Página 1 dos resultados de 21713 itens digitais encontrados em 0.057 segundos

Evaluation of the interactions of DNA with the textile dyes Disperse Orange 1 and Disperse Red 1 and their electrolysis products using an electrochemical biosensor

Uliana, Carolina Venturini; Garbellini, Gustavo Stoppa; Yamanaka, Hideko
Fonte: Universidade Estadual Paulista Publicador: Universidade Estadual Paulista
Tipo: Artigo de Revista Científica Formato: 627-635
ENG
Relevância na Pesquisa
55.81%
A disposable pencil graphite electrode modified with dsDNA was used in combination with square wave voltammetry in order to evaluate the interaction of DNA with the textile dyes Disperse Orange 1 (DO1) and Disperse Red 1 (DR1), and with the products of their electrolysis. Significant changes in the characteristic oxidation peaks of the guanine and adenine moieties of immobilized dsDNA were observed after incubation of the modified electrode for 180 s in solutions of the dyes in their original forms. The same was observed using the electrolysis products obtained by oxidation and reduction conversions. The oxidation peak currents of the guanine and adenine moieties decreased when the concentrations of DO1 and DR1 were increased up to 5.0 × 10 -6 and 1.0 × 10-6 mol L-1, respectively; the signal decreases were more pronounced after interaction with the oxidized dyes, compared to the reduced compounds. The interactions between DNA and DO1, DR1, and the electrolyzed dyes were further investigated by UV-vis spectrophotometry in solution, and different effects such as hypochromism and hyperchromism were observed in the resulting DNA spectra. The investigated interactions showed clear evidence of changes in the DNA structure, and suggested a predominant intercalation mode leading to damage in the biomolecule. © 2013 Elsevier B.V.

Gold nanoparticle-DNA conjugates for oligonucleotide vectorization towards gene silencing

Almeida, Carina Marisa dos Santos
Fonte: Faculdade de Ciências e Tecnologia Publicador: Faculdade de Ciências e Tecnologia
Tipo: Dissertação de Mestrado
Publicado em //2011 ENG
Relevância na Pesquisa
45.87%
Dissertação para obtenção do Grau de Mestre em Genética Molecular e Biomedicina; The main objective of the work presented in this thesis was to develop a gene silencing system by taking advantage of the nanovectorization capability and optical properties of gold nanoparticles. The idea is based on the construction of a DNA structure containing a therapeutic oligonucleotide with the ability to form Hoogsteen hydrogen bonds with double-stranded DNA, producing a DNA triple helix, besides silencing the gene of interest. Hoogsteen bonds, more unstable than the conventional Watson-Crick bonds, permit the achievement of lower melting temperatures. This attribute, coupled with the ability to generate heat by laser irradiation of the gold nanoparticles used, will allow the release of the therapeutic oligonucleotide and subsequent gene silencing without significant increase in the medium’s temperature. Thus, the thesis comprises three major sections: structure design and formation, vectorization, and gene expression silencing; the tasks involved in each of these sections were conducted in parallel. The design of the obtained structure took into account the desired melting temperature, stability at physiological conditions of the sequence-forming nucleotides...

An extended DNA structure through deoxyribose-base stacking induced by RecA protein

Nishinaka, Taro; Ito, Yutaka; Yokoyama, Shigeyuki; Shibata, Takehiko
Fonte: The National Academy of Sciences of the USA Publicador: The National Academy of Sciences of the USA
Tipo: Artigo de Revista Científica
Publicado em 24/06/1997 EN
Relevância na Pesquisa
45.88%
The family of proteins that are homologous to RecA protein of Escherichia coli is essential to homologous genetic recombination in various organisms including viruses, bacteria, lower eukaryotes, and mammals. In the presence of ATP (or ATPγS), these proteins form helical filaments containing single-stranded DNA at the center. The single-stranded DNA bound to RecA protein is extended 1.5 times relative to B-form DNA with the same sequence, and the extension is critical to pairing with homologous double-stranded DNA. This pairing reaction, called homologous pairing, is a key reaction in homologous recombination. In this NMR study, we determined a three-dimensional structure of the single-stranded DNA bound to RecA protein. The DNA structure contains novel deoxyribose-base stacking in which the 2′-methylene moiety of each deoxyribose is placed above the base of the following residue, instead of normal stacking of adjacent bases. As a result of this deoxyribose-base stacking, bases of the single-stranded DNA are spaced out nearly 5 Å. Thus, this novel structure well explains the axial extension of DNA in the RecA-filaments relative to B-form DNA and leads to a possible interpretation of the role of this extension in homologous pairing.

Homologous genetic recombination as an intrinsic dynamic property of a DNA structure induced by RecA/Rad51-family proteins: A possible advantage of DNA over RNA as genomic material

Shibata, Takehiko; Nishinaka, Taro; Mikawa, Tsutomu; Aihara, Hideki; Kurumizaka, Hitoshi; Yokoyama, Shigeyuki; Ito, Yutaka
Fonte: The National Academy of Sciences Publicador: The National Academy of Sciences
Tipo: Artigo de Revista Científica
Publicado em 17/07/2001 EN
Relevância na Pesquisa
45.9%
Heteroduplex joints are general intermediates of homologous genetic recombination in DNA genomes. A heteroduplex joint is formed between a single-stranded region (or tail), derived from a cleaved parental double-stranded DNA, and homologous regions in another parental double-stranded DNA, in a reaction mediated by the RecA/Rad51-family of proteins. In this reaction, a RecA/Rad51-family protein first forms a filamentous complex with the single-stranded DNA, and then interacts with the double-stranded DNA in a search for homology. Studies of the three-dimensional structures of single-stranded DNA bound either to Escherichia coli RecA or Saccharomyces cerevisiae Rad51 have revealed a novel extended DNA structure. This structure contains a hydrophobic interaction between the 2′ methylene moiety of each deoxyribose and the aromatic ring of the following base, which allows bases to rotate horizontally through the interconversion of sugar puckers. This base rotation explains the mechanism of the homology search and base-pair switch between double-stranded and single-stranded DNA during the formation of heteroduplex joints. The pivotal role of the 2′ methylene-base interaction in the heteroduplex joint formation is supported by comparing the recombination of RNA genomes with that of DNA genomes. Some simple organisms with DNA genomes induce homologous recombination when they encounter conditions that are unfavorable for their survival. The extended DNA structure confers a dynamic property on the otherwise chemically and genetically stable double-stranded DNA...

The non-B-DNA structure of d(CA/TG)n does not differ from that of Z-DNA.

Ho, P S
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 27/09/1994 EN
Relevância na Pesquisa
45.87%
A number of recent studies have shown that simple repetitive d(CA/TG) dinucleotide sequences adopt a left-handed non-B-DNA structure under negative superhelical stress. The pattern of chemical reactivities and the helical parameters observed for these sequences differ significantly from those of standard Z-DNA. In this study, the data for two naturally occurring d(CA/TG)n sequences are reevaluated by a statistical mechanics treatment of the B- to Z-DNA transition. The behavior of these sequences under negative superhelical stress is accurately simulated by this model, including the multiple and discrete transitions observed for the rat prolactin promoter. Furthermore, the average helical twist for the left-handed structure of d(CA/TG)n deviates < 2% from that expected for standard Z-DNA. Finally, the predicted distribution of the junctions between B- and Z-DNA are shown to account for differences observed in the patterns of chemical reactivity of d(CA/TG)n and d(CG)n. Thus, no new left-handed structure that differs from Z-DNA is needed to describe the supercoil-induced conformation in d(CA/TG)n sequences.

The Bloom’s and Werner’s syndrome proteins are DNA structure-specific helicases

Mohaghegh, Payam; Karow, Julia K.; Brosh Jr, Robert M.; Bohr, Vilhelm A.; Hickson, Ian D.
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
Publicado em 01/07/2001 EN
Relevância na Pesquisa
45.89%
BLM and WRN, the products of the Bloom’s and Werner’s syndrome genes, are members of the RecQ family of DNA helicases. Although both have been shown previously to unwind simple, partial duplex DNA substrates with 3′→5′ polarity, little is known about the structural features of DNA that determine the substrate specificities of these enzymes. We have compared the substrate specificities of the BLM and WRN proteins using a variety of partial duplex DNA molecules, which are based upon a common core nucleotide sequence. We show that neither BLM nor WRN is capable of unwinding duplex DNA from a blunt-ended terminus or from an internal nick. However, both enzymes efficiently unwind the same blunt-ended duplex containing a centrally located 12 nt single-stranded ‘bubble’, as well as a synthetic X-structure (a model for the Holliday junction recombination intermediate) in which each ‘arm’ of the 4-way junction is blunt-ended. Surprisingly, a 3′-tailed duplex, a standard substrate for 3′→5′ helicases, is unwound much less efficiently by BLM and WRN than are the bubble and X-structure substrates. These data show conclusively that a single-stranded 3′-tail is not a structural requirement for unwinding of standard B-form DNA by these helicases. BLM and WRN also both unwind a variety of different forms of G-quadruplex DNA...

A role for a bent DNA structure in E2F-mediated transcription activation.

Cress, W D; Nevins, J R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /05/1996 EN
Relevância na Pesquisa
45.86%
We examined the role of promoter architecture, as well as that of the DNA-bending capacity of the E2F transcription factor family, in the activation of transcription. DNA phasing analysis revealed that a consensus E2F site in the E2F1 promoter possesses an inherent bend with a net magnitude of 40 +/-2 degrees and with an orientation toward the major groove relative to the center of the E2F site. The inherent DNA bend is reversed upon binding of E2F, generating a net bend with a magnitude of 25 +/- 3 degrees oriented toward the minor groove relative to the center of the E2F site. We also found that three members of the E2F family, in conjunction with the DP1 protein, bend the DNA toward the minor groove, suggesting that DNA bending is a characteristic of the entire E2F family. The Rb-E2F complex, on the other hand, does not reverse the intrinsic DNA bend. Analysis of a series of E2F1 deletion mutants defined E2F1 sequences which are not required for DNA binding but are necessary for the DNA-bending capacity of E2F. An internal region of E2F1, previously termed the marked box, which is highly homologous among E2F family members, was particularly important in DNA bending. We also found that a bent DNA structure can be a contributory component in the activation of the E2F1 promoter but is not critical in the repression of that promoter in quiescent cells. This finding suggests that E2F exhibits characteristics typical of modular transcription factors...

The enhancer-blocking suppressor of Hairy-wing zinc finger protein of Drosophila melanogaster alters DNA structure.

Shen, B; Kim, J; Dorsett, D
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em /09/1994 EN
Relevância na Pesquisa
45.89%
Insertion of the gypsy retrotransposon of Drosophila melanogaster into a gene control region can repress gene expression. The zinc finger protein (SUHW) encoded by the suppressor of Hairy-wing [su(Hw)] gene binds to gypsy and prevents gene enhancers from activating transcription. SUHW blocks an enhancer only when positioned between the enhancer and promoter. Although position dependent, SUHW enhancer blocking is distance independent. These properties indicate that SUHW does not interact with the transcription activator proteins that bind to enhancers. To explore if DNA distortions are involved in enhancer blocking, the ability of SUHW to alter DNA structure was examined in gel mobility assays. Indeed, SUHW induces an unusual change in the structure of the binding-site DNA. The change is not a directed DNA bend but correlates with loss of sequence-directed bends in the unbound DNA. The DNA distortion requires a SUHW protein domain not required for DNA binding, and mutant proteins that fail to alter DNA structure also fail to eliminate the sequence-directed bends. These results suggest that SUHW increases DNA flexibility. The DNA distortion is not sufficient to block enhancers, and therefore it is suggested that increased DNA flexibility may help SUHW interact and interfere with proteins that support long-distance enhancer-promoter interactions.

Mutant p53 proteins bind DNA in a DNA structure-selective mode

Göhler, Thomas; Jäger, Stefan; Warnecke, Gabriele; Yasuda, Hideyo; Kim, Ella; Deppert, Wolfgang
Fonte: Oxford University Press Publicador: Oxford University Press
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
45.9%
Despite the loss of sequence-specific DNA binding, mutant p53 (mutp53) proteins can induce or repress transcription of mutp53-specific target genes. To date, the molecular basis for transcriptional modulation by mutp53 is not understood, but increasing evidence points to the possibility that specific interactions of mutp53 with DNA play an important role. So far, the lack of a common denominator for mutp53 DNA binding, i.e. the existence of common sequence elements, has hampered further characterization of mutp53 DNA binding. Emanating from our previous discovery that DNA structure is an important determinant of wild-type p53 (wtp53) DNA binding, we analyzed the binding of various mutp53 proteins to oligonucleotides mimicking non-B DNA structures. Using various DNA-binding assays we show that mutp53 proteins bind selectively and with high affinity to non-B DNA. In contrast to sequence-specific and DNA structure-dependent binding of wtp53, mutp53 DNA binding to non-B DNA is solely dependent on the stereo-specific configuration of the DNA, and not on DNA sequence. We propose that DNA structure-selective binding of mutp53 proteins is the basis for the well-documented interaction of mutp53 with MAR elements and for transcriptional activities mediates by mutp53.

CpG Island Methylation in Human Lymphocytes Is Highly Correlated with DNA Sequence, Repeats, and Predicted DNA Structure

Bock, Christoph; Paulsen, Martina; Tierling, Sascha; Mikeska, Thomas; Lengauer, Thomas; Walter, Jörn
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
45.87%
CpG island methylation plays an important role in epigenetic gene control during mammalian development and is frequently altered in disease situations such as cancer. The majority of CpG islands is normally unmethylated, but a sizeable fraction is prone to become methylated in various cell types and pathological situations. The goal of this study is to show that a computational epigenetics approach can discriminate between CpG islands that are prone to methylation from those that remain unmethylated. We develop a bioinformatics scoring and prediction method on the basis of a set of 1,184 DNA attributes, which refer to sequence, repeats, predicted structure, CpG islands, genes, predicted binding sites, conservation, and single nucleotide polymorphisms. These attributes are scored on 132 CpG islands across the entire human Chromosome 21, whose methylation status was previously established for normal human lymphocytes. Our results show that three groups of DNA attributes, namely certain sequence patterns, specific DNA repeats, and a particular DNA structure, are each highly correlated with CpG island methylation (correlation coefficients of 0.64, 0.66, and 0.49, respectively). We predicted, and subsequently experimentally examined 12 CpG islands from human Chromosome 21 with unknown methylation patterns and found more than 90% of our predictions to be correct. In addition...

DNA Structure Modulates the Oligomerization Properties of the AAV Initiator Protein Rep68

Mansilla-Soto, Jorge; Yoon-Robarts, Miran; Rice, William J.; Arya, Shailee; Escalante, Carlos R.; Linden, R. Michael
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
45.87%
Rep68 is a multifunctional protein of the adeno-associated virus (AAV), a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3) helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag) and bovine papillomavirus (BPV) E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID) and an AAA+ motor domain. The AAA+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However...

A Non-canonical DNA Structure Enables Homologous Recombination in Various Genetic Systems*

Masuda, Tokiha; Ito, Yutaka; Terada, Tohru; Shibata, Takehiko; Mikawa, Tsutomu
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
55.83%
Homologous recombination, which is critical to genetic diversity, depends on homologous pairing (HP). HP is the switch from parental to recombinant base pairs, which requires expansion of inter-base pair spaces. This expansion unavoidably causes untwisting of the parental double-stranded DNA. RecA/Rad51-catalyzed ATP-dependent HP is extensively stimulated in vitro by negative supercoils, which compensates for untwisting. However, in vivo, double-stranded DNA is relaxed by bound proteins and thus is an unfavorable substrate for RecA/Rad51. In contrast, Mhr1, an ATP-independent HP protein required for yeast mitochondrial homologous recombination, catalyzes HP without the net untwisting of double-stranded DNA. Therefore, we questioned whether Mhr1 uses a novel strategy to promote HP. Here, we found that, like RecA, Mhr1 induced the extension of bound single-stranded DNA. In addition, this structure was induced by all evolutionarily and structurally distinct HP proteins so far tested, including bacterial RecO, viral RecT, and human Rad51. Thus, HP includes the common non-canonical DNA structure and uses a common core mechanism, independent of the species of HP proteins. We discuss the significance of multiple types of HP proteins.

DNA Structure Specificity Conferred on a Replicative Helicase by Its Loader*

Gupta, Milind K.; Atkinson, John; McGlynn, Peter
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
55.9%
Prokaryotic and eukaryotic replicative helicases can translocate along single-stranded and double-stranded DNA, with the central cavity of these multimeric ring helicases being able to accommodate both forms of DNA. Translocation by such helicases along single-stranded DNA results in the unwinding of forked DNA by steric exclusion and appears critical in unwinding of parental strands at the replication fork, whereas translocation over double-stranded DNA has no well-defined role. We have found that the accessory factor, DnaC, that promotes loading of the Escherichia coli replicative helicase DnaB onto single-stranded DNA may also act to confer DNA structure specificity on DnaB helicase. When present in excess, DnaC inhibits DnaB translocation over double-stranded DNA but not over single-stranded DNA. Inhibition of DnaB translocation over double-stranded DNA requires the ATP-bound form of DnaC, and this inhibition is relieved during translocation over single-stranded DNA indicating that stimulation of DnaC ATPase is responsible for this DNA structure specificity. These findings demonstrate that DnaC may provide the DNA structure specificity lacking in DnaB, limiting DnaB translocation to bona fide replication forks. The ability of other replicative helicases to translocate along single-stranded and double-stranded DNA raises the possibility that analogous regulatory mechanisms exist in other organisms.

Gene Expression Divergence is Coupled to Evolution of DNA Structure in Coding Regions

Dai, Zhiming; Dai, Xianhua
Fonte: Public Library of Science Publicador: Public Library of Science
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
45.86%
Sequence changes in coding region and regulatory region of the gene itself (cis) determine most of gene expression divergence between closely related species. But gene expression divergence between yeast species is not correlated with evolution of primary nucleotide sequence. This indicates that other factors in cis direct gene expression divergence. Here, we studied the contribution of DNA three-dimensional structural evolution as cis to gene expression divergence. We found that the evolution of DNA structure in coding regions and gene expression divergence are correlated in yeast. Similar result was also observed between Drosophila species. DNA structure is associated with the binding of chromatin remodelers and histone modifiers to DNA sequences in coding regions, which influence RNA polymerase II occupancy that controls gene expression level. We also found that genes with similar DNA structures are involved in the same biological process and function. These results reveal the previously unappreciated roles of DNA structure as cis-effects in gene expression.

Methods to detect replication-dependent and replication-independent DNA structure-induced genetic instability

Wang, Guliang; Gaddis, Sally; Vasquez, Karen M.
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
EN
Relevância na Pesquisa
45.86%
DNA can adopt a variety of alternative secondary (i.e., non-B DNA) conformations that play important roles in cellular metabolism, including genetic instability, disease etiology, and evolution. While we still have much to learn, research in this field has expanded dramatically in the past decade. We have summarized in our previous Methods review (Wang et al., Methods, 2009) some commonly used techniques to determine non-B DNA structural conformations and non-B DNA-induced genetic instability in prokaryotes and eukaryotes. Since that time, we and others have further characterized mechanisms involved in DNA structure-induced mutagenesis and have proposed both replication-dependent and replication-independent models. Thus, in this review, we highlight some current methodologies to identify DNA replication-related and replication-independent mutations occurring at non-B DNA regions to allow for a better understanding of the mechanisms underlying DNA structure-induced genetic instability. We also describe a new web-based search engine to identify potential intramolecular triplex (H-DNA) and left-handed Z-DNA-forming motifs in entire genomes or at selected sequences of interest.

Prospects of nanoparticle-DNA binding and its implications in medical biotechnology

An, H.; Jin, B.
Fonte: Pergamon-Elsevier Science Ltd Publicador: Pergamon-Elsevier Science Ltd
Tipo: Artigo de Revista Científica
Publicado em //2012 EN
Relevância na Pesquisa
45.86%
Bio-nanotechnology is a new interdisciplinary R&D area that integrates engineering and physical science with biology through the development of multifunctional devices and systems, focusing biology inspired processes or their applications, in particular in medical biotechnology. DNA based nanotechnology, in many ways, has been one of the most intensively studied fields in recent years that involves the use and the creation of bio-inspired materials and their technologies for highly selective biosensing, nanoarchitecture engineering and nanoelectronics. Increasing researches have been offered to a fundamental understanding how the interactions between the nanoparticles and DNA molecules could alter DNA molecular structure and its biochemical activities. This minor review describes the mechanisms of the nanoparticle-DNA binding and molecular interactions. We present recent discoveries and research progresses how the nanoparticle-DNA binding could vary DNA molecular structure, DNA detection, and gene therapy. We report a few case studies associated with the application of the nanoparticle-DNA binding devices in medical detection and biotechnology. The potential impacts of the nanoparticles via DNA binding on toxicity of the microorganisms are briefly discussed. The nanoparticle-DNA interactions and their impact on molecular and microbial functionalities have only drown attention in recent a few years. The information presented in this review can provide useful references for further studies on biomedical science and technology.; Hongjie An...

Desenvolvimento de vacina genica veiculada em adjuvantes lipidicos para tratamento da tuberculose; Lipid adjuvants as carriers for tuberculosis DNA vaccine

Lucimara Gaziola de la Torre
Fonte: Biblioteca Digital da Unicamp Publicador: Biblioteca Digital da Unicamp
Tipo: Tese de Doutorado Formato: application/pdf
Publicado em 12/12/2006 PT
Relevância na Pesquisa
45.97%
Este trabalho visa o desenvolvimento tecnológico de uma vacina gênica, destinada ao combate à tuberculose, na qual o DNA encontra-se veiculado em lipossomas. Foram enfocados três aspectos principais: 1.A preparação e caracterização de estruturas lipídicas funcionais veiculando o DNA, projetadas para atenderem aos requisitos de imunização contra a tuberculose; 2. Complexação do DNA com peptídio sintético promotor de transporte nuclear e veiculação na estrutura lipossomal que se mostrou mais promissora nos ensaios in vitro e in vivo realizados no CPT-RP. 3. Análise do escalonamento da produção da estrutura lipossomal mais promissora para subsequente veiculação do DNA. Duas estruturas lipossomais foram compostas por lipídios com as seguintes funcionalidades: estrutural, de incorporação do DNA e atração eletrostática com a superfície das células, de intensificação da liberação do DNA no citoplasma celular. Foram preparadas pelo método da desidratação-rehidratação, gerando DRVs ('dehydrated-hydrated vesicles'). O DNA foi associado à essas estruturas, localizando-se no interior, [DRV(DNA)] ou prefencialmente na sua superfície [DRV-DNA]. A terceira estrutura, um agregado lipídico não lipossomal designado por lipoplexo...

The Human Oxidative DNA Glycosylase NEIL1 Excises Psoralen-induced Interstrand DNA Cross-links in a Three-stranded DNA Structure*S⃞

Couvé, Sophie; Macé-Aimé, Gaëtane; Rosselli, Filippo; Saparbaev, Murat K.
Fonte: American Society for Biochemistry and Molecular Biology Publicador: American Society for Biochemistry and Molecular Biology
Tipo: Artigo de Revista Científica
Publicado em 01/05/2009 EN
Relevância na Pesquisa
55.83%
Previously, we have demonstrated that human oxidative DNA glycosylase NEIL1 excises photoactivated psoralen-induced monoadducts but not genuine interstrand cross-links (ICLs) in duplex DNA. It has been postulated that the repair of ICLs in mammalian cells is mainly linked to DNA replication and proceeds via dual incisions in one DNA strand that bracket the cross-linked site. This process, known as “unhooking,” enables strand separation and translesion DNA synthesis through the gap, yielding a three-stranded DNA repair intermediate composed of a short unhooked oligomer covalently bound to the duplex. At present, the detailed molecular mechanism of ICL repair in mammalian cells remains unclear. Here, we constructed and characterized three-stranded DNA structures containing a single ICL as substrates for the base excision repair proteins. We show that NEIL1 excises with high efficiency the unhooked ICL fragment within a three-stranded DNA structure. Complete reconstitution of the repair of unhooked ICL shows that it can be processed in a short patch base excision repair pathway. The new substrate specificity of NEIL1 points to a preferential involvement in the replication-associated repair of ICLs. Based on these data...

The characterization of a mammalian DNA structure-specific endonuclease.

Harrington, J J; Lieber, M R
Fonte: PubMed Publicador: PubMed
Tipo: Artigo de Revista Científica
Publicado em 01/03/1994 EN
Relevância na Pesquisa
45.9%
The repair of some types of DNA double-strand breaks is thought to proceed through DNA flap structure intermediates. A DNA flap is a bifurcated structure composed of double-stranded DNA and a displaced single-strand. To identify DNA flap cleaving activities in mammalian nuclear extracts, we created an assay utilizing a synthetic DNA flap substrate. This assay has allowed the first purification of a mammalian DNA structure-specific nuclease. The enzyme described here, flap endonuclease-1 (FEN-1), cleaves DNA flap strands that terminate with a 5' single-stranded end. As expected for an enzyme which functions in double-strand break repair flap resolution, FEN-1 cleavage is flap strand-specific and independent of flap strand length. Furthermore, efficient flap cleavage requires the presence of the entire flap structure. Substrates missing one strand are not cleaved by FEN-1. Other branch structures, including Holliday junctions, are also not cleaved by FEN-1. In addition to endonuclease activity, FEN-1 has a 5'-3' exonuclease activity which is specific for double-stranded DNA. The endo- and exonuclease activities of FEN-1 are discussed in the context of DNA replication, recombination and repair.

Supramolecular Structure and Nuclear targeting efficiency Determine the Enhancement of Transfection by Modified Polylysines

Chan, Chee Kai; Senden, Timothy; Jans, David A
Fonte: Nature Publishing Group Publicador: Nature Publishing Group
Tipo: Artigo de Revista Científica
Relevância na Pesquisa
45.87%
Polylysine (ply) has been used as a DNA carrier in nonviral gene delivery systems because it forms complexes with plasmid DNA via charge interaction, and condenses it into a compact structure. We have recently shown that cross-linking nuclear localization sequences (NLSs) to ply can enhance transfection by conferring specific recognition by the cellular nuclear import 'receptor', the NLS-binding importin α/β heterodimer. The present study examines and correlates for the first time the effect of the lysine/nucleotide (Ly/Nu) ratio on transfection, recognition by importin α/β, and structure as determined using electron microscopy (EM) and atomic force microscopy (AFM), for ply-DNA complexes with and without NLSs or mutant versions thereof. Intriguingly, we observed two distinct peaks of transfection enhancement at Ly/Nu ratios of 0.4 and 4.0, attributable to specific NLS recognition by importins and DNA compaction, respectively. The results indicate a clear correlation between the ply-DNA structure, importin α/β recognition, and gene transfer efficiency, thus underlining the importance of using ply-DNA at the optimal Ly/Nu ratio.