Articles de revues : « Ss(ds-)-DNA »

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Chandran, Harish, Nikhil Gopalkrishnan, Bernard Yurke et John Reif. « Meta-DNA : synthetic biology via DNA nanostructures and hybridization reactions ». Journal of The Royal Society Interface 9, n^o 72 (11 janvier 2012) : 1637–53. http://dx.doi.org/10.1098/rsif.2011.0819.

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Can a wide range of complex biochemical behaviour arise from repeated applications of a highly reduced class of interactions? In particular, can the range of DNA manipulations achieved by protein enzymes be simulated via simple DNA hybridization chemistry? In this work, we develop a biochemical system which we call meta-DNA (abbreviated as mDNA), based on strands of DNA as the only component molecules. Various enzymatic manipulations of these mDNA molecules are simulated via toehold-mediated DNA strand displacement reactions. We provide a formal model to describe the required properties and operations of our mDNA, and show that our proposed DNA nanostructures and hybridization reactions provide these properties and functionality. Our meta-nucleotides are designed to form flexible linear assemblies (single-stranded mDNA ( ss mDNA)) analogous to single-stranded DNA. We describe various isothermal hybridization reactions that manipulate our mDNA in powerful ways analogous to DNA–DNA reactions and the action of various enzymes on DNA. These operations on mDNA include (i) hybridization of ss mDNA into a double-stranded mDNA ( ds mDNA) and heat denaturation of a ds mDNA into its component ss mDNA, (ii) strand displacement of one ss mDNA by another, (iii) restriction cuts on the backbones of ss mDNA and ds mDNA, (iv) polymerization reactions that extend ss mDNA on a template to form a complete ds mDNA, (v) synthesis of mDNA sequences via mDNA polymerase chain reaction, (vi) isothermal denaturation of a ds mDNA into its component ss mDNA, and (vii) an isothermal replicator reaction that exponentially amplifies ss mDNA strands and may be modified to allow for mutations.

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Liu, H., M. I. Boulton, C. L. Thomas, D. A. M. Prior, K. J. Oparka et J. W. Davies. « Maize Streak Virus Coat Protein Is Karyophyllic and Facilitates Nuclear Transport of Viral DNA ». Molecular Plant-Microbe Interactions® 12, n^o 10 (octobre 1999) : 894–900. http://dx.doi.org/10.1094/mpmi.1999.12.10.894.

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Transport of maize streak virus (MSV) DNA into the nucleus of host cells is essential for virus replication and the presence of virus particles in the nuclei of infected cells implies that coat protein (CP) must enter the nucleus. To see if CP is imported into the nucleus in the absence of other viral gene products, the MSV CP gene was expressed in insect cells with a baculovirus vector system, and also in tobacco protoplasts with a cauliflower mosaic virus (CaMV) 35S promoter-driven transient gene expression vector. Immunofluorescent staining showed that the CP accumulated in the nuclei of both insect and tobacco cells. Mutagenesis of a potential nuclear localization signal in the CP resulted in cytoplasmic accumulation of the mutant protein. We have shown previously that the CP binds to single-stranded (ss) and double-stranded (ds) viral DNA. To investigate if CP might also be involved in viral DNA nuclear transport, Escherichia coli-expressed CP, together with TOTO-1-labeled viral ss or ds DNA, was microinjected into maize and tobacco epidermal cells. Both ss and ds DNA moved into the nucleus when co-injected with the CP but not with E. coli proteins alone. These results suggest that, in addition to entering the nucleus where it is required for encapsidation of the viral ss DNA, the MSV CP facilitates the rapid transport of viral (ss or ds) DNA into the nucleus.

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Bastos, M., V. Castro, G. Mrevlishvili et J. Teixeira. « Hydration of ds-DNA and ss-DNA by Neutron Quasielastic Scattering ». Biophysical Journal 86, n^o 6 (juin 2004) : 3822–27. http://dx.doi.org/10.1529/biophysj.104.039586.

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De, Pallabi, Mandy M. Peak et Karla K. Rodgers. « DNA Cleavage Activity of the V(D)J Recombination Protein RAG1 Is Autoregulated ». Molecular and Cellular Biology 24, n^o 15 (1 août 2004) : 6850–60. http://dx.doi.org/10.1128/mcb.24.15.6850-6860.2004.

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ABSTRACT RAG1 and RAG2 catalyze the first DNA cleavage steps in V(D)J recombination. We demonstrate that the isolated central domain of RAG1 has inherent single-stranded (ss) DNA cleavage activity, which does not require, but is enhanced by, RAG2. The central domain, therefore, contains the active-site residues necessary to perform hydrolysis of the DNA phosphodiester backbone. Furthermore, the catalytic activity of this domain on ss DNA is abolished by addition of the C-terminal domain of RAG1. The inhibitory effects of this latter domain are suppressed on substrates containing double-stranded (ds) DNA. Together, the activities of the reconstituted domains on ss versus mixed ds-ss DNA approximate the activity of intact RAG1 in the presence of RAG2. We propose how the combined actions of the RAG1 domains may function in V(D)J recombination and also in aberrant cleavage reactions that may lead to genomic instability in B and T lymphocytes.

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Erben, Antonija, Josipa Matić, Nikola Basarić et Ivo Piantanida. « The Phenanthridine-modified Tyrosine Dipeptide ». Croatica chemica acta 92, n^o 2 (2019) : 249–58. http://dx.doi.org/10.5562/cca3542.

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Dipeptide 4 containing two unnatural amino acids, a modified tyrosine and a phenanthridine derivative, was synthesized. Binding of the dipeptide to a series of polynucleotides including ct-DNA, poly A - poly U, poly (dAdT)2, poly dG - poly dC and poly (dGdC)2 was investigated by thermal denaturation experiments, fluorescence spectroscopy and circular dichroism. Thermal denaturation experiments indicated that dipeptide 4 at pH 5.0, when phenanthridine is protonated, stabilizes ds-DNA, whereas it destabilizes ds-RNA. At pH 7.0, when the phenanthridine is not protonated, effects of 4 to the polynucleotide melting temperatures are negligible. At pH 5.0, dipeptide 4 stabilized DNA double helices, and the changes in the CD spectra suggest different modes of binding to ds-DNA, most likely the intercalation to poly dG- poly dC and non-specific binding in grooves of other DNA polynucleotides. At variance to ds-DNA, addition of 4 destabilized ds-RNA against thermal denaturation and CD results suggest that addition of 4 probably induced dissociation of ds-RNA into ss-RNA strands due to preferred binding to ss-RNA. Thus, 4 is among very rare small molecules that stabilize ds-DNA but destabilize ds-RNA. However, fluorescence titrations with all polynucleotides at both pH values gave similar binding affinity (log Ka ≈ 5), indicating nonselective binding. Preliminary photochemical experiments suggest that dipeptide 4 reacts in the photochemical reaction, which affects polynucleotides chirality, presumably via quinone methide intermediates that alkylate DNA.

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Hauck, Bernd, Wei Zhao, Katherine High et Weidong Xiao. « Intracellular Viral Processing, Not Single-Stranded DNA Accumulation, Is Crucial for Recombinant Adeno-Associated Virus Transduction ». Journal of Virology 78, n^o 24 (15 décembre 2004) : 13678–86. http://dx.doi.org/10.1128/jvi.78.24.13678-13686.2004.

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ABSTRACT Adeno-associated virus (AAV) is a unique gene transfer vector which takes approximately 4 to 6 weeks to reach its expression plateau. The mechanism for this slow-rise expression profile was proposed to be inefficient second-strand DNA synthesis from the input single-stranded (ss) DNA viral genome. In order to clarify the status of ss AAV genomes, we generated AAV vectors labeled with bromodeoxyuridine (BrdU), a nucleotide analog that can be incorporated into the AAV genome and packaged into infectious virions. Since BrdU-DNA can be detected only by an anti-BrdU antibody when DNA is in an ss form, not in a double-stranded (ds) form, ss AAV genomes with BrdU can be readily tracked in situ. Although ss AAV DNA was abundant by Southern blot analysis, free ss AAV genomes were not detectable after AAV transduction by this new detection method. Further Southern blot analysis of viral DNA and virions revealed that ss AAV DNA was protected within virions. Extracted cellular fractions demonstrated that viral particles in host cells remained infectious. In addition, a significant amount of AAV genomes was degraded after AAV transduction. Therefore, we conclude that the amount of free ss DNA is not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV ss DNA release (i.e., uncoating) before second-strand DNA synthesis can occur. AAV ss DNA released from viral uncoating is either converted into ds DNA efficiently or degraded by cellular DNA repair mechanisms as damaged DNA. This study elucidates a mechanism that can be exploited to develop new strategies to improve AAV vector transduction efficiency.

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Clausen, J., et S. A. Nielsen. « The Use of In Vitro Cultured Lymphocytes for Tracing Mutagenic Activities of Chemicals ». Alternatives to Laboratory Animals 14, n^o 3 (mars 1987) : 168–71. http://dx.doi.org/10.1177/026119298701400314.

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Lymphocytes from normal, non-smoking human individuals not taking drugs were isolated from the peripheral blood by means of the lymphoprep method. The cells were cultured in RPMI medium with 10% fetal calf serum and stimulated with Phytohemagglutinin. A mutagen such as 3-methylcholanthrene was added for varying periods of time. Then the subspecies of DNA, i.e. double and single stranded DNA (ds-DNA and ss-DNA), were separated by the alkaline elution technique and quantitated by fluorimetric estimation. The mutagen induced a significant rise in the level of ss-DNA, but no changes in ds-DNA could be traced. The time-dependent changes increased for at least four days of exposure, indicating that the repair enzymes were not able to compensate for the DNA damage.

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Veligura, Alina, Michael Koehler, Wolfgang Fritzsche, Peter Lytvyn, Alexandr Gorchinskyy et Eugenia Buzaneva. « Uv induced ds(ss)-DNA damage : optical and electrical recognition ». BMC Plant Biology 5, Suppl 1 (2005) : S32. http://dx.doi.org/10.1186/1471-2229-5-s1-s32.

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Amundsen, S. K., A. M. Neiman, S. M. Thibodeaux et G. R. Smith. « Genetic dissection of the biochemical activities of RecBCD enzyme. » Genetics 126, n^o 1 (1 septembre 1990) : 25–40. http://dx.doi.org/10.1093/genetics/126.1.25.

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Abstract RecBCD enzyme of Escherichia coli is required for the major pathway of homologous recombination following conjugation. The enzyme has an ATP-dependent DNA unwinding activity, ATP-dependent single-stranded (ss) and double-stranded (ds) DNA exonuclease activities, and an activity that makes a ss DNA endonucleolytic cut near Chi sites. We have isolated and characterized ten mutations that reduced recombination proficiency and inactivated some, but not all, activities of RecBCD enzyme. One class of mutants had weak ds DNA exonuclease activity and lacked Chi-dependent DNA cleavage activity, a second class lacked only Chi-dependent DNA cleavage activity, and a third class retained all activities tested. The properties of these mutants indicate that the DNA unwinding and ss DNA exonuclease activities of the RecBCD enzyme are not sufficient for recombination. Furthermore, they suggest that the Chi-dependent DNA cleavage activity or another, as yet unidentified activity or both are required for recombination. The roles of the RecBCD enzymatic activities in recombination and exclusion of foreign DNA are discussed in light of the properties of these and other recBCD mutations.

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Kirisawa, Rikio, Rika Kato, Koichi Furusaki et Takashi Onodera. « Universal Virucidal Activity of Calcium Bicarbonate Mesoscopic Crystals That Provides an Effective and Biosafe Disinfectant ». Microorganisms 10, n^o 2 (24 janvier 2022) : 262. http://dx.doi.org/10.3390/microorganisms10020262.

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We investigated the virucidal effects in solution of a new type of disinfectant, calcium bicarbonate mesoscopic crystals, designated CAC-717, against various types of virus. CAC-717 in solution is alkaline (pH 12.4) and has a self-electromotive force that generates pulsed electrical fields. Upon application to human skin, the pH of the solution becomes 8.4. CAC-717 contains no harmful chemicals and is thus non-irritating and harmless to humans and animals. Its virucidal effects were tested against six types of animal virus: enveloped double-strand (ds)-DNA viruses, non-enveloped ds-DNA viruses, non-enveloped single strand (ss)-DNA viruses, enveloped ss-RNA viruses, non-enveloped ss-RNA viruses, and non-enveloped ds-RNA viruses. The treatment resulted in a reduction in viral titer of at least 3.00 log10 to 6.38 log10. Fetal bovine serum was added as a representative organic substance. When its concentration was ≥20%, the virucidal effect of CAC-717 was reduced. Real-time PCR revealed that CAC-717 did not reduce the quantity of genomic DNA of most of the DNA viruses, but it greatly reduced that of the genomic RNA of most of the RNA viruses. CAC-717 may therefore be a useful biosafe disinfectant for use against a broad range of viruses.

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Chaudhury, AM, ES Dennis et RIS Brettell. « Gene-Expression Following T-DNA Transfer Into Plant Cells Is Aphidicolin-Sensitive ». Functional Plant Biology 21, n^o 2 (1994) : 125. http://dx.doi.org/10.1071/pp9940125.

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A transient assay for gene-expression was used to study the early events of T-DNA transfer. Particularly, it was asked if gene expression following T-DNA transfer required DNA replication in the host cell. A β-glucuronidase gene, linked to a CaMV 35S promoter (35S-GUS, engineered so that it was inactive in Agrobacterium tumefaciens) was introduced into Nicotiana plumbaginifolia protoplasts via a disarmed supervirulent strain of Agrobacterium tumefaciens. High β-glucuronidase activity appeared after 3 days of co-cultivation. The activity required the presence of the vir functions of agrobacteria. The activity was drastically reduced if the plant cells were treated with aphidicolin, an inhibitor of DNA replication in eukaryotic cells. While double-stranded (ds) 35S-GUS DNA, introduced by electroporation, showed undiminished expression in the presence of aphidicolin, gene expression from single-stranded (ss) 35S-GUS DNA was inhibited by aphidicolin. These results suggest that DNA replication in host cells is not required for gene expression if ds-DNA is introduced by electroporation, but is required if ss-DNA is introduced by electroporation, or if DNA is transferred via A. tumefaciens. The findings are consistent with a model of T-DNA transfer in which ss-DNA molecules, once introduced into plant cells, must pass through an aphidicolin sensitive step before they can be transcribed. The simplest interpretation is that the ss-DNA is replicated by the host cell's aphidicolin-sensitive DNA polymerase before being integrated into the host genome.

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Stojković, Marijana Radić, Marko Škugor, Łukasz Dudek, Jarosław Grolik, Julita Eilmes et Ivo Piantanida. « Molecular recognition of AT-DNA sequences by the induced CD pattern of dibenzotetraaza[14]annulene (DBTAA)–adenine derivatives ». Beilstein Journal of Organic Chemistry 10 (12 septembre 2014) : 2175–85. http://dx.doi.org/10.3762/bjoc.10.225.

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An investigation of the interactions of two novel and several known DBTAA–adenine conjugates with double-stranded DNA and RNA has revealed the DNA/RNA groove as the dominant binding site, which is in contrast to the majority of previously studied DBTAA analogues (DNA/RNA intercalators). Only DBTAA–propyladenine conjugates revealed the molecular recognition of AT-DNA by an ICD band pattern > 300 nm, whereas significant ICD bands did not appear for other ds-DNA/RNA. A structure–activity relation for the studied series of compounds showed that the essential structural features for the ICD recognition are a) the presence of DNA-binding appendages (adenine side chain and positively charged side chain) on both DBTAA side chains, and b) the presence of a short propyl linker, which does not support intramolecular aromatic stacking between DBTAA and adenine. The observed AT-DNA-ICD pattern differs from previously reported ss-DNA (poly dT) ICD recognition by a strong negative ICD band at 350 nm, which allows for the dynamic differentiation between ss-DNA (poly dT) and coupled ds-AT-DNA.

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Koa, Helena, Murray J. Fraser et Etta Käfer. « Endo-exonuclease of Aspergillus nidulans ». Biochemistry and Cell Biology 68, n^o 1 (1 janvier 1990) : 387–92. http://dx.doi.org/10.1139/o90-054.

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Endo-exonuclease (EE) has been found in both active and inactive, but trypsin-activatable, forms in Aspergillus nidulans. Active EE was present mainly in nuclei, mitochondria, and vacuoles, while trypsin-activatable EE was mainly in the cytosol. The active form accounts for over 90% of the neutral deoxyribonuclease activity extracted from mycelia. A single strand (ss) DNA-binding EE associated with a 28 kilodalton (kDa) polypeptide was partially purified and characterized. It was found to closely resemble, in size and enzymological properties, the ss-DNA-binding EE previously purified from Neurospora crassa. Aspergillus nidulans EE was also found to be immunochemically related to the N. crassa EE and, like that enzyme, was probably derived from a polypeptide of 90 kDa or larger through proteolysis during extraction and purification. It had divalent metal ion-dependent (Mg2+, Mn2+, or Zn2+) activity on both DNA and RNA, which ultimately produced small 5′-P-terminated oligonucleotides. The nuclease activity was mixed endo- and exo-nucleolytic with ss-DNA as substrate, but largely exonucleolytic with double strand (ds) DNA. Superhelical ΦX-174 DNA was nicked by EE to form relaxed circular and then linear ds-DNA, which was rapidly degraded to shorter fragments. Linearized pBR322 DNA was extensively nicked internally under conditions where there was relatively low exonuclease activity, but this nicking required that 5′-P-termini be present on the linear ds-DNA. The levels of active EE found in extracts of two recombination-deficient mutants of A. nidulans, uvsC and uvsE, dit not differ significantly from those in extracts of the wild type.Key words: endo-exonuclease, Aspergillus nidulans, purification, properties, inactive form.

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Grueso, Elia, Rosa M. Giráldez-Pérez, Pilar Perez-Tejeda, Emilio Roldán et R. Prado-Gotor. « What controls the unusual melting profiles of small AuNPs/DNA complexes ». Physical Chemistry Chemical Physics 21, n^o 21 (2019) : 11019–32. http://dx.doi.org/10.1039/c9cp01162e.

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The effect of the addition of low salt concentrations on ds-DNA and ss-DNA conformational changes induced by small N-(2-mercaptopropionyl)glycine gold nanoparticles (AuNPs) is studied in detail by using different techniques. The results are correlated with the unusual melting profiles of the AuNPs/DNA complexes.

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Itriago, Humberto, Rishi K. Jaiswal, Susanne Philipp et Marita Cohn. « The telomeric 5′ end nucleotide is regulated in the budding yeast Naumovozyma castellii ». Nucleic Acids Research 50, n^o 1 (15 décembre 2021) : 281–92. http://dx.doi.org/10.1093/nar/gkab1229.

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Abstract The junction between the double-stranded and single-stranded telomeric DNA (ds–ss junction) is fundamental in the maintenance of the telomeric chromatin, as it directs the assembly of the telomere binding proteins. In budding yeast, multiple Rap1 proteins bind the telomeric dsDNA, while ssDNA repeats are bound by the Cdc13 protein. Here, we aimed to determine, for the first time, the telomeric 5′ end nucleotide in a budding yeast. To this end, we developed a permutation-specific PCR-based method directed towards the regular 8-mer telomeric repeats in Naumovozyma castellii. We find that, in logarithmically growing cells, the 320 ± 30 bp long telomeres mainly terminate in either of two specific 5′ end permutations of the repeat, both corresponding to a terminal adenine nucleotide. Strikingly, two permutations are completely absent at the 5′ end, indicating that not all ds-ss junction structures would allow the establishment of the protective telomere chromatin cap structure. Using in vitro DNA end protection assays, we determined that binding of Rap1 and Cdc13 around the most abundant ds–ss junction ensures the protection of both 5′ ends and 3′ overhangs from exonucleolytic degradation. Our results provide mechanistic insights into telomere protection, and reveal that Rap1 and Cdc13 have complementary roles.

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STAINS, Joseph P., Fernando LECANDA, Dwight A. TOWLER et Roberto CIVITELLI. « Heterogeneous nuclear ribonucleoprotein K represses transcription from a cytosine/thymidine-rich element in the osteocalcin promoter ». Biochemical Journal 385, n^o 2 (7 janvier 2005) : 613–23. http://dx.doi.org/10.1042/bj20040680.

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HnRNP K (heterogeneous nuclear ribonucleoprotein K) was biochemically purified from a screen of proteins co-purifying with binding activity to the osteocalcin promoter. We identify hnRNP K as a novel repressor of osteocalcin gene transcription. Overexpression of hnRNP K lowers the expression of osteocalcin mRNA by 5-fold. Furthermore, luciferase reporter assays demonstrate that overexpression of hnRNP K represses osteocalcin transcription from a CT (cytosine/thymidine)-rich element in the proximal promoter. Electrophoretic mobility-shift analysis reveals that recombinant hnRNP K binds to the CT-rich element, but binds ss (single-stranded), rather than ds (double-stranded) oligonucleotide probes. Accordingly, hnRNP K antibody can supershift a binding activity present in nuclear extracts using ss sense, but not antisense or ds oligonucleotides corresponding to the CT-rich −95 to −47 osteocalcin promoter. Importantly, addition of recombinant hnRNP K to ROS 17/2.8 nuclear extract disrupts formation of a DNA–protein complex on ds CT element oligonucleotides. This action is mutually exclusive with hnRNP K's ability to bind ss DNA. These results demonstrate that hnRNPK, although co-purified with a dsDNA-binding activity, does not itself bind dsDNA. Rather, hnRNP K represses osteocalcin gene transcription by inhibiting the formation of a transcriptional complex on the CT element of the osteocalcin promoter.

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Nakai, Hiroyuki, Theresa A. Storm et Mark A. Kay. « Recruitment of Single-Stranded Recombinant Adeno-Associated Virus Vector Genomes and Intermolecular Recombination Are Responsible for Stable Transduction of Liver In Vivo ». Journal of Virology 74, n^o 20 (15 octobre 2000) : 9451–63. http://dx.doi.org/10.1128/jvi.74.20.9451-9463.2000.

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ABSTRACT Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Following portal-vein administration of rAAV vectors in vivo, single-stranded (ss) rAAV genomes become double stranded (ds), circularized, and/or concatemerized concomitant with a slow rise and, eventually, steady-state levels of transgene expression. Over time, at least some of the stabilized genomes become integrated into mouse chromosomal DNA. The mechanism(s) of formation of stable ds rAAV genomes from input ss DNA molecules has not been delineated, although second-strand synthesis and genome amplification by a rolling-circle model has been proposed. To begin to delineate a mechanism, we produced rAAV vectors in the presence of bacterial PaeR7 or Dam methyltransferase or constructed rAAV vectors labeled with different restriction enzyme recognition sites and introduced them into mouse hepatocytes in vivo. A series of molecular analyses demonstrated that second-strand synthesis and rolling-circle replication did not appear to be the major processes involved in the formation of stable ds rAAV genomes. Rather, recruitment of complementary plus and minus ss genomes and subsequent random head-to-head, head-to-tail, and tail-to-tail intermolecular joining were primarily responsible for the formation of ds vector genomes. These findings contrast with the previously described mechanism(s) of transduction based on in vitro studies. Understanding the mechanistic process responsible for vector transduction may allow the development of new strategies for improving rAAV-mediated gene transfer in vivo.

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Boulter, Nicky, Gregor Tevz, Betty Yu, Michelle Chan, David Murray, Erin L. Symonds, Graeme P. Young et Susanne Kartin Pedersen. « An improved method for detection of methylated circulating tumor DNA in colorectal cancer. » Journal of Clinical Oncology 37, n^o 15_suppl (20 mai 2019) : e15120-e15120. http://dx.doi.org/10.1200/jco.2019.37.15_suppl.e15120.

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e15120 Background: Assays detecting methylated circulating tumor DNA (ctDNA) hold promise for colorectal cancer (CRC) screening, but there is a need to develop more accurate assays for early stages. In this study, we examined modifications to COLVERA (an IKZF1 and BCAT1 methylation marker qPCR assay) that added a third biomarker (IRF4) as well as introducing double stranded detection. Methods: Bisulfite converted DNA extracted from plasma collected from subjects undergoing diagnosis for CRC was assayed using either a double-stranded (ds) specific multiplexed qPCR for BCAT1 and IKZF1 detection (Assay 1) and/or a single-stranded (ss) specific BCAT1/IRF4, ds-specific IKZF1 multiplexed qPCR (Assay 2). The unmodified COLVERA test (Clinical Genomics) was used for comparison. Detection of any marker was considered positive. Results: Assay 1 performance evaluated against COLVERA on bisulfite converted DNA from 1453 samples showed an improved sensitivity for CRC detection (93/134 (69.4%) vs 79/134 (59.0%) COLVERA, p = 0.02), but specificity was suboptimal (no neoplasia: 861/1023 (84.2%) vs 946/1023 (92.5%) COLVERA, p < 0.0001). The decrease in specificity resulted primarily from modifications in the BCAT1 assay component (1023 no neoplasia: 60 positive with ss- BCAT1 vs 146 positive with ds- BCAT1, p < 0.0001). Thus, a novel configuration, Assay 2 (ss- BCAT1, ss- IRF4 and ds- IKZF1) was compared to Assay 1 using 189 previously untested specimens. Assay 2 showed a notable improvement in specificity (99/104 (95.2%) vs 93/104 (89.4%) Assay 1; p = 0.0351). Both assays exhibited comparable sensitivities for CRC (41/49 (83.7%) vs 39/49 (79.6%), Assay 1, p = 0.3633). Assay 2 was positive in: adenomas, 9/36 (25.0%); Stage I, 3/7 (42.9%); Stage II, 17/19 (89.5%); Stage III, 6/6 (100%); Stage IV, 7/7 (100%) and 8/10 (80%) unstaged CRCs. Prior COLVERA sensitivity estimates were 9% adenoma, 41% Stage I and 76% Stage II. Conclusions: Targeting both of the non-complementary bisulfite converted strands of selective regions of interest markedly improve ctDNA assay sensitivity for cancer including early lesions while achieving good specificity. Prospective evaluation in a true CRC screening population is now underway. Clinical trial information: 12611000318987.

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Xiao, Gefei, Yanling Zhao, Wuyan Huang, Liqing Hu, Guoqing Wang et Huayu Luo. « Health economic evaluation of noninvasive prenatal testing and serum screening for down syndrome ». PLOS ONE 17, n^o 4 (14 avril 2022) : e0266718. http://dx.doi.org/10.1371/journal.pone.0266718.

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Background Down syndrome (DS), also known as trisomy 21 (T21), is the most common genetic disorder associated with intellectual disability. There are two methods commonly used for prenatal testing of DS: serum screening (SS) for biomarkers in maternal serum and noninvasive prenatal testing (NIPT) for aneuploidy by cell-free DNA (cfDNA) in maternal plasma. However, cost-effectiveness analyses of these two methods are mostly based on data derived from simulations with various models, with theoretical values calculated. In this study, we statistically analyzed clinical DS screening data and pregnancy outcomes during the follow-up of pregnant women in Zhuhai City, China. The economics of the two mainstream prenatal DS screening methods was evaluated from a public health perspective. Methods A retrospective analysis was performed on the data of 17,363 pregnant women who received SS and NIPT during gestation in Zhuhai from 2018 to 2019, and a cost-effectiveness analysis was performed with four screening strategies. In strategy I, all pregnant women received SS, and those with T21 risk ≥1/270 had invasive prenatal diagnosis (IPD). In strategy II, all pregnant women received SS, those with T21 risk ≥ 1/270 had IPD, and those with 1/270 > T21 risk ≥ 1/1,000 had NIPT; then, women at high risk based on NIPT also had IPD. In strategy III, all pregnant women received SS, and those with T21 risk ≥1,000 had NIPT; then, women at high risk based on NIPT results had IPD. In strategy IV, all pregnant women received NIPT and those at high risk based on NIPT results had IPD. Finally, to assess the cost and effectiveness of DS screening, the total costs were calculated as the sum of screening and diagnosis as well as the direct and indirect economic burden during the average life cycle of DS patients. Results A total of 22 of the 17,363 (1/789) pregnant women had DS, of which only one woman was over 35 years of age. SS detected 1,024 cases at high risk of T21 (≥1/270), 8 cases were true positive, with a positive predictive value of 0.78% and a detection rate of 36.4%. NIPT detected 27 cases at high risk of T21 (Z ≥ 3) and 22 cases of DS, with a positive predictive value of 81.5% and a detection rate of 100%. Strategy I had the largest total cost of 65.54 million CNY, strategy II and III had similar total costs of 40 million CNY, and strategy IV had the lowest total cost of 14.91 million CNY. By comparison, the screening strategy with NIPT alone had the highest health economic value for DS. Conclusions SS was greatly affected by nuchal translucency and the accuracy of gestational age measured by ultrasonography. Unstandardized ultrasonography was an important reason for the low DS detection rate with SS. The influence of interfering factors on NIPT was much lower than in SS. NIPT can be used as an alternative to SS and as a primary screening strategy of prenatal DS screening for secondary prevention and control of birth defects. NIPT greatly decreased the frequency of IPD and the miscarriages associated with IPD, saved the limited medical and health resources, and greatly increased DS detection rate. Therefore, NIPT has great social and economic benefits.

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Yang, Bo, Xiao-Dan Zhang, Jian Li, Jia Tian, Yi-Peng Wu, Fa-Xing Yu, Ruibing Wang et al. « In Situ Loading and Delivery of Short Single- and Double-Stranded DNA by Supramolecular Organic Frameworks ». CCS Chemistry 1, n^o 2 (juin 2019) : 156–65. http://dx.doi.org/10.31635/ccschem.019.20180011.

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Short DNA represents an important class of biomacromolecules that are widely applied in gene therapy, editing, and modulation. However, the development of simple and reliable methods for their intracellular delivery remains a challenge. Herein, we describe that seven water-soluble, homogeneous supramolecular organic frameworks (SOFs) with a well-defined pore size and high stability in water that can accomplish in situ inclusion of single-stranded (ss) and double-stranded (ds) DNA (21, 23, and 58 nt) and effective intracellular delivery (including two noncancerous and six cancerous cell lines). Fluorescence quenching experiments for single and double end-labeled ss- and ds-DNA support that the DNA sequences can be completely enveloped by the SOFs. Confocal laser scanning microscopy and flow cytometry reveal that five of the SOFs exhibit excellent delivery efficiencies that, in most of the studied cases, outperform the commercial standard Lipo2000, even at low SOF–nucleic acid ratios. In addition to high delivery efficiencies, the water-soluble, self-assembled SOF carriers have a variety of advantages, including convenient preparation, high stability, and in situ DNA inclusion, which are all critical for practical applications in nucleic acid delivery.

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Schonenberg-Meinema, D., S. Bergkamp, A. Nassar-Sheikh Rashid, M. Gruppen, A. E. Hak, P. C. E. Hissink Muller, M. Middelkamp et al. « POS1311 SCLERODERMA PATTERN IN NAILFOLD CAPILLARIES OF (CHILDHOOD-ONSET) SYSTEMIC LUPUS ERYTHEMATOSUS : LESSONS FROM LONGITUDINAL FOLLOW-UP ». Annals of the Rheumatic Diseases 80, Suppl 1 (19 mai 2021) : 937.2–938. http://dx.doi.org/10.1136/annrheumdis-2021-eular.2195.

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Background:It has been suggested that a capillary scleroderma pattern in patients with systemic lupus erythematosus (SLE)-patients predisposes for clinical signs of systemic sclerosis (SSc) or overlap disease (1). However, this was previously shown not to be the case in a cross-sectional study in childhood-onset SLE (cSLE) (2).Objectives:To assess if nailfold capillary patterns in cSLE change over time and if a capillary scleroderma pattern is associated with prospective development of clinical SSc-features, higher SLE disease activity or –damage.Methods:Prospective clinical and capillaroscopy data were used from a longitudinal cohort of cSLE patients. Patients with a disease onset before the age of 18 years and diagnosis according to SLICC 2012 criteria were included. Disease activity was defined by SLEDAI score and disease damage by the SLICC damage index. Nailfold images from eight fingers (excluding thumbs) were obtained by videocapillaroscopy. A scleroderma pattern was defined according to the ‘fast track algorithm’ (3). An abnormal capillary pattern that did not match the criteria for a scleroderma pattern was defined as ‘microangiopathy’.Results:Our longitudinal study cohort consisted of n=53 cSLE patients with a median disease onset of 14 years (IQR 12.5-15.5 years) and a median SLEDAI score at diagnosis of 11 (IQR 8-15.5). Clinical follow-up data were available from 0.5-16 years after disease onset. Median disease duration at first capillaroscopy was 17 months (IQR 5.5-51.5 months) and 17/53 (32.1%) of patients had Raynaud’s phenomenon. In total, n=9 (17%) showed a scleroderma pattern (at some point in time), n=37 (70%) had microangiopathy and n=7 (13%) showed a normal capillary pattern. N=27 patients had follow up with capillaroscopy (1-7 times over 1-5 years). In most patients (23/27) we did not observe any change in capillary pattern during follow-up. Two patients showed changes from microangiopathy to a scleroderma pattern, one patient from a scleroderma pattern to microangiopathy and one patient from microangiopathy to a normal pattern. Raynaud’s phenomenon was equally distributed among patients with different capillaroscopy patterns (p=0.487), as was median SLEDAI score at diagnosis (p=0.285). Follow-up patients with a capillary scleroderma pattern did not show any clinical features for SSc over time (follow-up over 1-5 years, Table 1 below. Patients with a capillary scleroderma pattern showed significantly more disease damage (Chi-square, p=0.008), also indicated by a survival analysis for disease damage (Figure 1, p=0.042).Conclusion:Our study indicates that a capillary scleroderma pattern in cSLE correlates with disease damage but not with clinical SSc features during follow-up period.References:[1]S. Pavlov-Dolijanovic et al. Is there a difference in systemic lupus erythematosus with and without Raynaud’s phenomenon? Rheum Int 2013;33: 859–865[2]D. Schonenberg-Meinema et al. Nailfold capillary abnormalities in childhood-onset systemic lupus erythematosus. Revised manuscript after review re-submitted to Lupus in January 2021[3]V. Smith et al. Fast track algorithm. Autoimm Rev 2019 Nov;18(11): 102394Table 1.Clinical characteristics of cSLE-patients (n=9/53) with a nailfold capillary scleroderma patternButterfly rashPhotosensitive rashLupus nephritisAutoimmune cytopeniaPositive CoombsLow C3/C4SerositisDamageType of auto-antibodiesFollow-up in years1++-+++-+ANA, Anti-ds-DNA, anti-Sm, anti-RNP, anti-Ro52, anti-SS-A52++-+++-+ANA, Anti-ds-DNA, anti-Sm, anti-RNP, anti-SS-A53-+++++++ANA, Anti-ds-DNA, anti-Sm, anti-RNP, anti-SS-A104-++-++++ANA, Anti-ds-DNA, anti-Sm, anti-RNP, anti-Ro52, anti-SS-A95-+--+-+-ANA1 (lost)6-++++++-ANA, Anti-ds-DNA, anti-c1q37---+----ANA, Anti-ds-DNA, anti-Sm, anti-RNP18++-+----ANA99-+++++++ANA, Anti-ds-DNA, anti-RNP, anti-SS-A8Figure 1.Occurrence of disease damage (with survival as ‘no damage’) in cSLE patients with microangiopathy (blue) and a capillary scleroderma pattern (red)Disclosure of Interests:None declared

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Fraccari, Raquel L., Marco Carminati, Giacomo Piantanida, Tina Leontidou, Giorgio Ferrari et Tim Albrecht. « High-bandwidth detection of short DNA in nanopipettes ». Faraday Discussions 193 (2016) : 459–70. http://dx.doi.org/10.1039/c6fd00109b.

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Glass or quartz nanopipettes have found increasing use as tools for studying the biophysical properties of DNA and proteins, and as sensor devices. The ease of fabrication, favourable wetting properties and low capacitance are some of the inherent advantages, for example compared to more conventional, silicon-based nanopore chips. Recently, we have demonstrated high-bandwidth detection of double-stranded (ds) DNA with microsecond time resolution in nanopipettes, using custom-designed electronics. The electronics design has now been refined to include more sophisticated control features, such as integrated bias reversal and other features. Here, we exploit these capabilities and probe the translocation of short dsDNA in the 100 bp range, in different electrolytes. Single-stranded (ss) DNA of similar length are in use as capture probes, so label-free detection of their ds counterparts could therefore be of relevance in disease diagnostics.

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Nishihara, Tadashi, Fumikiyo Nagawa, Hirofumi Nishizumi, Masami Kodama, Satoshi Hirose, Reiko Hayashi et Hitoshi Sakano. « In Vitro Processing of the 3′-Overhanging DNA in the Postcleavage Complex Involved in V(D)J Joining ». Molecular and Cellular Biology 24, n^o 9 (1 mai 2004) : 3692–702. http://dx.doi.org/10.1128/mcb.24.9.3692-3702.2004.

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ABSTRACT The postcleavage complex involved in V(D)J joining is known to possess a transpositional strand transfer activity, whose physiological role is yet to be clarified. Here we report that RAG1 and RAG2 proteins in the signal end (SE) complex cleave the 3′-overhanging structure of the synthetic coding-end (CE) DNA in two successive steps in vitro. The 3′-overhanging structure is attacked by the SE complex imprecisely, near the double-stranded/single-stranded (ds/ss) junction, and transferred to the SE. The transferred overhang is then resolved and cleaved precisely at the ds/ss junction, generating either the linear or the circular cleavage products. Thus, the blunt-end structure is restored for the SE and variably processed ends are generated for the synthetic CE. This 3′-processing activity is observed not only with the core RAG2 but also with the full-length protein.

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Mavlyanova, Sh Z., M. S. Mutavaliev, A. I. Ismogilov, J. B. Mullakhanov et Yu A. Alimukhamedova. « ASSESSMENT OF IgG AUTOANTIBODIES TO SINGLE-STRANDED DNA (ss-DNA) AND DOUBLE-STRANDED DNA (ds-DNA) IN PATIENTS WITH ACANTHOLYTIC PEMPHIGUS ». Juvenis Scientia 6, n^o 4 (2020) : 57–62. http://dx.doi.org/10.32415/jscientia_2020_6_4_57-62.

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OU-YANG, ZHONG-CAN. « THE ELASTIC THEORY OF SINGLE-MOLECULE DNA ». International Journal of Modern Physics B 17, n^o 01n02 (20 janvier 2003) : 69–75. http://dx.doi.org/10.1142/s0217979203017102.

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Our recent work on the elastic responses of double- (ds) and single-stranded (ss) DNA at external force fields is reviewed. By constructing as elastic model of dsDNA in which the base-pair stacking interaction is included, we demonstrate that dsDNA entropic elasticity, cooperative extensibility, and supercoiling property can all be understood from a unified viewpoint. The base-pair stacking interaction is also found to determine the cooperativity of the stretch-induced hairpin-coil transition is ssDNA.

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Chen, Youqiang, et Gaoquan Shi. « Fluorescence Detection and Discrimination of ss- and ds-DNA with a Water Soluble Oligopyrene Derivative ». Sensors 9, n^o 6 (2 juin 2009) : 4164–77. http://dx.doi.org/10.3390/s90604164.

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Edberg, J. C., G. A. Kujala et R. P. Taylor. « Clearance kinetics and immunochemistry in rabbits of soluble antibody/DNA immune complexes. Effects of antibody class and DNA conformation. » Journal of Immunology 139, n^o 1 (1 juillet 1987) : 180–87. http://dx.doi.org/10.4049/jimmunol.139.1.180.

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Abstract We examined the clearance kinetics in rabbits of soluble antibody/DNA immune complexes (IC) containing either IgG or IgM anti-DNA antibodies. Differences in the complement-mediated binding of these IC to rabbit blood cells (platelets) were also studied. Complexation of either double-stranded (ds) or single-stranded (ss) DNA with IgG anti-DNA tends to preclude in vivo DNA recognition mechanisms; the DNA is cleared as part of an IC at a rate slower than that of free DNA. Binding of ds- or ssDNA by IgM anti-DNA antibodies leads to formation of IC which are cleared more like free DNA, and this effect is most evident for ssDNA. However, although both IgG- and IgM-containing IC bound rapidly to blood cells in vivo, significant differences in their immunochemistry were apparent. For example, the DNA in IgM-containing IC was more susceptible to both in vivo and in vitro degradation. In addition, the binding of IgM-containing IC to rabbit platelets and human red blood cells was considerably more labile. Based on this systematic investigation of the soluble antibody/DNA IC that can potentially form in the circulation of a patient with systemic lupus erythematosus, it should be possible to formulate predictions regarding the relative pathogenic potential of these IC.

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Karwowski. « The Influence of (5′R)- and (5′S)-5′,8-Cyclo-2′-Deoxyadenosine on UDG and hAPE1 Activity. Tandem Lesions are the Base Excision Repair System’s Nightmare ». Cells 8, n^o 11 (23 octobre 2019) : 1303. http://dx.doi.org/10.3390/cells8111303.

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DNA lesions are formed continuously in each living cell as a result of environmental factors, ionisation radiation, metabolic processes, etc. Most lesions are removed from the genome by the base excision repair system (BER). The activation of the BER protein cascade starts with DNA damage recognition by glycosylases. Uracil-DNA glycosylase (UDG) is one of the most evolutionary preserved glycosylases which remove the frequently occurring 2′-deoxyuridine from single (ss) and double-stranded (ds) oligonucleotides. Conversely, the unique tandem lesions (5′R)- and (5′S)-5′,8-cyclo-2′-deoxyadenosine (cdA) are not suitable substrates for BER machinery and are released from the genome by the nucleotide excision repair (NER) system. However, the cyclopurines appearing in a clustered DNA damage structure can influence the BER process of other lesions like dU. In this article, UDG inhibition by 5′S- and 5′R-cdA is shown and discussed in an experimental and theoretical manner. This phenomenon was observed when a tandem lesion appears in single or double-stranded oligonucleotides next to dU, on its 3′-end side. The cdA shift to the 5′-end side of dU in ss-DNA stops this effect in both cdA diastereomers. Surprisingly, in the case of ds-DNA, 5′S-cdA completely blocks uracil excision by UDG. Conversely, 5′R-cdA allows glycosylase for uracil removal, but the subsequently formed apurinic/apyrimidinic (AP) site is not suitable for human AP-site endonuclease 1 (hAPE1) activity. In conclusion, the appearance of the discussed tandem lesion in the structure of single or double-stranded DNA can stop the entire base repair process at its beginning, which due to UDG and hAPE1 inhibition can lead to mutagenesis. On the other hand, the presented results can cast some light on the UDG or hAPE1 inhibitors being used as a potential treatment.

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Pavlovic, Mirjana, Anna Kats, Michelle Cavallo, Ran Chen, James X. Hartmann et Yehuda Shoenfeld. « Pathogenic and Epiphenomenal Anti-DNA Antibodies in SLE ». Autoimmune Diseases 2010 (2010) : 1–18. http://dx.doi.org/10.4061/2010/462841.

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The discoveries of natural and the development of manufactured highly efficient catalytic antibodies (abzymes) opens the door to many practical applications. One of the most fascinating is the use of such antibodies in human therapy and prevention (vaccination), of cancer, AIDS, autoimmune diseases. A special entity of naturally occurring DNA hydrolytic anti-DNA antibodies is emerging within past decades linked to autoimmune and lymphoproliferative disorders, such as systemic lupus erythematosus (SLE), multiple sclerosis (MS), Sjogren Syndrome (SS), B - Chronic lymphocytic leucosis (B-CLL), and Multiple Myeloma (MM). The origin of the antibodies is unknown. The underlying mechanisms of these activities are suggested to be penetration into the living cells and translocation in the nucleus, with recognition of the specific binding sites at particular (ss or ds) DNA. There are controversies in the literature whether hydrolysis is a sequence-specific event. The interplay between anti-DNA antibodies and DNA is not yet elucidated. This molecular “twist” also suggests that anti-DNA antibodies with DNA hydrolytic capacity could be the organism's immune response to a microbial attack, with microbial DNA, or specific genes within microbial DNA sequence, as a target for neutralization. The catalytic antibody-based approach can become a key tool in selective chemotherapeutic strategies.

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Zhou, Xiaohuai, Irene Zolotukhin, Dong-Soo Im et Nicholas Muzyczka. « Biochemical Characterization of Adeno-Associated Virus Rep68 DNA Helicase and ATPase Activities ». Journal of Virology 73, n^o 2 (1 février 1999) : 1580–90. http://dx.doi.org/10.1128/jvi.73.2.1580-1590.1999.

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ABSTRACT The adeno-associated virus (AAV) nonstructural proteins Rep68 and Rep78 are site-specific DNA binding proteins, ATP-dependent site-specific endonucleases, helicases, and ATPases. These biochemical activities are required for viral DNA replication and control of viral gene expression. In this study, we characterized the biochemical properties of the helicase and ATPase activities of homogeneously pure Rep68. The enzyme exists as a monomer in solution at the concentrations used in this study (<380 nM), as judged by its mobility in sucrose density gradients. Using a primed single-stranded (ss) circular M13 substrate, the helicase activity had an optimum pH of 7 to 7.5, an optimum temperature of 45°C, and an optimal divalent-cation concentration of 5 mM MgCl2. Several nucleoside triphosphates could serve as cofactors for Rep68 helicase activity, and the order of preference was ATP = GTP > CTP = dATP > UTP > dGTP. The Km values for ATP in both the DNA helicase reaction and the site-specific trsendonuclease reaction were essentially the same, approximately 180 μM. Both reactions were sigmoidal with respect to ATP concentration, suggesting that a dimer or higher-order multimer of Rep68 is necessary for both DNA helicase activity and terminal resolution site (trs) nicking activity. Furthermore, when the enzyme itself was titrated in the trs endonuclease and ATPase reactions, both activities were second order with respect to enzyme concentration. This suggests that a dimer of Rep68 is the active form for both the ATPase and nicking activities. In contrast, DNA helicase activity was linear with respect to enzyme concentration. When bound to ssDNA, the enzyme unwound the DNA in the 3′-to-5′ direction. DNA unwinding occurred at a rate of approximately 345 bp per min per monomeric enzyme molecule. The ATP turnover rate was approximately 30 to 50 ATP molecules per min per enzyme molecule. Surprisingly, the presence of DNA was not required for ATPase activity. We estimated that Rep translocates processively for more than 1,300 bases before dissociating from its substrate in the absence of any accessory proteins. DNA helicase activity was not significantly stimulated by substrates that have the structure of a replication fork and contain either a 5′ or 3′ tail. Rep68 binds only to ssDNA, as judged by inhibition of the DNA helicase reaction with ss or double-stranded (ds) DNA. Consistent with this observation, no helicase activity was detected on blunt-ended ds oligonucleotide substrates unless they also contained an ss 3′ tail. However, if a blunt-ended ds oligonucleotide contained the 22-bp Rep binding element sequence, Rep68 was capable of unwinding the substrate. This means that Rep68 can function both as a conventional helicase for strand displacement synthesis and as a terminal-repeat-unwinding protein which catalyzes the conversion of a duplex end to a hairpin primer. Thus, the properties of the Rep DNA helicase activity suggest that Rep is involved in all three of the key steps in AAV DNA replication: terminal resolution, reinitiation, and strand displacement.

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Heussman, Dylan J., Patrick J. Herbert, Tom Steinberg, Peter H. von Hippel et Andrew H. Marcus. « Characterizing local conformations and conformational disorder near single-stranded (ss) - double stranded (ds) DNA replication junctions ». Biophysical Journal 121, n^o 3 (février 2022) : 170a. http://dx.doi.org/10.1016/j.bpj.2021.11.1871.

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Drappa, Jorn, Lynn A. Kamen, Elena Chan, Maria Georgiev, Dalit Ashany, Francesc Marti et Philip D. King. « Impaired T Cell Death and Lupus-like Autoimmunity in T Cell–specific Adapter Protein–deficient Mice ». Journal of Experimental Medicine 198, n^o 5 (1 septembre 2003) : 809–21. http://dx.doi.org/10.1084/jem.20021358.

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T cell–specific adaptor protein (TSAd) is a T lineage–restricted signaling adaptor molecule that is thought to participate in the assembly of intracellular signaling complexes in T cells. Previous studies of TSAd-deficient mice have revealed a role for TSAd in the induction of T cell interleukin 2 secretion and proliferation. We now show that TSAd-deficient mice are susceptible to lupus-like autoimmune disease. On the nonautoimmune-prone C57BL/6 genetic background, TSAd deficiency results in hypergammaglobulinemia that affects all immunoglobulin (Ig)G subclasses. Older C57BL/6 TSAd-deficient mice (1 yr of age) accumulate large numbers of activated T and B cells in spleen, produce autoantibodies against a variety of self-targets including single stranded (ss) and double stranded (ds) DNA, and, in addition, develop glomerulonephritis. We further show that immunization of younger C57BL/6 TSAd-deficient mice (at age 2 mo) with pristane, a recognized nonspecific inflammatory trigger of lupus, results in more severe glomerulonephritis compared with C57BL/6 controls and the production of high titer ss and ds DNA antibodies of the IgG subclass that are not normally produced by C57BL/6 mice in this model. The development of autoimmunity in TSAd-deficient mice is associated with defective T cell death in vivo. These findings illustrate the role of TSAd as a critical regulator of T cell death whose absence promotes systemic autoimmunity.

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Safarzadeh, Mina, et Genhua Pan. « Detection of a Double-Stranded MGMT Gene Using Electrochemically Reduced Graphene Oxide (ErGO) Electrodes Decorated with AuNPs and Peptide Nucleic Acids (PNA) ». Biosensors 12, n^o 2 (5 février 2022) : 98. http://dx.doi.org/10.3390/bios12020098.

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The ability to detect double-stranded DNA (dsDNA) as a biomarker without denaturing it to single-stranded DNA (ss-DNA) continues to be a major challenge. In this work, we report a sandwich biosensor for the detection of the ds-methylated MGMT gene, a potential biomarker for brain tumors and breast cancer. The purpose of this biosensor is to achieve simultaneous recognition of the gene sequence, as well as the presence of methylation. The biosensor is based on reduced graphene oxide (rGO) electrodes decorated with gold nanoparticles (AuNPs) and uses Peptide Nucleic Acid (PNA) that binds to the ds-MGMT gene. The reduction of GO was performed in two ways: electrochemically (ErGO) and thermally (TrGO). XPS and Raman spectroscopy, as well as voltammetry techniques, showed that the ErGO was more efficiently reduced, had a higher C/O ratio, showed a smaller crystallite size of the sp2 lattice, and was more stable during measurement. It was also revealed that the electro-deposition of the AuNPs was more successful on the ErGO surface due to the higher At% of Au on the ErGO electrode. Therefore, the ErGO/AuNPs electrode was used to develop biosensors to detect the ds-MGMT gene. PNA, which acts as a bio-recognition element, was used to form a self-assembled monolayer (SAM) on the ErGO/AuNPs surface via the amine-AuNPs interaction, recognizing the ds-MGMT gene sequence by its invasion of the double-stranded DNA and the formation of a triple helix. The methylation was then detected using biotinylated-anti-5mC, which was then measured using the amperometric technique. The selectivity study showed that the proposed biosensor was able to distinguish between blank, non-methylated, non-complementary, and target dsDNA spiked in mouse plasma. The LOD was calculated to be 0.86 pM with a wide linear range of 1 pM to 50 µM. To the best of our knowledge, this is the first report on using PNA to detect ds-methylated DNA. This sandwich design can be modified to detect other methylated genes, making it a promising platform to detect ds-methylated biomarkers.

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Ye, Wei Wei, et Mo Yang. « Optimal Surface Functionalization of Nanoporous Alumina Membrane for DNA Detection ». Advanced Materials Research 631-632 (janvier 2013) : 572–75. http://dx.doi.org/10.4028/www.scientific.net/amr.631-632.572.

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This study shows the study of optimal surface functionalization of nanoporous alumina membrane for "label-free" DNA detection. Single stranded DNA was first covalently immobilized on the nanopore walls via silane-PEG-NHS linker. The remained NHS group was hydrolyzed to form PEG layer to minimize the unspecific DNA binding during hybridization process. Optimal PEG-silane linker was achieved for better DNA immobilization efficiency. Using this optofluidic device, both ss-DNA immobilization and ds-DNA hybridization were successfully monitored via UV-Vis spectrum montoring. The nanopore size effect on DNA binding efficiency of membranes were also studied. With the increase of nanopore size, the DNA binding efficiency increased due to the increased reacted surface area. This portable optofluidic device integrated with nanoporos alumina membrane has the potential for nucleic acid in field detection in the application of food screening and environmental monitoring with high sensitivity

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Xu, Hui, Hui Li, Elisabeth Suri-Payer, Richard R. Hardy et Martin Weigert. « Regulation of Anti-DNA B Cells in Recombination-activating Gene–deficient Mice ». Journal of Experimental Medicine 188, n^o 7 (5 octobre 1998) : 1247–54. http://dx.doi.org/10.1084/jem.188.7.1247.

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Anti-DNA antibodies are regulated in normal individuals but are found in high concentration in the serum of systemic lupus erythematosus (SLE) patients and the MRL lpr/lpr mouse model of SLE. We previously studied the regulation of anti–double-stranded (ds)DNA and anti–single-stranded (ss)DNA B cells in a nonautoimmune background by generating mice carrying immunoglobulin transgenes coding for anti-DNAs derived from MRL lpr/lpr. Anti-dsDNA B cells undergo receptor editing, but anti-ssDNA B cells seem to be functionally silenced. Here we have investigated how anti-DNA B cells are regulated in recombination- activating gene (RAG)-2−/− mice. In this setting, anti-dsDNA B cells are eliminated by apoptosis in the bone marrow and anti-ssDNA B cells are partially activated.

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Qi, Rui, Ke Bian, Fangyi Chen, Qi Tang, Xianhao Zhou et Deyu Li. « Sequence Dependent Repair of 1,N6-Ethenoadenine by DNA Repair Enzymes ALKBH2, ALKBH3, and AlkB ». Molecules 26, n^o 17 (31 août 2021) : 5285. http://dx.doi.org/10.3390/molecules26175285.

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Mutation patterns of DNA adducts, such as mutational spectra and signatures, are useful tools for diagnostic and prognostic purposes. Mutational spectra of carcinogens derive from three sources: adduct formation, replication bypass, and repair. Here, we consider the repair aspect of 1,N6-ethenoadenine (εA) by the 2-oxoglutarate/Fe(II)-dependent AlkB family enzymes. Specifically, we investigated εA repair across 16 possible sequence contexts (5′/3′ flanking base to εA varied as G/A/T/C). The results revealed that repair efficiency is altered according to sequence, enzyme, and strand context (ss- versus ds-DNA). The methods can be used to study other aspects of mutational spectra or other pathways of repair.

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Ben-Eli, Hadas, Abraham Solomon, Doron J. Aframian, Eldad Ben-Chetrit, Dror Mevorach, Geffen Kleinstern, Tim Waterboer, Martina Willhauck-Fleckenstein, Michael Pawlita et Ora Paltiel. « Serological and hematological characteristics of Sjogren’s syndrome and dry eye syndrome patients using a novel immune serology technique ». PLOS ONE 15, n^o 12 (31 décembre 2020) : e0244712. http://dx.doi.org/10.1371/journal.pone.0244712.

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Objectives To compare hematologic and serological parameters among patients with Sjogren’s syndrome (SS), dry eye syndrome (DES) and controls, and validate a novel multiplex-serology method for identifying auto-antibodies in these populations. Methods In a clinic-based case-control study a total of 422 participants were recruited, including 91 with SS, 120 DES, and 211 controls (age and sex frequency-matched). We measured blood counts, anti-nuclear-antibodies (ANA), anti-SSA/SSB, anti-ribonucleoprotein (RNP), anti-double-stranded-DNA (DS-DNA), and rheumatoid factor (RF) using the “Immunodot” qualitative-ELISA assay. Immunoglobulins, C3 and C4 were measured by immune-fluorescence. Autoantibodies were also quantified with a newly-developed method using glutathione-S-transferase fusion proteins of SSA/Ro 52 and 60kD and SSB/La (multiplex-serology), measuring median fluorescence intensity (MFI). Results Among DES patients, only 2% (95%CI: 0.36–6.3) had positive immune serology. SS patients had lower lymphocyte, hemoglobin and C3 levels but higher prevalence of RF, ANA, anti-SSA/B and higher IgG and MFI levels, compared to DES and controls (P<0.001). Presence of anti-SSA/Ro-52kD was associated with SS [odds ratio (OR) = 2.05, 95% confidence interval (CI): 1.46–2.88]. Anti-SSB/La was inversely associated with DES (OR = 0.81, 95%CI: 0.65–1.00) compared to controls. Positivity to RF (adjusted for age, gender and ethnicity OR = 5.03, 95%CI: 1.78–14.21), ANA (OR = 14.75, 95%CI: 4.09–53.17), or combination of anti-SSA/B (OR = 20.97, 95%CI: 4.60–95.54) were more likely in SS compared to DES. The novel multiplex-serology method correctly identified anti-SSA/B autoantibodies by ELISA among SS, DES patients and controls (sensitivity = 1.0, negative-predictive-value = 1.0). Conclusions Serologic parameters distinguish SS from DES patients and controls. A newly-developed multiplex-serology technique may be useful to detect autoantibodies in large epidemiologic studies.

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He, Feng, Kevin DuPrez, Eduardo Hilario, Zhenhang Chen et Li Fan. « Structural basis of the XPB helicase–Bax1 nuclease complex interacting with the repair bubble DNA ». Nucleic Acids Research 48, n^o 20 (28 septembre 2020) : 11695–705. http://dx.doi.org/10.1093/nar/gkaa801.

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Abstract Nucleotide excision repair (NER) removes various DNA lesions caused by UV light and chemical carcinogens. The DNA helicase XPB plays a key role in DNA opening and coordinating damage incision by nucleases during NER, but the underlying mechanisms remain unclear. Here, we report crystal structures of XPB from Sulfurisphaera tokodaii (St) bound to the nuclease Bax1 and their complex with a bubble DNA having one arm unwound in the crystal. StXPB and Bax1 together spirally encircle 10 base pairs of duplex DNA at the double-/single-stranded (ds–ss) junction. Furthermore, StXPB has its ThM motif intruding between the two DNA strands and gripping the 3′-overhang while Bax1 interacts with the 5′-overhang. This ternary complex likely reflects the state of repair bubble extension by the XPB and nuclease machine. ATP binding and hydrolysis by StXPB could lead to a spiral translocation along dsDNA and DNA strand separation by the ThM motif, revealing an unconventional DNA unwinding mechanism. Interestingly, the DNA is kept away from the nuclease domain of Bax1, potentially preventing DNA incision by Bax1 during repair bubble extension.

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Landry, Aaron P., et Huangen Ding. « The N-Terminal Domain of Human DNA Helicase Rtel1 Contains a Redox Active Iron-Sulfur Cluster ». BioMed Research International 2014 (2014) : 1–8. http://dx.doi.org/10.1155/2014/285791.

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Human telomere length regulator Rtel1 is a superfamily II DNA helicase and is essential for maintaining proper length of telomeres in chromosomes. Here we report that the N-terminal domain of human Rtel1 (RtelN) expressed inEscherichia colicells produces a protein that contains a redox active iron-sulfur cluster with the redox midpoint potential of −248 ± 10 mV (pH 8.0). The iron-sulfur cluster in RtelN is sensitive to hydrogen peroxide and nitric oxide, indicating that reactive oxygen/nitrogen species may modulate the DNA helicase activity of Rtel1 via modification of its iron-sulfur cluster. Purified RtelN retains a weak binding affinity for the single-stranded (ss) and double-stranded (ds) DNAin vitro. However, modification of the iron-sulfur cluster by hydrogen peroxide or nitric oxide does not significantly affect the DNA binding activity of RtelN, suggesting that the iron-sulfur cluster is not directly involved in the DNA interaction in the N-terminal domain of Rtel1.

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Xiong, Ying, Bao Gao, Kesheng Wu, Yunqing Wu, Yinjiao Chai, Xiaolin Huang et Yonghua Xiong. « Fluorescence immunoassay based on the enzyme cleaving ss-DNA to regulate the synthesis of histone-ds-poly(AT) templated copper nanoparticles ». Nanoscale 10, n^o 42 (2018) : 19890–97. http://dx.doi.org/10.1039/c8nr06175k.

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For the first time we report a novel competitive fluorescence immunoassay for the ultrasensitive detection of aflatoxin B₁ (AFB₁) using histone-ds-poly(AT) templated copper nanoparticles (His-pAT CuNPs) as fluorescent indicators.

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Vardevanyan, Poghos O., Valeri B. Arakelyan, Marine A. Parsadanyan, Ara P. Antonyan, Gohar G. Hovhannisyan et Mariam A. Shahinyan. « Analysis of experimental binding curves of EtBr with single- and double-stranded DNA at small fillings ». Modern Physics Letters B 28, n^o 22 (30 août 2014) : 1450178. http://dx.doi.org/10.1142/s0217984914501784.

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In this paper, a method that allows to analyze the binding curves of ligand ( EtBr ) with single-stranded (ss) and double-stranded (ds) DNA, when there are at least two modes of ligand binding to DNA at small fillings has been proposed. The obtained experimental binding curves for EtBr –ssDNA and EtBr –dsDNA have two clearly expressed linear regions. These curves were analyzed by two modes: Experimental points on linear regions were described by two different lines and all experimental points were described by single curve. It was revealed that the description by single curve permits obtaining more precise data of binding parameters (i.e. binding constant and number of base pairs that bind one ligand molecule). Moreover, the proposed method permits determining the value of proportion of binding sites of each binding mode.

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Kysil, Olena, Iryna Sporysh, Eugenia Buzaneva, Tobias Erb, Gerhard Gobsch, Uwe Ritter et Peter Scharff. « The C60 fullerene molecules integration by ds-, ss-DNA molecules in fluids : Optical spectroscopy characterization of the biointerface organization ». Materials Science and Engineering : B 169, n^o 1-3 (mai 2010) : 85–88. http://dx.doi.org/10.1016/j.mseb.2010.01.063.

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Crnolatac, Ivo, Iva Rogan, Boris Majić, Sanja Tomić, Todor Deligeorgiev, Gordan Horvat, Damjan Makuc, Janez Plavec, Gennaro Pescitelli et Ivo Piantanida. « Small molecule probes finely differentiate between various ds- and ss-DNA and RNA by fluorescence, CD and NMR response ». Analytica Chimica Acta 940 (octobre 2016) : 128–35. http://dx.doi.org/10.1016/j.aca.2016.08.021.

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Wloch, M. K., S. H. Clarke et G. S. Gilkeson. « Influence of VH CDR3 arginine and light chain pairing on DNA reactivity of a bacterial DNA-induced anti-DNA antibody from a BALB/c mouse. » Journal of Immunology 159, n^o 12 (15 décembre 1997) : 6083–90. http://dx.doi.org/10.4049/jimmunol.159.12.6083.

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Abstract Anti-DNA induced in BALB/c mice by immunization with bacterial (Escherichia coli) DNA resemble spontaneous anti-DNA from lupus mice in V gene use and cross-reactivity with other nuclear Ags, but lack the high V(H) CDR3 arginine content seen in anti-DNA from lupus mice. Moreover, the induced anti-DNA bind bacterial and mammalian single-stranded (ss) DNA and bacterial double-stranded (ds) DNA, but do not bind mammalian dsDNA. This reactivity profile is in contrast to that of the spontaneously arising anti-DNA of lupus mice, among which mammalian dsDNA reactive Abs are prominent. In this study we demonstrate that the addition of arginine to V(H) CDR3 of an induced anti-DNA confers the mammalian dsDNA binding characteristic of anti-DNA from lupus mice. The ability to confer mammalian dsDNA binding is dependent on both the position of the arginine in V(H) CDR3 and the light chain with which the heavy chain is paired, suggesting the light chain plays a more substantial role in DNA binding by this Ab than has previously been reported for other anti-DNA. Our data support the argument that V(H) CDR3 arginines tend to confer antimammalian dsDNA reactivity, leading to censure of B cells expressing these Abs and provides an explanation for the absence of arginine-rich V(H) CDR3 in the bacterial DNA-induced response.

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Maurer, Jack W., Claire Albrecht, Patrick J. Herbert, Dylan Heussman, Peter H. von Hippel et Andrew H. Marcus. « Using single molecule polarization-sweep spectroscopy to monitor site-specific fluctuations of internal (iCy3)2 dimer-labeled DNA constructs at ss-ds DNA junctions ». Biophysical Journal 122, n^o 3 (février 2023) : 212a. http://dx.doi.org/10.1016/j.bpj.2022.11.1268.

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Heussman, Dylan, Patrick J. Herbert, Jack W. Maurer, Justin Kittell, Amr Tamimi, Tom Steinberg, Peter H. von Hippel et Andrew H. Marcus. « Determining Local Conformations and Conformational Disorder at and Near Single-Stranded (Ss) and Double-Stranded (Ds) DNA Forks and Junctions ». Biophysical Journal 120, n^o 3 (février 2021) : 219a. http://dx.doi.org/10.1016/j.bpj.2020.11.1470.

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Zasedateleva, O. A., A. S. Krylov, D. V. Prokopenko, M. A. Skabkin, L. P. Ovchinnikov, A. Kolchinsky et A. D. Mirzabekov. « Specificity of Mammalian Y-box Binding Protein p50 in Interaction with ss and ds DNA Analyzed with Generic Oligonucleotide Microchip ». Journal of Molecular Biology 324, n^o 1 (novembre 2002) : 73–87. http://dx.doi.org/10.1016/s0022-2836(02)00937-3.

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OU-YANG, Z. C., H. ZHOU et Y. ZHANG. « THE ELASTIC THEORY OF A SINGLE DNA MOLECULE ». Modern Physics Letters B 17, n^o 01 (10 janvier 2003) : 1–10. http://dx.doi.org/10.1142/s0217984903004877.

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We review our recent efforts in understanding the elastic properties of double-stranded (ds) and single-stranded (ss) DNA macromolecules. A simple geometric model of dsDNA was constructed and solved by path integral methods. The good agreement with experiments on dsDNA's entropic elasticity, cooperative extensibility, and supercoiling property suggested that the short-ranged base-pair stacking interaction is crucial for the stability and the high deformability of dsDNA. For ssDNA at high ionic conditions, base-pairing and base-pair stacking interactions cause the polymer to fold into compact hairpin configurations. The force-induced hairpin-coil transition was studied with the generating function method. In accordance with experiment, this transition was found to be highly cooperative when the average stacking potential is higher than some critical level. At low ionic conditions, the electrostatic repulsive interaction along the ssDNA becomes dominant, and ssDNA can be regarded as model polyelectrolytes. Our MC simulation results suggested an exponential relation between external force and the total extension. This prediction was confirmed experimentally.

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Ackermann, Hans W. « Bacteriophage taxonomy ». Microbiology Australia 32, n^o 2 (2011) : 90. http://dx.doi.org/10.1071/ma11090.

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Bacteriophages or ?phages? are viruses of prokaryotes. At least 5,360 tailed and 179 cubic, filamentous, and pleomorphic bacterial viruses have been examined in the electron microscope since the introduction of negative staining in 1959. Since at least 100 novel bacterial viruses are described every year1, the approximate number of viruses under consideration is over 6,000. Numerically, this makes bacteriophages the largest virus group known. Phages are presently classified in a hierarchical and holistic system with one order and 10 families. Over 96% of phages are tailed and contain dsDNA. The seven families of cubic, filamentous and pleomorphic phages are small and well defined. They contain ds or ss DNA or RNA. The most important developments are reclassifications of the Podoviridae and Myoviridae families of tailed phages.

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Mrevlishvili, George M., Ana Paula S. M. C. Carvalho et Manuel A. V. Ribeiro da Silva. « Low-temperature DSC study of the hydration of ss-DNA and ds-DNA and the role of hydrogen-bonded network to the duplex transition thermodynamics ». Thermochimica Acta 394, n^o 1-2 (octobre 2002) : 73–82. http://dx.doi.org/10.1016/s0040-6031(02)00240-x.

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