life Sciences Research Program, especially to the Structural Biology, the Human Genome and the Health Effects Programs. The mission of the latter Program includes understanding the biological effects and consequences of DNA damages produced by toxic agents in the many DOE waste sites so that cleanup can be accomplished in a safe, effective and timely manner.
Structural features of DNA interaction with caffeine and theophylline
2008-01-01
Caffeine and theophylline are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. However, there has been no information on the interactions of these xanthine derivatives with individual DNA at molecular level. The aim of this study was to examine the stability and structural features of calf-thymus DNA complexes with caffeine and theophylline in aqueous solution, using constant DNA concentration (6.25mM) and various caffeine or theophylline/DNA(P) ratios of 1/80, 1/40, 1/20, 1/10, 1/5, 1/2 and 1/1. FTIR, UV-visible spectroscopic methods were used to determine the ligand external binding modes, the binding constant and the stability of caffeine, theophylline-DNA complexes in aqueous sol...
Structural analysis of DNA complexation with cationic lipids
2009-01-01
Complexes of cationic liposomes with DNA are promising tools to deliver genetic information into cells for gene therapy and vaccines. Electrostatic interaction is thought to be the major force in lipid–DNA interaction, while lipid-base binding and the stability of cationic lipid–DNA complexes have been the subject of more debate in recent years. The aim of this study was to examine the complexation of calf-thymus DNA with cholesterol (Chol), 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), dioctadecyldimethylammoniumbromide (DDAB) and dioleoylphosphatidylethanolamine (DOPE), at physiological condition, using constant DNA concentration and various lipid contents. Fourier transform infrared (FTIR), UV-visible, circular dichroism spectroscopic methods and atomic force microsc...
Small Sample Whole-Genome Amplification
2005-09-20
Many challenges arise when trying to amplify and analyze human samples collected in the field due to limitations in sample quantity, and contamination of the starting material. Tests such as DNA fingerprinting and mitochondrial typing require a certain sample size and are carried out in large volume reactions; in cases where insufficient sample is present whole genome amplification (WGA) can be used. WGA allows very small quantities of DNA to be amplified in a way that enables subsequent DNA-based tests to be performed. A limiting step to WGA is sample preparation. To minimize the necessary sample size, we have developed two modifications of WGA: the first allows for an increase in amplified product from small, nanoscale, purified samples with the use of carrier DNA while the second is a single-step method for cleaning and amplifying samples all in one column. Conventional DNA cleanup involves binding the DNA to silica, washing away impurities, and then releasing the DNA for subsequent testing. We have eliminated losses associated with incomplete sample release, thereby decreasing the required amount of starting template for DNA testing. Both techniques address the limitations of sample size by providing ample copies of genomic samples. Carrier DNA, included in our WGA reactions, can be used when amplifying samples with the standard purification method, or can be used in conjunction with our single-step DNA purification technique to potentially further decrease the amount of starting sample necessary for future forensic DNA-based assays.
Selection of DNA aptamers using atomic force microscopy
2010-03-01
Full Text Available.Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind to their targets with very high affinity. Therefore, we propose the Systematic Evolution of Ligands by an EXponential enrichment (SELEX) method using AFM to obtain aptamers that have a strong affinity for target molecules. In this study, thrombin was chosen as the target molecule, and an ‘AFM-SELEX’ cycle was performed. As a result, selected cycles were completed with only three rounds, and many of the obtained aptamers had a higher affinity to thrombin than the conventional thrombin aptamer. Moreover, one type of obtained aptamer had a high affinity to thrombin as well as the anti-thrombin antibody. AFM-SELEX is, therefore, considered to be an available method for the selection of DNA aptamers that have a high affinity for their target molecules.
Selection of DNA aptamers using atomic force microscopy
2010-03-01
Atomic force microscopy (AFM) can detect the adhesion or affinity force between a sample surface and cantilever, dynamically. This feature is useful as a method for the selection of aptamers that bind...Full Text Available
S-Phase Cells Are More Sensitive to High-Linear Energy Transfer Radiation
2009-01-01
Purpose: S-phase cells are more resistant to low-linear energy transfer (LET) ionizing radiation (IR) than nonsynchronized and G1-phase cells, because both nonhomologous end-joining (NHEJ) and homologous recombination repair can repair DNA double-strand breaks (DSBs) in the S phase. Although it was reported 3 decades ago that S-phase cells did not show more resistance to high-LET IR than cells in other phases, the mechanism remains unclear. We therefore attempted to study the phenotypes and elucidate the mechanism involved. Methods and Materials: Wild-type and NHEJ-deficient cell lines were synchronized using the double-thymidine approach. A clonogenic assay was used to detect the sensitivity of nonsynchronized, synchronized S-phase, and G2-phase cells to high- and low-LET IR. The amounts of Ku bound to DSBs in the high- and ... >>
Regulation of Gene Expression in Protozoa Parasites
2010-01-01
Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites,...Full Text Available
Regulation of Gene Expression in Protozoa Parasites
2010-01-01
Full Text Available.Infections with protozoa parasites are associated with high burdens of morbidity and mortality across the developing world. Despite extensive efforts to control the transmission of these parasites, the spread of populations resistant to drugs and the lack of effective vaccines against them contribute to their persistence as major public health problems. Parasites should perform a strict control on the expression of genes involved in their pathogenicity, differentiation, immune evasion, or drug resistance, and the comprehension of the mechanisms implicated in that control could help to develop novel therapeutic strategies. However, until now these mechanisms are poorly understood in protozoa. Recent investigations into gene expression in protozoa parasites suggest that they possess many of the canonical machineries employed by higher eukaryotes for the control of gene expression at transcriptional, posttranscriptional, and epigenetic levels, but they also contain exclusive mechanisms. Here, we review the current understanding about the regulation of gene expression in Plasmodium sp., Trypanosomatids, Entamoeba histolytica and Trichomonas vaginalis.
2009-01-01
Purpose: Concurrent temozolomide (TMZ) and radiation therapy (RT) followed by adjuvant TMZ is standard treatment for patients with glioblastoma multiforme (GBM), although the relative contribution of concurrent versus adjuvant TMZ is unknown. In this study, the efficacy of TMZ/RT was tested with a panel of 20 primary GBM xenografts. Methods and Materials: Mice with intracranial xenografts were treated with TMZ, RT, TMZ/RT, or placebo. Survival ratio for a given treatment/line was defined as the ratio of median survival for treatment vs. placebo. Results: The median survival ratio was significantly higher for O6-methylguanine-DNA methyltransferase (MGMT) methylated tumors versus unmethylated tumors following treatment with TMZ (median survival ratio, 3.6 vs. 1.5, respectively. P = 0.008) or TMZ/RT (5.7 vs. 2.3, respectively. P = 0.001) but not RT alone (1.7 vs. 1.6. P ... >>
Radiation-induced electron migration along DNA
1994-04-01
Radiation-induced electron migration along DNA is a mechanism by which randomly produced stochastic energy deposition events can lead to nonrandom types of damage along DNA manifested distal to the sites of the initial energy deposition. Electron migration along DNA is significantly influenced by the DNA base sequence and DNA conformation. Migration along 7 base pairs in oligonucleotides containing guanine bases was observed for oligonucleotides irradiated in solution which compares to average migration distances of 6 to 10 bases for Escherichia coli DNA irradiated in solution and 5.5 base pairs for Escherichia coli DNA irradiated in cells. Evidence also suggests that electron migration can occur preferentially in the 5{prime} to 3{prime} direction along DNA. Our continued efforts will provide information regarding the contribution of electron transfer along DNA to formation of locally multiply damaged sites created in DNA by exposure to ionizing radiation.
2008-01-28
Several hybridization-based methods used to delineate single copy or repeated DNA sequences in larger genomic intervals take advantage of the increased resolution and sensitivity of free chromatin, i.e., chromatin released from interphase cell nuclei. Quantitative DNA fiber mapping (QDFM) differs from the majority of these methods in that it applies FISH to purified, clonal DNA molecules which have been bound with at least one end to a solid substrate. The DNA molecules are then stretched by the action of a receding meniscus at the water-air interface resulting in DNA molecules stretched homogeneously to about 2.3 kb/{micro}m. When non-isotopically, multicolor-labeled probes are hybridized to these stretched DNA fibers, their respective binding sites are visualized in the fluorescence microscope, their relative distance can be measured and converted into kilobase pairs (kb). The QDFM technique has found useful applications ranging from the detection and delineation of deletions or overlap between linked clones to the construction of high-resolution physical maps to studies of stalled DNA replication and transcription.
Precise targeted integration by a chimaeric transposase zinc-finger fusion protein
2010-03-01
Transposons of the Tc1/mariner family have been used to integrate foreign DNA stably into the genome of a large variety of different cell types and organisms. Integration is at TA dinucleotides...Full Text Available
Precise targeted integration by a chimaeric transposase zinc-finger fusion protein
2010-03-01
Full Text Available.Transposons of the Tc1/mariner family have been used to integrate foreign DNA stably into the genome of a large variety of different cell types and organisms. Integration is at TA dinucleotides located essentially at random throughout the genome, potentially leading to insertional mutagenesis, inappropriate activation of nearby genes, or poor expression of the transgene. Here, we show that fusion of the zinc-finger DNA-binding domain of Zif268 to the C-terminus of ISY100 transposase leads to highly specific integration into TA dinucleotides positioned 6-17 bp to one side of a Zif268 binding site. We show that the specificity of targeting can be changed using Zif268 variants that bind to sequences from the HIV-1 promoter, and demonstrate a bacterial genetic screen that can be used to select for increased levels of targeted transposition. A TA dinucleotide flanked by two Zif268 binding sites was efficiently targeted by our transposase-Zif268 fusion, suggesting the possibility of designer ‘Z-transposases’ that could deliver transgenic cargoes to chosen genomic locations.
Population Genomics of Parallel Adaptation in Threespine Stickleback using Sequenced RAD Tags
2010-02-01
Next-generation sequencing technology provides novel opportunities for gathering genome-scale sequence data in natural populations, laying the empirical foundation for the evolving field of population...Full Text Available
Population Genomics of Parallel Adaptation in Threespine Stickleback using Sequenced RAD Tags
2010-02-01
Full Text Available.Next-generation sequencing technology provides novel opportunities for gathering genome-scale sequence data in natural populations, laying the empirical foundation for the evolving field of population genomics. Here we conducted a genome scan of nucleotide diversity and differentiation in natural populations of threespine stickleback (Gasterosteus aculeatus). We used Illumina-sequenced RAD tags to identify and type over 45,000 single nucleotide polymorphisms (SNPs) in each of 100 individuals from two oceanic and three freshwater populations. Overall estimates of genetic diversity and differentiation among populations confirm the biogeographic hypothesis that large panmictic oceanic populations have repeatedly given rise to phenotypically divergent freshwater populations. Genomic regions exhibiting signatures of both balancing and divergent selection were remarkably consistent across multiple, independently derived populations, indicating that replicate parallel phenotypic evolution in stickleback may be occurring through extensive, parallel genetic evolution at a genome-wide scale. Some of these genomic regions co-localize with previously identified QTL for stickleback phenotypic variation identified using laboratory mapping crosses. In addition, we have identified several novel regions showing parallel differentiation across independent populations. Annotation of these regions revealed numerous genes that are candidates for stickleback phenotypic evolution and will form the basis of future genetic analyses in this and other organisms. This study represents the first high-density SNP–based genome scan of genetic diversity and differentiation for populations of threespine stickleback in the wild. These data illustrate the complementary nature of laboratory crosses and population genomic scans by confirming the adaptive significance of previously identified genomic regions, elucidating the particular evolutionary and demographic history of such regions in natural populations, and identifying new genomic regions and candidate genes of evolutionary significance.
Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase
2010-03-01
RNA helicases function in numerous aspects of RNA biology. These enzymes are RNA-stimulated ATPases that translocate on RNA and unwind or remodel structured RNA in an ATP-dependent fashion. How ATP...Full Text Available
Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase
2010-03-01
Full Text Available.RNA helicases function in numerous aspects of RNA biology. These enzymes are RNA-stimulated ATPases that translocate on RNA and unwind or remodel structured RNA in an ATP-dependent fashion. How ATP and the ATPase cycle fuel the work performed by helicases is not completely clear. The hepatitis C virus RNA helicase, NS3, is an important model system for this class of enzymes. NS3 binding to a single-/double-strand RNA or DNA junction leads to ATP-independent melting of the duplex and formation of a complex capable of ATP-dependent unwinding by using a spring-loaded mechanism. We have established an RNA substrate for NS3 that can be unwound in a single sub-step. Our studies are consistent with a model in which a single ATP binding and/or hydrolysis event sets the unwinding spring and phosphate dissociation contributes to release of the spring, thereby driving the power stroke used for unwinding.
PhiC31 integrase induces a DNA damage response and chromosomal rearrangements in human adult fibroblasts
2009-01-01
BACKGROUND: PhiC31 integrase facilitates efficient integration of transgenes into human and mouse genomes and is considered for clinical gene therapy. However recent studies have shown that the enzyme can induce various chromosomal abnormalities in primary human embryonic cells and mammalian cell lines. The mechanisms involved are unknown, but it has been proposed that PhiC31 attachment sites in the host genome recombine leading to chromosomal translocations. RESULTS: We have studied possible effects of the PhiC31 integrase expression in human adult fibroblasts by karyotyping. All control cells were cytogenetically normal, whereas cells expressing PhiC31 integrase show chromosomal abnormalities confirming our previous results using primary embryonic fibroblasts. In order to study the early mechanisms involved we measured H2AX phosphorylation - a primary event in the response to DNA double-strand-breaks. Transient transfection with PhiC31 integrase encoding plasmids lead to an elevated number of cells positive for H2AX phosphorylation detected by immunofluorescence. Western blot analysis confirmed the upregulated H2AX phosphorylation, whereas markers for apoptosis as well as p53 and p21 were not induced. Cells transfected with plasmids encoding the Sleeping Beauty transposase remained cytogenetically normal, and in these cells less upregulation of H2AX phosphorylation could be detected. CONCLUSION: In primary human fibroblasts expression of PhiC31 integrase leads to a DNA damage response and chromosomal aberrations.
Patterning quantum dot arrays using DNA replication principles.
2004-11-01
The convergence of nanoscience and biotechnology has opened the door to the integration of a wide range of biological molecules and processes with synthetic materials and devices. A primary biomolecule of interest has been DNA based upon its role as information storage in living systems, as well as its ability to withstand a wide range of environmental conditions. DNA also offers unique chemistries and interacts with a range of biomolecules, making it an ideal component in biological sensor applications. The primary goal of this project was to develop methods that utilize in vitro DNA synthesis to provide spatial localization of nanocrystal quantum dots (nQDs). To accomplish this goal, three specific technical objectives were addressed: (1) attachment of nQDs to DNA nucleotides, (2) demonstrating the synthesis of nQD-DNA strands in bulk solution, and (3) optimizing the ratio of unlabeled to nQD-labeled nucleotides. DNA nucleotides were successfully attached to nQDs using the biotin-streptavidin linkage. Synthesis of 450-nm long, nQD-coated DNA strands was demonstrated using a DNA template and the polymerase chain reaction (PCR)-based method of DNA amplification. Modifications in the synthesis process and conditions were subsequently used to synthesize 2-{micro}m long linear nQD-DNA assemblies. In the case of the 2-{micro}m structures, both the ratio of streptavidin-coated nQDs to biotinylated dCTP, and streptavidin-coated nQD-dCTPs to unlabeled dCTPs affected the ability to synthesize the nQD-DNA assemblies. Overall, these proof-of-principles experiments demonstrated the successful synthesis of nQD-DNA using DNA templates and in vitro replication technologies. Continued development of this technology may enable rapid, spatial patterning of semiconductor nanoparticles with Angstrom-level resolution, as well as optically active probes for DNA and other biomolecular analyses.
Oxidative DNA damage background estimated by a system model of base excision repair
2004-05-13
Human DNA can be damaged by natural metabolism through free radical production. It has been suggested that the equilibrium between innate damage and cellular DNA repair results in an oxidative DNA damage background that potentially contributes to disease and aging. Efforts to quantitatively characterize the human oxidative DNA damage background level based on measuring 8-oxoguanine lesions as a biomarker have led to estimates varying over 3-4 orders of magnitude, depending on the method of measurement. We applied a previously developed and validated quantitative pathway model of human DNA base excision repair, integrating experimentally determined endogenous damage rates and model parameters from multiple sources. Our estimates of at most 100 8-oxoguanine lesions per cell are consistent with the low end of data from biochemical and cell biology experiments, a result robust to model limitations and parameter variation. Our results show the power of quantitative system modeling to interpret composite experimental data and make biologically and physiologically relevant predictions for complex human DNA repair pathway mechanisms and capacity.
2010-03-01
RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of...Full Text Available
2010-03-01
Full Text Available.RAD51, a key protein in the homologous recombinational DNA repair (HRR) pathway, is the major strand-transferase required for mitotic recombination. An important early step in HRR is the formation of single-stranded DNA (ss-DNA) coated by RPA (a ss-DNA-binding protein). Displacement of RPA by RAD51 is highly regulated and facilitated by a number of different proteins known as the ‘recombination mediators’. To assist these recombination mediators, a second group of proteins also is required and we are defining these proteins here as ‘recombination co-mediators’. Defects in either recombination mediators or co-mediators, including BRCA1 and BRCA2, lead to impaired HRR that can genetically be complemented for (i.e. suppressed) by overexpression of RAD51. Defects in HRR have long been known to contribute to genomic instability leading to tumor development. Since genomic instability also slows cell growth, precancerous cells presumably require genomic re-stabilization to gain a growth advantage. RAD51 is overexpressed in many tumors, and therefore, we hypothesize that the complementing ability of elevated levels of RAD51 in tumors with initial HRR defects limits genomic instability during carcinogenic progression. Of particular interest, this model may also help explain the high frequency of TP53 mutations in human cancers, since wild-type p53 represses RAD51 expression.
2010-03-01
Full Text Available.DNA microarray measurements are susceptible to error caused by non-specific hybridization between a probe and a target (cross-hybridization), or between two targets (bulk-hybridization). Search algorithms such as BLASTN can quickly identify potentially hybridizing sequences. We set out to improve BLASTN accuracy by modifying the substitution matrix and gap penalties. We generated gene expression microarray data for samples in which 1 or 10% of the target mass was an exogenous spike of known sequence. We found that the 10% spike induced 2-fold intensity changes in 3% of the probes, two-third of which were decreases in intensity likely caused by bulk-hybridization. These changes were correlated with similarity between the spike and probe sequences. Interestingly, even very weak similarities tended to induce a change in probe intensity with the 10% spike. Using this data, we optimized the BLASTN substitution matrix to more accurately identify probes susceptible to non-specific hybridization with the spike. Relative to the default substitution matrix, the optimized matrix features a decreased score for A–T base pairs relative to G–C base pairs, resulting in a 5–15% increase in area under the ROC curve for identifying affected probes. This optimized matrix may be useful in the design of microarray probes, and in other BLASTN-based searches for hybridization partners.
2010-03-01
DNA microarray measurements are susceptible to error caused by non-specific hybridization between a probe and a target (cross-hybridization), or between two targets (bulk-hybridization). Search algorithms...Full Text Available
2010-03-01
Full Text Available.CpG dinucleotides are mutational hotspots associated with cancer and genetic diseases. Thymine DNA glycosylase (TDG) plays an integral role in CpG maintenance by excising mispaired thymine and uracil in a CpG context and also participates in transcriptional regulation via gene-specific CpG demethylation and functional interactions with the transcription machinery. Here, we report that protein kinase C α (PKCα) interacts with TDG and phosphorylates amino-terminal serine residues adjacent to lysines acetylated by CREB-binding protein (CBP) and p300 (CBP/p300). We establish that acetylation and phosphorylation are mutually exclusive, and their interplay dramatically alters the DNA mispair-processing functions of TDG. Remarkably, acetylation of the amino-terminal region abrogates high-affinity DNA binding and selectively prevents processing of G:T mispairs. In contrast, phosphorylation does not markedly alter DNA interactions, but may preserve G:T processing in vivo by preventing CBP-mediated acetylation. Mutational analysis suggests that the acetyl-acceptor lysines are not directly involved in contacting DNA, but may constitute a conformationally sensitive interface that modulates DNA interactions. These findings reveal opposing roles of CBP/p300 and PKCα in regulating the DNA repair functions of TDG and suggest that the interplay of these modifications in vivo may be critically important in the maintenance of CpG dinucleotides and epigenetic regulation.
2010-03-01
CpG dinucleotides are mutational hotspots associated with cancer and genetic diseases. Thymine DNA glycosylase (TDG) plays an integral role in CpG maintenance by excising mispaired thymine and uracil...Full Text Available
Mutational analysis of the 5′-OH oligonucleotide phosphate acceptor site of T4 polynucleotide kinase
2010-03-01
T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5′-kinase and 3′-phosphatase activities that function in nucleic acid repair. The N-terminal...Full Text Available
Mutational analysis of the 5′-OH oligonucleotide phosphate acceptor site of T4 polynucleotide kinase
2010-03-01
Full Text Available.T4 polynucleotide kinase/phosphatase (Pnkp) exemplifies a family of bifunctional enzymes with 5′-kinase and 3′-phosphatase activities that function in nucleic acid repair. The N-terminal kinase domain belongs to the P-loop phosphotransferase superfamily. The kinase is distinguished by a tunnel-like active site with separate entrances on opposite sides of the protein for the NTP phosphate donor and a 5′-OH single-stranded oligonucleotide phosphate acceptor. Here, we probed by mutagenesis the roles of individual amino acids that comprise the acceptor binding site. We thereby identified Glu57 as an important residue, by virtue of its participation in a salt bridge network with two catalytic residues identified previously: Arg38, which binds the 3′-phosphate of the terminal 5′-OH nucleotide, and the putative general base Asp35 that contacts the nucleophilic 5′-OH group. The 5′-OH nucleoside fits into a pocket lined by aliphatic amino acids (Val131, Pro132 and Val135) that make van der Waals contacts to the nucleobase. Whereas subtraction of these contacts by single alanine substitutions for Val131 or Val135 and glycine for Pro132 had modest effects on kinase activity, the introduction of bulkier phenylalanines for Val131 and Val135 were deleterious, especially V131F, which severely impeded both substrate binding (increasing Km by 15-fold) and catalysis (decreasing kcat by 300-fold).
Molecular dynamics simulations of xDNA
2009-01-01
xDNA is a modified DNA, which contains natural as well as expanded bases. Expanded bases are generated by the addition of a benzene spacer to the natural bases. A set of AMBER force-field parameters were derived for the expanded bases and the structural dynamics of the xDNA decamer (xT5prime G xT A xC xG C xA xG T3prime) (xA5prime C T xG C G xT A xC A3prime) was explored using a 22 ns molecular dynamics simulation in explicit solvent. During the simulation, the duplex retained its Watson-Crick base-pairing and double helical structure, with deviations from the starting B-form geometry towards A-form; the deviations are mainly in the backbone torsion angles and in the helical parameters. The sugar pucker of the residues were distributed among a variety of modes; C2prime endo, C1prime exo, O...
Methods for interpreting lists of affected genes obtained in a DNA microarray experiment
2009-01-01
The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence) and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding) workshop focusing on post analysis of microarray data. The participating groups were provided with identical lists of microarray probes, including test statistics for three different contrasts, and the normalised log-ratios for each array, to be used as the starting point for interpreting the affected probes. The data originated from a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria
Methods for interpreting lists of affected genes obtained in a DNA microarray experiment
2009-01-01
The aim of this paper was to describe and compare the methods used and the results obtained by the participants in a joint EADGENE (European Animal Disease Genomic Network of Excellence) and SABRE (Cutting Edge Genomics for Sustainable Animal Breeding) workshop focusing on post analysis of microarray data. The participating groups were provided with identical lists of microarray probes, including test statistics for three different contrasts, and the normalised log-ratios for each array, to be used as the starting point for interpreting the affected probes. The data originated from a microarray experiment conducted to study the host reactions in broilers occurring shortly after a secondary challenge with either a homologous or heterologous species of Eimeria
Mechanisms for radiation damage in DNA. Progress report, June 1, 1994--May 31, 1995
1994-11-01
In this project we have proposed several mechanisms for radiation damage to DNA and its constituents, and have detailed a series of experiments utilizing electron spin resonance spectroscopy, HPLC, GC-mass spectroscopy and ab initio molecular orbital calculations to test the proposed mechanisms. The results from these various techniques have resulted in an understanding of consequences of radiation damage to DNA from the early ionization event to the production of non-radical lesions (discussed in detail in Comprehensive Report). In this year`s work we have found the hydroxyl radical in DNA`s hydration layer. This is an important result which impacts the hole transfer hypothesis and the understanding of the direct vs. indirect effect in DNA. Further we have found the first ESR evidence for sugar radicals as a result of direct radiation damage to DNA nucleotides in an aqueous environment. This is significant as it impacts the biological endpoint of radiation damage to DNA and suggests future work in DNA. Work with DNA-polypeptides show clear evidence for electron transfer to DNA from the polypeptide which we believe is a radioprotective mechanism. Our work with ab initio molecular orbital theory has gain insight into the initial events of radiation damage to DNA. Ab initio calculations have provided an understanding of the energetics involved in anion and cation formation, ion radical transfer in DNA as well as proton transfer with DNA base pair radical ions. This has been extended in this year`s work to new, more accurate values for the electron affinities of the DNA bases, understanding of the relative stability of all possible sugar radicals formed by hydrogen abstraction on the deoxyribose group, hydration effects on, thiol radioprotectors, and an ongoing study of radical intermediates formed from initial DNA ion radicals. During this fiscal year five articles have been published, three are in press, two are submitted and several more are in preparation.
Mechanisms for radiation damage in DNA. Progress report, June 1, 1992--May 31, 1993
1992-12-01
In this project we have proposed several mechanisms for radiation damage and recently radiation protection to DNA and its constituents, and have detailed a series of experiments utilizing electron spin resonance spectroscopy, HPLC, GC-mass spectroscopy and ab initio molecular orbital calculations to test the proposed mechanisms. In this years work we have performed experiments which elucidate the role of hydration water on DNA radiation damage, continued the investigation of the localization of the initial charges on DNA and employed ab initio molecular orbital theory to gain insight into the initial events of radiation damage to DNA. Ab initio calculations have provided an understanding of the energetics envolved in anion and cation formation, ion radical transfer in DNA as well as proton transfer with DNA base pair radical ions. This information has aided the formation of new radiation models for the effect of radiation on DNA. During this fiscal year four articles have been published, two are in press, two are submitted and several more are in preparation. Six papers have been presented at scientific meetings. This years effort includes a review article on the ``Chemical Consequences of Radiation Damage to DNA``. This review presents an overview of this field at this time.
Mechanisms for radiation damage in DNA
1992-12-01
In this project we have proposed several mechanisms for radiation damage and recently radiation protection to DNA and its constituents, and have detailed a series of experiments utilizing electron spin resonance spectroscopy, HPLC, GC-mass spectroscopy and ab initio molecular orbital calculations to test the proposed mechanisms. In this years work we have performed experiments which elucidate the role of hydration water on DNA radiation damage, continued the investigation of the localization of the initial charges on DNA and employed ab initio molecular orbital theory to gain insight into the initial events of radiation damage to DNA. Ab initio calculations have provided an understanding of the energetics envolved in anion and cation formation, ion radical transfer in DNA as well as proton transfer with DNA base pair radical ions. This information has aided the formation of new radiation models for the effect of radiation on DNA. During this fiscal year four articles have been published, two are in press, two are submitted and several more are in preparation. Six papers have been presented at scientific meetings. This years effort includes a review article on the Chemical Consequences of Radiation Damage to DNA''. This review presents an overview of this field at this time.
Macrophages and Adipocytes in Human Obesity Adipose Tissue Gene Expression and Insulin Sensitivity During Calorie Restriction and Weight Stabilization
2009-01-01
OBJECTIVE-We investigated the regulation of adipose tissue gene expression during different phases of a dietary weight loss program and its relation with insulin sensitivity. RESEARCH DESIGN AND METHODS-Twenty-two obese women followed a dietary intervention program composed of an energy restriction phase with a 4-week very-low-calorie diet and a weight stabilization period composed of a 2-month low-calorie diet followed by 3-4 months of a weight maintenance diet. At each time point, a euglycemic-hyperinsulinemic clamp and subcutaneous adipose tissue biopsies were performed. Adipose tissue gene expression profiling was performed using a DNA microarray in a subgroup of eight women. RT-quantitative PCR was used for determination of mRNA levels of 31 adipose tissue macrophage markers (n = 22). RESULTS-Body weight, fat mass, and C-reactive protein level decreased and glucose disposal rate increased during the dietary intervention program. Transcriptome profiling revealed two main patterns of variations. The first involved 464 mostly adipocyte genes involved in metabolism that were downregulated during energy restriction, upregulated during weight stabilization, and unchanged during the dietary intervention. The second comprised 511 mainly macrophage genes involved in inflammatory pathways that were not changed or upregulated during energy restriction and downregulated during weight stabilization and dietary intervention. Accordingly, macrophage markers were upregulated during energy restriction and downregulated during weight stabilization and dietary intervention. The increase in glucose disposal rates in each dietary phase was associated with variation in expression of sets of 80-110 genes that differed among energy restriction, weight stabilization, and dietary intervention. CONCLUSIONS-Adipose tissue macrophages and adipocytes show distinct patterns of gene regulation and association with insulin sensitivity during the various phases of a dietary weight loss program. Diabetes 58:1558-1567, 2009
2010-02-01
The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate...Full Text Available
2010-02-01
Full Text Available.The repair of DNA double-strand breaks (DSBs) is critical for the maintenance of genomic integrity and viability for all organisms. Mammals have evolved at least two genetically discrete ways to mediate DNA DSB repair: homologous recombination (HR) and non-homologous end joining (NHEJ). In mammalian cells, most DSBs are preferentially repaired by NHEJ. Recent work has demonstrated that NHEJ consists of at least two sub-pathways—the main Ku heterodimer-dependent or “classic” NHEJ (C-NHEJ) pathway and an “alternative” NHEJ (A-NHEJ) pathway, which usually generates microhomology-mediated signatures at repair junctions. In our study, recombinant adeno-associated virus knockout vectors were utilized to construct a series of isogenic human somatic cell lines deficient in the core C-NHEJ factors (Ku, DNA-PKcs, XLF, and LIGIV), and the resulting cell lines were characterized for their ability to carry out DNA DSB repair. The absence of DNA-PKcs, XLF, or LIGIV resulted in cell lines that were profoundly impaired in DNA DSB repair activity. Unexpectedly, Ku86-null cells showed wild-type levels of DNA DSB repair activity that was dominated by microhomology joining events indicative of A-NHEJ. Importantly, A-NHEJ DNA DSB repair activity could also be efficiently de-repressed in LIGIV-null and DNA-PKcs-null cells by subsequently reducing the level of Ku70. These studies demonstrate that in human cells C-NHEJ is the major DNA DSB repair pathway and they show that Ku is the critical C-NHEJ factor that regulates DNA NHEJ DSB pathway choice.
2005-12-29
Cisplatin and carboplatin are platinum-based drugs that are widely used in cancer chemotherapy. The cytotoxicity of these drugs is mediated by platinum-DNA monoadducts and intra- and interstrand diadducts, which are formed following uptake of the drug into the nucleus of cells. The pharmacodynamics of carboplatin display fewer side effects than for cisplatin, albeit with less potency, which may be due to differences in rates of DNA adduct formation. We report the use of accelerator mass spectrometry (AMS), a sensitive detection method often used for radiocarbon quantitation, to measure both the kinetics of [{sup 14}C]carboplatin-DNA adduct formation with genomic DNA and drug uptake and DNA binding in T24 human bladder cancer cells. Only carboplatin-DNA monoadducts contain radiocarbon in the platinated DNA, which allowed for calculation of kinetic rates and concentrations within the system. The percent of radiocarbon bound to salmon sperm DNA in the form of monoadducts was measured by AMS over 24 h. Knowledge of both the starting concentration of the parent carboplatin and the concentration of radiocarbon in the DNA at a variety of time points allowed calculation of the rates of Pt-DNA monoadduct formation and conversion to toxic cross-links. Importantly, the rate of carboplatin-DNA monoadduct formation was approximately 100-fold slower than that reported for the more potent cisplatin analogue, which may explain the lower toxicity of carboplatin. T24 human bladder cancer cells were incubated with a subpharmacological dose of [{sup 14}C]carboplatin, and the rate of accumulation of radiocarbon in the cells and nuclear DNA was measured by AMS. The lowest concentration of radiocarbon measured was approximately 1 amol/10 {micro}g of DNA. This sensitivity may allow the method to be used for clinical applications.
2009-01-01
Ion beam biotechnology has been developed for induction of DNA transfer into biological cells. To separately investigate effects of ion interaction with plant cell envelope for understanding relevant mechanisms, this study used chitosan and cellulose membranes to simulate the cell envelope and characterize behavior of the membranes modified by ion beam. Chitosan and cellulose membranes were bombarded with argon and nitrogen ion beams at energy of 15-25 keV to fluencies in orders of 1015 ion/cm2. Modifications of the membrane surface morphology, contact angle and electric characteristics were investigated. Results showed that subjected to ion bombardment, the membrane surface was roughened, the contact angle of the membrane surface was varied, the membrane impedance was decreased and the conductance and capacitance were increased. These ... >>
Interaction of zanamivir with DNA and RNA: Models for drug-DNA and drug-RNA bindings
2007-01-01
Zanamivir (ZAN) is the first of a new generation of influenza virus-specific drugs known as neuraminidase inhibitors, which acts by interfering with life cycles of influenza viruses A and B. It prevents the virus spreading infection to other cells by blocking the neuraminidase enzyme present on the surface of the virus. The aim of this study was to examine the stability and structural features of calf thymus DNA and yeast RNA complexes with zanamivir in aqueous solution, using constant DNA or RNA concentration (12.5mM) and various zanamivir/polynucleotide (P) ratios of 1/20, 1/10, 1/4, and 1/2. FTIR and UV-visible spectroscopy are used to determine the drug external binding modes, the binding constant and the stability of zanamivir-DNA and RNA complexes in aqueous solution. Structural anal...
2009-01-01
Purpose: To determine whether glioma cells irradiated with a microplanar X-ray beam exert bystander effects. Methods and Materials: Microplanar beam irradiation of glioma cells in vitro was done using the SPring-8 synchrotron radiation facility. The amount of DNA double-strand breaks (dsbs) was measured by the fluorescence intensity of phosphorylated H2AX or the number of 53BP1 foci. The dose distribution in a cell population exposed to a single microplanar beam was determined by the amount of phosphorylated H2AX-positive cells. Bystander effects were determined by counting the number of 53BP1 foci in nonirradiated cells treated with conditioned medium from cultures of irradiated cells. Results: More DNA dsbs were detected in cells adjacent to an area irradiated by the single beam than in cells in distant, nonirradiated areas as a result of bystander effects caused by ... >>
2009-01-01
Purpose: The aim of this study was to evaluate the induction and rejoining of DNA double strand breaks (DSBs) in melanoma cells exposed to low and high linear energy transfer (LET) radiation. Methods and Materials: DSBs and survival were determined as a function of dose in melanoma cells (B16-F0) irradiated with monoenergetic proton and lithium beams and with a gamma source. Survival curves were obtained by clonogenic assay and fitted to the linear-quadratic model. DSBs were evaluated by the detection of phosphorylated histone H2AX (gammaH2AX) foci at 30 min and 6 h post-irradiation. Results: Survival curves showed the increasing effectiveness of radiation as a function of LET. gammaH2AX labeling showed an increase in the number of foci vs. dose for all the radiations evaluated. A decrease in the number of foci was found at 6 h post-irradiation for low ... >>
Identification of a New G-Quadruplex Motif in the KRAS Promoter and Design of Pyrene-Modified G4-Decoys with Antiproliferative Activity in Pancreatic Cancer Cells
2009-01-01
A new quadruplex motif located in the promoter of the human KRAS gene, within a nuclease hypersensitive element (NHE), has been characterized. Oligonucleotides mimicking this quadruplex are found to compete with a DNA-protein complex between NHE and a nuclear extract from pancreatic cancer cells. When modified with (R)-1-O-[4-1-(1-pyrenylethynyl) phenylmethyl]glycerol insertions (TINA), the quadruplex oligonucleotides showed a dramatic increase of the Tm (ÎTm from 22 to 32 °C) and a strong antiproliferative effects in Panc-1 cells.
Identification and Characterization of a Human DNA Double-Strand Break Repair Complex
1999-07-12
The authors have used atomic force microscopy (AFM) to characterize the assembly and structure of the macromolecular assemblies involved in DNA repair. They have demonstrated using AFM that the DNA-dependent protein kinase can play a structural role in the repair of DNA double-strand breaks (DSBs) by physically holding DNA ends together. They have extended these studies to include other DNA damage response proteins, these efforts have resulted in important and novel findings regarding the ATM protein. Specifically, the work has demonstrated, for the first time, that the ATM protein binds with specificity to a DNA end. This finding is the first to implicate the ATM protein in the detection of DNA damage by direct physical interaction with DSBs.
Guiding Properties of Silica/Air Hollow-Core Bragg Fibers
2008-01-01
The guiding properties of realistic silica/air hollow-core Bragg fibers have been investigated by calculating the dispersion curves, the confinement loss spectrum and the field distribution of the guided modes through a full-vector modal solver based on the finite element method. In particular, the silica bridge influence on the fundamental mode has been analyzed, by comparing the properties of an ideal structure, without the silica nano-supports, and of two realistic fibers, with squared off and rounded air-holes. Simulation results have demonstrated the presence of anti-crossing points in the dispersion curves, associated to the transition of the fundamental mode into a surface one. It has been shown that surface modes are responsible of the sharp loss peaks, also experimentally measured, which pollute the loss spectrum of the fundamental mode and of the higher-order ones. Then, the influence on the guiding properties of each geometric characteristic in the hollow-core Bragg fiber cross-section has been deeply investigated, thus showing which parameter it is better to change in order to properly modify the loss values or its spectral behaviour. Moreover, in order to improve the loss properties of hollow-core Bragg fibers, the number of silica and air layers in the fiber cladding has been increased, and the layer thickness has been modified. Results have shown that the first change is more effective for the loss reduction, while the second is useful for a spectral shift. Finally, among the different possible applications, the feasibility of a DNA bio-sensor based on a hollow-core Bragg fiber has been demonstrated.
Gel Electrophoresis of Gold-DNA Nano-Conjugates
2006-01-10
Single stranded DNA of different lengths and different amounts was attached to colloidal phosphine stabilized Au nanoparticles. The resulting conjugates were investigated in detail by a gel electrophoresis study based on 1200 gels. We demonstrate how these experiments help to understand the binding of DNA to Au particles. In particular we compare specific attachment of DNA via gold-thiol bonds with nonspecific adsorption of DNA. The maximum number of DNA molecules that can be bound per particle was determined. We also compare several methods to used gel electrophoresis for investigating the effective diameter of DNA-Au conjugates, such as using a calibration curve of particles with known diameters and Ferguson plots.
GRAS Proteins Form a DNA Binding Complex to Induce Gene Expression during Nodulation Signaling in Medicago truncatula
2009-01-01
Fusion of a viral antigen to invariant chain leads to augmented T-cell immunity and improved protection in gene-gun DNA-vaccinated mice
2009-01-01
It has recently been demonstrated that a recombinant replication-deficient human adenovirus 5 (Ad5) vector expressing lymphocytic choriomeningitis virus (LCMV) glycoprotein (GP) fused to the p31 invariant (Ii) chain confers broad, long-lasting T-cell immunity that completely protects C57BL/6 mice against lethal peripheral challenge. The current study questioned whether the same strategy, i.e. linkage of GP to an Ii chain, could be applied to a naked DNA vaccine. Following gene-gun immunization with the linked construct (DNA-IiGP), GP-specific CD4(+) T cells could not be detected by flow cytometry. However, inclusion of the Ii chain augmented the priming of GP-specific CD8(+) T cells directed towards both immunodominant (GP(33-41)) and subdominant (GP(276-286) and GP(92-101)) epitopes, and vaccination with DNA-IiGP conferred significantly improved protection against systemic LCMV infection compared with the unlinked construct. In contrast, substantial protection against peripheral challenge was not observed. Additional experiments with T-cell subset-depleted or perforin-deficient mice revealed that virus control in vaccinated mice depends critically on cytotoxic CD8(+) T cells. Finally, priming with the naked DNA vaccine was shown to augment the immune response raised by subsequent immunization with the Ad5 vector. In conclusion, this study showed that the immunoenhancing effect of Ii chain linkage is not limited to the Ad5 vector, but is also relevant with a DNA platform. Furthermore, given the fact that the Ii chain enhances the presentation of more than one epitope, this suggests that Ii-chain-based DNA vaccines may be promising candidates for various heterologous prime-boost regimes.
Final Report on Grant DE-FG-02ER63350
2005-06-10
Research funded by grant DE-FG-02ER63350 focused on DNA bending measured NMR spectroscopy and modeled by classical molecular dynamics (MD) simulation. Bending is a structural aspect of DNA that is plays a key role in its function. The most important finding of our research was that oxidation of guanine, a ubiquitous DNA lesion caused by endogenous and environmental oxidative stress, changes DNA bending dynamics in a way that favors binding of glycosylases, repair enzymes that remove damaged bases from DNA. Hence, the effect of 8-oxoguanine on DNA bending contributes to its recognition and removal by the base excision repair system.
2010-03-01
The bacterial activator protein NorR binds to enhancer-like elements, upstream of the promoter site, and activates σ54-dependent transcription of genes that encode nitric oxide detoxifying...Full Text Available
2010-03-01
Full Text Available.The bacterial activator protein NorR binds to enhancer-like elements, upstream of the promoter site, and activates σ54-dependent transcription of genes that encode nitric oxide detoxifying enzymes (NorVW), in response to NO stress. Unique to the norVW promoter in Escherichia coli is the presence of three enhancer sites associated with a binding site for σ54-RNA polymerase. Here we show that all three sites are required for NorR-dependent catalysis of open complex formation by σ54-RNAP holoenzyme (Eσ54). We demonstrate that this is essentially due to the need for all three enhancers for maximal ATPase activity of NorR, energy from which is used to remodel the closed Eσ54 complex and allow melting of the promoter DNA. We also find that site-specific DNA binding per se promotes oligomerisation but the DNA flanking the three sites is needed to further stabilise the functional higher order oligomer of NorR at the enhancers.
Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs
2010-03-01
Type IIS restriction endonuclease BtsCI (GGATG 2/0) is a neoschizomer of FokI (GGATG 9/13) and cleaves closer to the recognition sequence. Although M.BtsCI shows 62% amino acid sequence identity to...Full Text Available
Engineering Nt.BtsCI and Nb.BtsCI nicking enzymes and applications in generating long overhangs
2010-03-01
Full Text Available.Type IIS restriction endonuclease BtsCI (GGATG 2/0) is a neoschizomer of FokI (GGATG 9/13) and cleaves closer to the recognition sequence. Although M.BtsCI shows 62% amino acid sequence identity to M.FokI, BtsCI and FokI restriction endonucleases do not share significant amino acid sequence similarity. BtsCI belongs to a group of Type IIS restriction endonucleases, BsmI, Mva1269I and BsrI, that carry two different catalytic sites in a single polypeptide. By inactivating one of the catalytic sites through mutagenesis, we have generated nicking variants of BtsCI that specifically nick the bottom-strand or the top-strand of the target site. By treating target DNA sequentially with the appropriate combinations of FokI and BtsCI nicking variants, we are able to generate long overhangs suitable for fluorescent labeling through end-filling or other techniques based on annealing of complementary DNA sequences.
Electrochemical control of a DNA Holliday junction nanoswitch by Mg2+ ions
2008-01-01
Electrochemical control of a DNA Holliday Junction nanoswitch by Mg2+ ions
2008-01-01
The molecular conformation of a synthetic branched, 4-way DNA Holliday junction (HJ) was electrochemically switched between the open and closed (stacked) conformers. Switching was achieved by electrochemically induced quantitative release of Mg(2+) ions from the oxidised poly(N-methylpyrrole) film (PPy), which contained polyacrylate as an immobile counter anion and Mg(2+) ions as charge compensating mobile cations. This increase in the Mg(2+) concentration screened the electrostatic repulsion between the widely separated arms in the open HJ configuration, inducing switching to the closed conformation. Upon electrochemical reduction of PPy, entrapment of Mg(2+) ions back into the PPy film induced the reverse HJ switching from the closed to open state. The conformational transition was monitored using fluorescence resonance energy transfer (FRET) between donor and acceptor dyes each located at the terminus of one of the arms. The demonstrated electrochemical control of the conformation of the used probe-target HJ complex, previously reported as a highly sequence specific nanodevice for detecting of unlabelled target [Buck, A.H., Campbell, C.J., Dickinson, P., Mountford, C.P., Stoquert, H.C., Terry, J.G., Evans, S.A.G., Keane, L., Su, T.J., Mount, A.R., Walton, A.J., Beattie, J.S., Crain, J., Ghazal, P., 2007. Anal. Chem., 79, 4724-4728], allows the development of electronically addressable DNA nanodevices and label-free gene detection assays.
Disruption of the NHR4 domain structure in AML1-ETO abrogates SON binding and promotes leukemogenesis.
2008-01-01
AML1-ETO is generated from t(8;21)(q22;q22), which is a common form of chromosomal translocation associated with development of acute myeloid leukemia (AML). Although full-length AML1-ETO alone fails to promote leukemia because of its detrimental effects on cell proliferation, an alternatively spliced isoform, AML1-ETO9a, without its C-terminal NHR3/NHR4 domains, strongly induces leukemia. However, full-length AML1-ETO is a major form of fusion product in many t(8;21) AML patients, suggesting additional molecular mechanisms of t(8;21)-related leukemogenesis. Here, we report that disruption of the zinc-chelating structure in the NHR4 domain of AML1-ETO by replacing only one critical amino acid leads to rapid onset of leukemia, demonstrating that the NHR4 domain with the intact structure generates inhibitory effects on leukemogenesis. Furthermore, we identified SON, a DNA/RNA-binding domain containing protein, as a novel NHR4-interacting protein. Knock-down of SON by siRNA resulted in significant growth arrest, and disruption of the interaction between AML1-ETO and endogenous SON rescued cells from AML1-ETO-induced growth arrest, suggesting that SON is an indispensable factor for cell growth, and AML1-ETO binding to SON may trigger signals inhibiting leukemogenesis. In t(8;21) AML patient-derived primary leukemic cells and cell lines, abnormal cytoplasmic localization of SON was detected, which may keep cells proliferating in the presence of full-length AML1-ETO. These results uncovered the crucial role of the NHR4 domain in determination of cellular fate during AML1-ETO-associated leukemogenesis.
Defining interactions between DNA-PK and ligase IV/XRCC4
2001-04-10
Non-homologous end joining (NHEJ) is a major pathway for the repair of DNA double-strand breaks in mammalian cells. DNA-dependent protein kinase (DNA-PK), ligase IV, and XRCC4 are all critical components of the NHEJ repair pathway. DNA-PK is composed of a heterodimeric DNA-binding component, Ku, and a large catalytic subunit, DNA-PKcs. Ligase IV and XRCC4 associate to form a multimeric complex that is also essential for NHEJ. DNA-PK and ligase IV/XRCC4 interact at DNA termini which results in stimulated ligase activity. Here we define interactions between the components of these two essential complexes, DNA-PK and ligase IV/XRCC4. We find that ligase IV/XRCC4 associates with DNA-PK in a DNA-independent manner. The specific protein-protein interactions that mediate the interaction between these two complexes are further identified. Direct physical interactions between ligase IV and Ku as well as between XRCC4 and DNA-PKcs are shown. No direct interactions are observed between ligase IV and DNA-PKcs or between XRCC4 and Ku. Our data defines the specific protein pairs involved in the association of DNA-PK and ligase IV/XRCC4, and suggests a molecular mechanism for coordinating the assembly of the DNA repair complex at DNA breaks.
Deciphering the Structural Properties That Confer Stability to a DNA Nanocage
2009-01-01
2010-03-01
Full Text Available.Common fragile sites (CFS) are chromosomal regions that exhibit instability during DNA replication stress. Although the mechanism of CFS expression has not been fully elucidated, one known feature is a severely delayed S-phase. We used an in vitro primer extension assay to examine the progression of DNA synthesis through various sequences within FRA16D by the replicative human DNA polymerases δ and α, and with human cell-free extracts. We found that specific cis-acting sequence elements perturb DNA elongation, causing inconsistent DNA synthesis rates between regions on the same strand and complementary strands. Pol δ was significantly inhibited in regions containing hairpins and microsatellites, [AT/TA]24 and [A/T]19–28, compared with a control region with minimal secondary structure. Pol δ processivity was enhanced by full length Werner Syndrome protein (WRN) and by WRN fragments containing either the helicase domain or DNA-binding C-terminal domain. In cell-free extracts, stalling was eliminated at smaller hairpins, but persisted in larger hairpins and microsatellites. Our data support a model whereby CFS expression during cellular stress is due to a combination of factors—density of specific DNA secondary-structures within a genomic region and asymmetric rates of strand synthesis.
2010-03-01
Common fragile sites (CFS) are chromosomal regions that exhibit instability during DNA replication stress. Although the mechanism of CFS expression has not been fully elucidated, one known feature is...Full Text Available
DNA fragment sizing and sorting by laser-induced fluorescence
1992-12-31
A method is provided for obtaining DNA fingerprints using high speed detection systems, such as flow cytometry to determine unique characteristics of DNA pieces from a selected sample. In one characterization the DNA piece is fragmented at preselected sites to produce a plurality of DNA fragments. The DNA piece or the resulting DNA fragments are treated with a dye effective to stain stoichiometrically the DNA fragments. The fluorescence from the dye in the stained fragments is then examined to generate an output functionally related to the number of nucleotides in each one of the DNA fragments. In one embodiment, the intensity of the fluorescence emissions from each fragment is directly proportional to the fragment length. Additional dyes can be bound to the DNA piece and DNA fragments to provide information additional to length information. Oligonucleotide specific dyes and/or hybridization probes can be bound to the DNA fragments to provide information on oligonucleotide distribution or probe hybridization to DNA fragments of different sizes.
DNA Compaction by Yeast Mitochondrial Protein ABF2p
2003-05-09
We used high resolution Atomic Force Microscopy (AFM) to image compaction of linear and circular DNA by the yeast mitochondrial protein ABF2p , which plays a major role in maintaining mitochondrial DNA. AFM images show that protein binding induces drastic bends in the DNA backbone for both linear and circular DNA. At high concentration of ABF2p DNA collapses into a tight globular structure. We quantified the compaction of linear DNA by measuring the end-to-end distance of the DNA molecule at increasing concentrations of ABF2p. We also derived a polymer statistical mechanics model that gives quantitative description of compaction observed in our experiments. This model shows that a number of sharp bends in the DNA backbone is often sufficient to cause DNA compaction. Comparison of our model with the experimental data showed excellent quantitative correlation and allowed us to determine binding characteristics for ABF2. Our studies indicate that ABF2 compacts DNA through a novel mechanism that involves bending of DNA backbone. We discuss the implications of such a mechanism for mitochondrial DNA maintenance.
DNA Adducts with Antioxidant Flavonoids: Morin, Apigenin, and Naringin
2008-01-01
Flavonoids have recently attracted a great interest as potential therapeutic drugs against a wide range of free-radical-mediated diseases. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. While the antioxidant activity of these natural polyphenolic compounds is well known, their bindings to DNA are not fully investigated. This study was designed to examine the interactions of morin (Mor), naringin (Nar), and apigenin (Api) with calf thymus DNA in aqueous solution at physiological conditions, using constant DNA concentration (6.25 mM) and various drug/DNA(phosphate) ratios of 1/40 to 1. FTIR and UV-Vis spectroscopic methods were used to determine the ligand binding modes, the binding constant, and the stability of DNA in flavono...
Characterization of the mammalian DNA polymerase gene and protein. Annual progress report
1993-01-01
Methods were developed to purify the DNA polymerases of the {alpha}-family from Chinese hamster cells and their mutants selected as resistant to aphidicolin or specific inhibitor of DNA polymerases of the {alpha}-family. The wild type and mutant DNA polymerases were characterized with respect to their biochemical properties. A methodology was also developed to identify the replication intermediates and aphidicolin was found to inhibit a replication intermediate of the 24Kb size indicating the fact that aphidicolin inhibits the elongation process during DNA replication. This is the first demonstration of such role of aphidicolin in the eukaryotic DNA replication.
1996-08-01
This project has focused on the DNA polymerase of phage T7 for use in DNA sequencing. A complex of T7 DNA polymerase and E. coli thioredoxin form a highly processive DNA polymerase. The exonuclease activity of the enzyme can be reduced by chemical or genetic modifications resulting in an enzyme that has several properties useful in sequencing including high processivity and lack of discrimination against dideoxynucleotides. Manganese ion eliminates all discrimination against ddNTPs allowing sequence determination based on band intensity. A single tyrosine residue in the active site of T7 DNA polymerase is responsible for the efficient incorporation of ddNMPs. Replacement of the phenylalanine at this position in Klenow or Taq DNA polymerase with tyrosine eliminates discrimination against ddNTPs, a property that has advantages for cycle sequencing. Pyrophosphorolysis catalyzed by a polymerase results in the hydrolysis of specific fragments in DNA sequencing reactions, a problem that is eliminated by the addition of pyrophosphatase. The thioredoxin domain of gene 5 protein has been identified and transferred to Klenow DNA polymerase to make it processive. We have crystallized a complex of T7 DNA polymerase/thioredoxin bound to a primer-template in the presence of a dNTP.
2010-02-01
Full Text Available.Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination by dismantling Rad51 filaments and is also required for double-strand break (DSB) repair. Here we examine the biological significance of Cdk1-dependent phosphorylation of Srs2, using mutants that constitutively express the phosphorylated or unphosphorylated protein isoforms. We found that Cdk1 targets Srs2 to repair DSB and, in particular, to complete synthesis-dependent strand annealing, likely controlling the disassembly of a D-loop intermediate. Cdk1-dependent phosphorylation controls turnover of Srs2 at the invading strand; and, in absence of this modification, the turnover of Rad51 is not affected. Further analysis of the recombination phenotypes of the srs2 phospho-mutants showed that Srs2 phosphorylation is not required for the removal of toxic Rad51 nucleofilaments, although it is essential for cell survival, when DNA breaks are channeled into homologous recombinational repair. Cdk1-targeted Srs2 displays a PCNA–independent role and appears to have an attenuated ability to inhibit recombination. Finally, the recombination defects of unphosphorylatable Srs2 are primarily due to unscheduled accumulation of the Srs2 protein in a sumoylated form. Thus, the Srs2 anti-recombination function in removing toxic Rad51 filaments is genetically separable from its role in promoting recombinational repair, which depends exclusively on Cdk1-dependent phosphorylation. We suggest that Cdk1 kinase counteracts unscheduled sumoylation of Srs2 and targets Srs2 to dismantle specific DNA structures, such as the D-loops, in a helicase-dependent manner during homologous recombinational repair.
2010-02-01
Cdk1 kinase phosphorylates budding yeast Srs2, a member of UvrD protein family, displays both DNA translocation and DNA unwinding activities in vitro. Srs2 prevents homologous recombination...Full Text Available
2009-01-01
Purpose: Current biodosimetric techniques for determining radiation exposure have inherent delays, as well as quantitation and interpretation limitations. We have identified a new technique with the advantage of directly measuring circulating DNA by amplifying inter-B1 regions in the mouse genome, providing a sensitive method for quantitating plasma DNA. Methods and Materials: Real-time quantitative polymerase chain reaction (PCR) was used to detect levels of DNA by amplifying inter-B1 genomic DNA in plasma samples collected at 0-48 h from mice receiving 0-10 Gy total- or partial-body irradiation (137Cs gamma-ray source at approx1.86 Gy/min. Homogeneity: +- 6.5%). Results: The correlation coefficient between DNA levels and the threshold cycle value (CT) was 0.996, and the average recoveries of DNA ... >>
B-DNA Helix Stability in a Solvent-Free Environment
2007-01-01
B-DNA is the most common DNA helix conformation under physiological conditions. However, when the amount of water in a DNA solution is decreased, B-to-A helix transitions have been observed. To understand what type of helix conformations exist in a solvent-free environment, a series of poly d(CG)n and mixed sequence DNA duplexes from 18 to 30 bp were examined with circular dichroism (CD), ESI-MS, ion mobility, and molecular dynamics. From the CD spectra, it was observed that all sequences had B-form helices in solution. However, the solvent-free results were more complex. For the poly d(CG)n series, the 18 bp duplex had an A-form helix conformation, both A- and B-helices were present for the 22 bp duplex, and only B-helices were observed for the 26 and 30 bp duplexes. Since these sequences...
2010-03-01
MicroRNA (miR)390 cleaves the non-coding TAS3 precursor RNA for the production of tasiRNA-ARF, a group of an endogenous...Full Text Available
2010-03-01
Full Text Available.MicroRNA (miR)390 cleaves the non-coding TAS3 precursor RNA for the production of tasiRNA-ARF, a group of an endogenous trans-acting small-interfering RNAs which cleave the transcripts of auxin response factor (ARF) 3/4. miR390-cleaved TAS3 RNA is polymerized and diced into tasiRNA-ARF by RNA-dependent RNA polymerase6 (RDR6) and Dicer-like4 (DCL4), respectively. tasiRNA-ARF-dependent post-transcriptional gene silencing (PTGS) of ARF3/4 is involved in auxin-mediated polarity establishment in the development of aerial lateral organs, such as leaf and flower. To understand how auxin regulates ARF4 expression, we examined auxin responsiveness of miR390 expression, which comprises a regulatory step for the biogenesis pathway of tasiRNA-ARF (the tasiRNA-ARF pathway), in Arabidopsis thaliana lateral root (LR) development. The results of this study provide evidence that miR390 expression is sensitive to TIR1-dependent transcriptional regulation and auxin concentration, and also that mutual negative-regulation between the tasiRNA-ARF pathway and ARF4 modulates the spatiotemporal expression of ARF4. We propose that, together with auxin concentration sensing through miR390 transcription, the tasiRNA-ARF pathway mediates the auxin response and ARF4-mediated LR developmental processes.
Assembling semiconductor nanocomposites using DNA replication technologies.
2005-11-01
Deoxyribonucleic acid (DNA) molecules represent Nature's genetic database, encoding the information necessary for all cellular processes. From a materials engineering perspective, DNA represents a nanoscale scaffold with highly refined structure, stability across a wide range of environmental conditions, and the ability to interact with a range of biomolecules. The ability to mass-manufacture functionalized DNA strands with Angstrom-level resolution through DNA replication technology, however, has not been explored. The long-term goal of the work presented in this report is focused on exploiting DNA and in vitro DNA replication processes to mass-manufacture nanocomposite materials. The specific objectives of this project were to: (1) develop methods for replicating DNA strands that incorporate nucleotides with ''chemical handles'', and (2) demonstrate attachment of nanocrystal quantum dots (nQDs) to functionalized DNA strands. Polymerase chain reaction (PCR) and primer extension methodologies were used to successfully synthesize amine-, thiol-, and biotin-functionalized DNA molecules. Significant variability in the efficiency of modified nucleotide incorporation was observed, and attributed to the intrinsic properties of the modified nucleotides. Noncovalent attachment of streptavidin-coated nQDs to biotin-modified DNA synthesized using the primer extension method was observed by epifluorescence microscopy. Data regarding covalent attachment of nQDs to amine- and thiol-functionalized DNA was generally inconclusive; alternative characterization tools are necessary to fully evaluate these attachment methods. Full realization of this technology may facilitate new approaches to manufacturing materials at the nanoscale. In addition, composite nQD-DNA materials may serve as novel recognition elements in sensor devices, or be used as diagnostic tools for forensic analyses. This report summarizes the results obtained over the course of this 1-year project.
Arginine methylation regulates the p53 response
2008-01-01
Activation of the p53 tumour suppressor protein in response to DNA damage leads to apoptosis or cell-cycle arrest. Enzymatic modifications are widely believed to affect and regulate p53 activity. We describe here a level of post-translational control that has an important functional consequence on the p53 response. We show that the protein arginine methyltransferase (PRMT) 5, as a co-factor in a DNA damage responsive co-activator complex that interacts with p53, is responsible for methylating p53. Arginine methylation is regulated during the p53 response and affects the target gene specificity of p53. Furthermore, PRMT5 depletion triggers p53-dependent apoptosis. Thus, methylation on arginine residues is an underlying mechanism of control during the p53 response.
2009-01-01
Saffron is the red dried stigmas of Crocus sativus L. flowers and used both as a spice and as a drug in traditional medicine. Its numerous applications as an antioxidant and anticancer agent are due to its secondary metabolites and their derivatives (safranal, crocetin, dimethylcrocetin). In this work we are comparing the spectroscopic results and antioxidant activities of saffron components safranal, crocetin (CRT) and dimethylcrocetin (DMCRT) complexes with calf-thymus DNA (ctDNA) and transfer RNA (tRNA) in aqueous solution at physiological conditions Intercalative and external binding modes of saffron compounds to DNA and RNA were observed with overall binding constants of Ksafranal=1.24x103M-1, KCRT=6.20x103M-1 and KDMCRT=1.85x105M-1, for DNA adducts and Ksafranal=6.80x103M-1, KCRT=1.4...
An Overview of DNA and RNA Bindings to Antioxidant Flavonoids
2007-01-01
In this report we are examining how the antioxidant flavonoids can prevent DNA damage and what mechanism of action is involved in the process. Flavonoids are strong antioxidants that prevent DNA damage. The anticancer and antiviral activities of these natural products are implicated in their mechanism of actions. We study the interactions of quercetin (que), kaempferol (kae), and delphinidin (del) with DNA and transfer RNA in aqueous solution at physiological conditions, using constant DNA or RNA concentration 6.25Â mmol (phosphate) and various pigment/polynucleotide(phosphate) ratios of 1/65 to 1 (DNA) and 1/48 to 1/8 (tRNA). The structural analysis showed quercetin, kaempferol, and delphinidin intercalate DNA and RNA duplexes with minor external binding to the major or minor groove and t...
Full Text Available.BackgroundThe recD mutants of the Antarctic Pseudomonas syringae Lz4W are sensitive to DNA-damaging agents and fail to grow at 4°C. Generally, RecD associates with two other proteins (RecB and RecC) to produce RecBCD enzyme, which is involved in homologous recombination and DNA repair in many bacteria, including Escherichia coli. However, RecD is not essential for DNA repair, nor does its deletion cause any growth defects in E. coli. Hence, the assessment of the P. syringae RecBCD pathway was imperative.Methodology/Principal FindingsMutational analysis and genetic complementation studies were used to establish that the individual null-mutations of all three genes, recC, recB, and recD, or the deletion of whole recCBD operon of P. syringae, lead to growth inhibition at low temperature, and sensitivity to UV and mitomycin C. Viability of the mutant cells dropped drastically at 4°C, and the mutants accumulated linear chromosomal DNA and shorter DNA fragments in higher amounts compared to 22°C. Additional genetic data using the mutant RecBCD enzymes that were inactivated either in the ATPase active site of RecB (RecBK29Q) or RecD (RecDK229Q), or in the nuclease center of RecB (RecBD1118A and RecBΔnuc) suggested that, while the nuclease activity of RecB is not so critical in vivo, the ATP-dependent functions of both RecB and RecD are essential. Surprisingly, E. coli recBCD or recBC alone on plasmid could complement the defects of the ΔrecCBD strain of P. syringae.Conclusions/SignificanceAll three subunits of the RecBCDPs enzyme are essential for DNA repair and growth of P. syringae at low temperatures (4°C). The RecD requirement is only a function of the RecBCD complex in the bacterium. The RecBCD pathway protects the Antarctic bacterium from cold-induced DNA damages, and is critically dependent on the helicase activities of both RecB and RecD subunits, but not on the nuclease of RecBCDPs enzyme.
BackgroundThe recD mutants of the Antarctic Pseudomonas syringae Lz4W are sensitive to DNA-damaging agents and fail to grow at 4°C. Generally,...Full Text Available
ATM-Dependent Hyper-Radiosensitivity in Mammalian Cells Irradiated by Heavy Ions
2009-01-01
Purpose: Low-dose hyper-radiosensitivity (HRS) and the later appearing radioresistance (termed induced radioresistance [IRR]) was mainly studied in low linear energy transfer (LET) radiation with survival observation. The aim of this study was to find out whether equivalent hypersensitivity occurred in high LET radiation, and the roles of ataxia telangiectasia mutated (ATM) kinase. Methods and Materials: Survival and mutation were measured by clonogenic assay and HPRT mutation assay. ATM Ser1981 activation was detected by Western blotting and immunofluorescent staining. Pretreatment of specific ATM inhibitor (10 muM KU55933) and activator (20 mug/mL chloroquine) before carbon radiation were adopted to explore the involvement of ATM. The roles of ATM were also investigated in its G2/M checkpoint function with histone H3 ... >>
ALIS-FLP: Amplified ligation selected fragment-length polymorphism method for microbial genotyping
2008-01-01
A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs in that only one specific restriction enzyme (TspRI) is used. The cohesive ends of the DNA fragments are ligated with two types of oligonucleotide. A long oligonucleotide containing the primer site and the specific 9 nt 3 prime end, which is complementary to specific 9 nt, cohesive 3 prime end of the TspRI genomic DNA fragment, and a short, degenerated, oligonucleotide covering the remaining TspRI cohesive ends. Other cohesive ends are covered by a short degenerated oligonucleotide lacking the primer site. The ligation mixture is used as a template for amplification using a single primer corresponding to the 5 prime end of the long, specific oligonucleotide. The selection of TspRI digested genomic DNA fragments for amplification is achieved by sequence selective ligation of the specific long oligonucleotide carrying the primer site to both ends of the specific target fragment. This technique allows for differentiation of the organisms without previous knowledge of their DNA sequence. The usefulness of the method is confirmed by genotyping of 70 previously characterized clinical E. coli isolates. The grouping obtained was identical to the results of REA-PFGE. Versatility of the method is highlighted, i.e. its combining the advantages of the AFLP technique with a simple, rapid and cheap polymerase chain reaction product detection method.
A native chromatin purification system for epigenomic profiling in Caenorhabditis elegans
2010-03-01
High-resolution mapping of chromatin features has emerged as an important strategy for understanding gene regulation and epigenetic inheritance. We describe an in vivo tagging system...Full Text Available
A native chromatin purification system for epigenomic profiling in Caenorhabditis elegans
2010-03-01
Full Text Available.High-resolution mapping of chromatin features has emerged as an important strategy for understanding gene regulation and epigenetic inheritance. We describe an in vivo tagging system coupled to chromatin purification for genome-wide epigenetic profiling in Caenorhabditis elegans. In this system, we coexpressed the Escherichia coli biotin ligase enzyme (BirA), together with the C. elegans H3.3 gene fused to BioTag, a 23-amino-acid peptide serving as a biotinylation substrate for BirA, in vivo in worms. We found that the fusion BioTag::H3.3 was efficiently biotinylated in vivo. We developed methods to isolate chromatin under different salt extraction conditions, followed by affinity purification of biotinylated chromatin with streptavidin and genome-wide profiling with microarrays. We found that embryonic chromatin is differentially extracted with increasing salt concentrations. Interestingly, chromatin that remains insoluble after washing in 600 mM salt is enriched at 5′ and 3′ ends, suggesting the presence of large protein complexes that render chromatin insoluble at transcriptional initiation and termination sites. We also found that H3.3 landscapes from these salt fractions display consistent features that correlate with gene activity: the most highly expressed genes contain the most H3.3. This versatile two-component approach has the potential of facilitating genome-wide chromatin dynamics and regulatory site identification in C. elegans.
A RNA transcript (Heg) in mononuclear cells is negatively correlated with CD14 mRNA and TSH receptor autoantibodies
2008-01-01
During a study of gene expression of foxp3 in blood mononuclear cells we observed a DNA product of an unknown RNA fragment. The area of this peak correlated with CD14 mRNA in a small group of subjects. The sequence was localized to chromosome 1. We tested the hypothesis that gene expression of the poly A(-) transcript (designated Heg) in mononuclear cells was correlated with CD14 mRNA in normal subjects and with CD14 mRNA and TSH receptor autoantibodies in patients with acute and untreated Graves' disease. mRNA was expressed in amol/mu g DNA. The main study groups were: (i) normal subjects; (ii) patients with early and untreated Graves' disease; and (iii) patients with Graves' disease studied after treatment. In 18 normal subjects and in 20 patients with treated Graves' disease CD14 mRNA was negatively correlated with Heg (P
A Major Role for the Plasmodium falciparum ApiAP2 Protein PfSIP2 in Chromosome End Biology
2010-02-01
The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric...Full Text Available
A Major Role for the Plasmodium falciparum ApiAP2 Protein PfSIP2 in Chromosome End Biology
2010-02-01
Full Text Available.The heterochromatic environment and physical clustering of chromosome ends at the nuclear periphery provide a functional and structural framework for antigenic variation and evolution of subtelomeric virulence gene families in the malaria parasite Plasmodium falciparum. While recent studies assigned important roles for reversible histone modifications, silent information regulator 2 and heterochromatin protein 1 (PfHP1) in epigenetic control of variegated expression, factors involved in the recruitment and organization of subtelomeric heterochromatin remain unknown. Here, we describe the purification and characterization of PfSIP2, a member of the ApiAP2 family of putative transcription factors, as the unknown nuclear factor interacting specifically with cis-acting SPE2 motif arrays in subtelomeric domains. Interestingly, SPE2 is not bound by the full-length protein but rather by a 60kDa N-terminal domain, PfSIP2-N, which is released during schizogony. Our experimental re-definition of the SPE2/PfSIP2-N interaction highlights the strict requirement of both adjacent AP2 domains and a conserved bipartite SPE2 consensus motif for high-affinity binding. Genome-wide in silico mapping identified 777 putative binding sites, 94% of which cluster in heterochromatic domains upstream of subtelomeric var genes and in telomere-associated repeat elements. Immunofluorescence and chromatin immunoprecipitation (ChIP) assays revealed co-localization of PfSIP2-N with PfHP1 at chromosome ends. Genome-wide ChIP demonstrated the exclusive binding of PfSIP2-N to subtelomeric SPE2 landmarks in vivo but not to single chromosome-internal sites. Consistent with this specialized distribution pattern, PfSIP2-N over-expression has no effect on global gene transcription. Hence, contrary to the previously proposed role for this factor in gene activation, our results provide strong evidence for the first time for the involvement of an ApiAP2 factor in heterochromatin formation and genome integrity. These findings are highly relevant for our understanding of chromosome end biology and variegated expression in P. falciparum and other eukaryotes, and for the future analysis of the role of ApiAP2-DNA interactions in parasite biology.
A DNA nanoswitch incorporating the fluorescent base analogue 2-aminopurine detects single nucleotide mismatches in unlabelled targets
2009-01-01
A rationally designed tyrosine hydroxylase DNA vaccine induces specific antineuroblastoma immunity.
2008-01-01
Therapeutic vaccination against tumor antigens without induction of autoimmunity remains a major challenge in cancer immunotherapy. Here, we show for the first time effective therapeutic vaccination followed by suppression of established spontaneous neuroblastoma metastases using a tyrosine hydroxylase (TH) DNA minigene vaccine. We identified three novel mouse TH (mTH3) derived peptides with high predicted binding affinity to MHC class I antigen H2-K(k) according to the prediction program SYFPEITHI and computer modeling of epitopes into the MHC class I antigen binding groove. Subsequently, a DNA minigene vaccine was generated based on the expression vector pCMV-F3Ub encoding mutated ubiquitin (Gly(76) to Ala(76)) and mTH3. Prophylactic and therapeutic efficacies of this vaccine were established following oral delivery with attenuated Salmonella typhimurium SL7207. Only mice immunized with mTH3 were free of spontaneous liver metastases. This effect was clearly dependent on ubiquitin and high affinity of the mTH epitopes to MHC class I antigens. Specifically, we showed a crucial role for minigene expression as a stable ubiquitin-Ala(76) fusion peptide for vaccine efficacy. The immune response following the mTH3 DNA minigene vaccination was mediated by CD8(+) T cells as indicated by infiltration of primary tumors and TH-specific cytolytic activity in vitro. Importantly, no cell infiltration was detectable in TH-expressing adrenal medulla, indicating the absence of autoimmunity. In summary, we show effective therapeutic vaccination against neuroblastoma with a novel rationally designed TH minigene vaccine without induction of autoimmunity providing an important baseline for future clinical application of this strategy.
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