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Helicase
Kinds of Helicase Terms modified by Helicase Selected AbstractsmRNA Encoding a Putative RNA Helicase of the DEAD-Box Gene Family is Up-Regulated in Trypomastigotes of Trypanosoma cruziTHE JOURNAL OF EUKARYOTIC MICROBIOLOGY, Issue 6 2000ALBERTO M. DÍAZ AÑEL ABSTRACT. Differential display of mRNAs from Trypanosoma cruzi epimastigote and metacyclic trypomastigote stages showed several mRNA species differing in their expression level. The cDNA corresponding to one of these mRNAs was used as a probe in Northern blots and identified a RNA product of 2.6 kb with an expression level eight or more times higher in trypomastigotes than in epimastigotes. This probe was also used to screen a genomic library of T. cruzi CL Brener clone prepared in lambda FIX. A clone of about 15 kb was selected that, after partial sequencing, revealed an open reading frame of 688 amino acids encoding a deduced protein with similarity to RNA helicases of the DEAD-box gene family. The presence of the eight conserved motifs characteristic of the DEAD protein family was observed in the T. cruzi sequence, indicating that it corresponds to a putative RNA helicase gene, which we named HelTc. Southern blot analysis indicated that HelTc is a single-copy gene. Pulsed-field gel electrophoresis separation of chromosomes of several isolates of T. cruzi showed that this gene was localized in one or two chromosomal bands. [source] A Graphene-Based Platform for the Assay of Duplex-DNA Unwinding by Helicase,ANGEWANDTE CHEMIE, Issue 33 2010Hongje Jang Mit bloßen Auge zu erkennen: Mithilfe von Graphenoxid (GO) kann die Helicase-abhängige Entwindung doppelsträngiger DNA (dsDNA) quantitativ und in Echtzeit verfolgt werden. GO bindet selektiv an entwundene fluoreszenzmarkierte einzelsträngige DNA und löscht die Fluoreszenz (siehe Bild). Die Helicaseaktivität wird über die Fluoreszenzänderung registriert. [source] Assessing the link between BACH1/FANCJ and MLH1 in DNA crosslink repairENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 6 2010Sharon B. Cantor Abstract FANCJ (also known as BRIP1 or BACH1) is a DNA helicase that was originally identified by its direct interaction with the hereditary breast cancer protein, BRCA1. Similar to BRCA1, FANCJ function is essential for DNA repair and breast cancer suppression. FANCJ is also mutated in the cancer prone syndrome Fanconi anemia, for which patient cells are characterized by extreme sensitivity to agents that generate DNA interstand crosslinks. Unexpectedly, correction of the interstrand crosslink sensitivity of FANCJ-null patient cells did not require the FANCJ/BRCA1 interaction. Instead, FANCJ binding to the mismatch repair protein, MLH1 was required. Given this finding, we address the role of FANCJ and MLH1 in DNA crosslink processing and how their functions could be linked in checkpoint and/or recombination pathways. We speculate that after DNA crosslink processing and repair, the FANCJ/MLH1 interaction is critical for recovery and restart of replication. These ideas are considered and summarized in this review. Environ. Mol. Mutagen., 2010. © 2010 Wiley-Liss, Inc. [source] An evolutionary transition of vasa regulation in echinodermsEVOLUTION AND DEVELOPMENT, Issue 5 2009Celina E. Juliano SUMMARY Vasa, a DEAD box helicase, is a germline marker that may also function in multipotent cells. In the embryo of the sea urchin Strongylocentrotus purpuratus, Vasa protein is posttranscriptionally enriched in the small micromere lineage, which results from two asymmetric cleavage divisions early in development. The cells of this lineage are subsequently set aside during embryogenesis for use in constructing the adult rudiment. Although this mode of indirect development is prevalent among echinoderms, early asymmetric cleavage divisions are a derived feature in this phylum. The goal of this study is to explore how vasa is regulated in key members of the phylum with respect to the evolution of the micromere and small micromere lineages. We find that although striking similarities exist between the vasa mRNA expression patterns of several sea urchins and sea stars, the time frame of enriched protein expression differs significantly. These results suggest that a conserved mechanism of vasa regulation was shifted earlier in sea urchin embryogenesis with the derivation of micromeres. These data also shed light on the phenotype of a sea urchin embryo upon removal of the Vasa-positive micromeres, which appears to revert to a basal mechanism used by extant sea stars and pencil urchins to regulate Vasa protein accumulation. Furthermore, in all echinoderms tested here, Vasa protein and/or message is enriched in the larval coelomic pouches, the site of adult rudiment formation, thus suggesting a conserved role for vasa in undifferentiated multipotent cells set aside during embryogenesis for use in juvenile development. [source] Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domainsFEBS JOURNAL, Issue 2 2007Anne R. Karow RNA helicases mediate structural rearrangements of RNA or RNA,protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis,Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication. [source] Leishmania infantum LeIF protein is an ATP-dependent RNA helicase and an eIF4A-like factor that inhibits translation in yeastFEBS JOURNAL, Issue 22 2006Mourad Barhoumi LeIF, a Leishmania protein similar to the eukaryotic initiation factor eIF4A, which is a prototype of the DEAD box protein family, was originally described as a Th1-type natural adjuvant and as an antigen that induces an IL12-mediated Th1 response in the peripheral blood mononuclear cells of leishmaniasis patients. This study aims to characterize this protein by comparative biochemical and genetic analysis with eIF4A in order to assess its potential as a target for drug development. We show that a His-tagged, recombinant, LeIF protein of Leishmania infantum, which was purified from Escherichia coli, is both an RNA-dependent ATPase and an ATP-dependent RNA helicase in vitro, as described previously for other members of the DEAD box helicase protein family. In vivo experiments show that the LeIF gene cannot complement the deletion of the essential TIF1 and TIF2 genes in the yeast Saccharomyces cerevisiae that encode eIF4A. In contrast, expression of LeIF inhibits yeast growth when endogenous eIF4A is expressed off only one of its two encoding genes. Furthermore, in vitro binding assays show that LeIF interacts with yeast eIF4G. These results show an unproductive interaction of LeIF with translation initiation factors in yeast. Furthermore, the 25 amino terminal residues were shown to enhance the ability of LeIF to interfere with the translation machinery in yeast. [source] Monomeric solution structure of the helicase-binding domain of Escherichia coli DnaG primaseFEBS JOURNAL, Issue 21 2006Xun-Cheng Su DnaG is the primase that lays down RNA primers on single-stranded DNA during bacterial DNA replication. The solution structure of the DnaB-helicase-binding C-terminal domain of Escherichia coli DnaG was determined by NMR spectroscopy at near-neutral pH. The structure is a rare fold that, besides occurring in DnaG C-terminal domains, has been described only for the N-terminal domain of DnaB. The C-terminal helix hairpin present in the DnaG C-terminal domain, however, is either less stable or absent in DnaB, as evidenced by high mobility of the C-terminal 35 residues in a construct comprising residues 1,171. The present structure identifies the previous crystal structure of the E. coli DnaG C-terminal domain as a domain-swapped dimer. It is also significantly different from the NMR structure reported for the corresponding domain of DnaG from the thermophile Bacillus stearothermophilus. NMR experiments showed that the DnaG C-terminal domain does not bind to residues 1,171 of the E. coli DnaB helicase with significant affinity. [source] Human ATP-dependent RNA/DNA helicase hSuv3p interacts with the cofactor of survivin HBXIPFEBS JOURNAL, Issue 19 2005Michal Minczuk The human SUV3gene encodes an NTP-dependent DNA/RNA DExH box helicase predominantly localized in mitochondria. Its orthologue in yeast is a component of the mitochondrial degradosome complex involved in the mtRNA decay pathway. In contrast to this, the physiological function of human SUV3 remains to be elucidated. In this report we demonstrate that the hSuv3 protein interacts with HBXIP, previously identified as a cofactor of survivin in suppression of apoptosis and as a protein that binds the HBx protein encoded by the hepatitis B virus. Using deletion analysis we identified the region within the hSuv3 protein, which is responsible for binding to HBXIP. The HBXIP binding domain was found to be important for mitochondrial import and stability of the Suv3 protein in vivo. We discuss the possible involvement of the hSuv3p,HBXIP interaction in the survivin-dependent antiapoptotic pathway. [source] Structural insight of human DEAD-box protein rck/p54 into its substrate recognition with conformational changesGENES TO CELLS, Issue 4 2006Tsutomu Matsui Human rck/p54, a product of the gene cloned at the breakpoint of t(11; 14) (q23;q32) chromosomal translocation on 11q23 in B-cell lymphoma, is a member of the DEAD-box RNA helicase family. Here, the crystal structure of Nc-rck/p54, the N-terminal core domain of rck/p54, revealed that the P-loop in motif I formed a closed conformation, which was induced by Asn131, a residue unique to the RCK subfamily. It appears that ATP does not bind to the P-loop. The results of dynamic light scattering revealed to ATP-induced conformational change of rck/p54. It was demonstrated that free rck/p54 is a distended molecule in solution, and that the approach between N-terminal core and C-terminal domains for ATP binding would be essential when unwinding RNA. The results from helicase assay using electron micrograph, ATP hydrolytic and luciferase assay showed that c-myc IRES RNA, whose secondary structure regulates IRES-dependant translation, was unwound by rck/p54 and indicated that it is a good substrate for rck/p54. Over-expression of rck/p54 in HeLa cells caused growth inhibition and cell cycle arrest at G2/M with down-regulation of c-myc expression. These findings altogether suggest that rck/p54 may affect the IRES-dependent translation of c-myc even in the cells. [source] The archaeal Hjm helicase has recQ-like functions, and may be involved in repair of stalled replication forkGENES TO CELLS, Issue 2 2006Ryosuke Fujikane The archaeal Hjm is a structure-specific DNA helicase, which was originally identified in the hyperthermophilic archaeon, Pyrococcus furiosus, by in vitro screening for Holliday junction migration activity. Further biochemical analyses of the Hjm protein from P. furiosus showed that this protein preferably binds to fork-related Y-structured DNAs and unwinds their double-stranded regions in vitro, just like the E. coli RecQ protein. Furthermore, genetic analyses showed that Hjm produced in E. coli cells partially complemented the defect of functions of RecQ in a recQ mutant E. coli strain. These results suggest that Hjm may be a functional counterpart of RecQ in Archaea, in which it is necessary for the maintenance of genome integrity, although the amino acid sequences are not conserved. The functional interaction of Hjm with PCNA for its helicase activity further suggests that the Hjm works at stalled replication forks, as a member of the reconstituted replisomes to restart replication. [source] Rep helicase suppresses short-homology-dependent illegitimate recombination in Escherichia coliGENES TO CELLS, Issue 11 2005Kouya Shiraishi To study roles of Rep helicase in short-homology-dependent illegitimate recombination, we examined the effect of a rep mutation on illegitimate recombination and found that the frequency of spontaneous illegitimate recombination is enhanced by the rep mutation. In addition, illegitimate recombination was synergistically enhanced by the rep mutation and UV irradiation, showing that Rep helicase plays a role in suppression of spontaneous as well as UV-induced illegitimate recombination. The defect in RecQ helicase also has a synergistic effect on the increased illegitimate recombination in the rep mutant. It was also found that the illegitimate recombination induced by the rep mutation is independent of the RecA function with or without UV irradiation. Nucleotide sequence analyses of the recombination junctions showed that the illegitimate recombination induced by the rep mutation mostly takes place between short homologous sequences. Based on the fact that the defect of Rep helicase induces replication arrest during replication, resulting in the formation of DNA double-strand breaks, we propose a model for illegitimate recombination, in which double-strand breaks caused by defect of Rep helicase promotes illegitimate recombination via short-homology-dependent-end-joining. In addition, the mechanism of synergistic action between the rep mutation and UV irradiation on illegitimate recombination is discussed. [source] Control of DNA replication licensing in a cell cycleGENES TO CELLS, Issue 6 2002Hideo Nishitani To maintain genome integrity in eukaryotes, DNA must be duplicated precisely once before cell division occurs. A process called replication licensing ensures that chromosomes are replicated only once per cell cycle. Its control has been uncovered by the discovery of the CDKs (cyclin dependent kinases) as master regulators of the cell cycle and the initiator proteins of DNA replication, such as the Origin Recognition Complex (ORC), Cdc6/18, Cdt1 and the MCM complex. At the end of mitosis, the MCM complex is loaded on to chromatin with the aid of ORC, Cdc6/18 and Cdt1, and chromatin becomes licensed for replication. CDKs, together with the Cdc7 kinase, trigger the initiation of replication, recruiting the DNA replicating enzymes on sites of replication. The activated MCM complex appears to play a key role in the DNA unwinding step, acting as a replicating helicase and moves along with the replication fork, at the same time bringing the origins to the unlicensed state. The cycling of CDK activity in the cell cycle separates the two states of replication origins, the licensed state in G1-phase and the unlicensed state for the rest of the cell cycle. Only when CDK drops at the completion of mitosis, is the restriction on licensing relieved and a new round of replication is allowed. Such a CDK-regulated licensing control is conserved from yeast to higher eukaryotes, and ensures that DNA replication takes place only once in a cycle. Xenopus laevis and mammalian cells have an additional system to control licensing. Geminin, whose degradation at the end of mitosis is essential for a new round of licensing, has been shown to bind Cdt1 and negatively regulate it, providing a new insight into the regulation of DNA replication in higher eukaryotes. [source] Modulation of dendritic cell maturation and function with mono- and bifunctional small interfering RNAs targeting indoleamine 2,3-dioxygenaseIMMUNOLOGY, Issue 1pt2 2009Gro F. Flatekval Summary Antigen-presenting cells expressing indoleamine 2,3-dioxygenase (IDO) play a critical role in maintaining peripheral tolerance. Strategies to inhibit IDO gene expression and enhance antigen-presenting cell function might improve anti-tumour immunity. Here we have designed highly effective anti-IDO small interfering (si) RNAs that function at low concentrations. When delivered to human primary immune cells such as monocytes and dendritic cells (DCs), they totally inhibited IDO gene expression without impairing DC maturation and function. Depending on the design and chemical modifications, we show that it is possible to design either monofunctional siRNAs devoid of immunostimulation or bifunctional siRNAs with gene silencing and immunostimulatory activities. The latter are able to knockdown IDO expression and induce cytokine production through either endosomal Toll-like receptor 7/8 or cytoplasmic retinoid acid-inducible gene 1 helicase. Inhibition of IDO expression with both classes of siRNAs inhibited DC immunosuppressive function on T-cell proliferation. Immature monocyte-derived DCs that had been transfected with siRNA-bearing 5,-triphosphate activated T cells, indicating that, even in the absence of external stimuli such as tumour necrosis factor-,, those DCs were sufficiently mature to initiate T-cell activation. Collectively, our data highlight the potential therapeutic applications of this new generation of siRNAs in immunotherapy. [source] Changes in Drosophila melanogaster midgut proteins in response to dietary Bowman,Birk inhibitorINSECT MOLECULAR BIOLOGY, Issue 5 2007H.-M. Li Abstract The midgut proteome of Drosophila melanogaster was compared in larvae fed dietary Bowman,Birk inhibitor (BBI) vs. larvae fed a control diet. By using two-dimensional gel electrophoresis, nine differentially expressed proteins were observed, which were associated with enzymes or transport functions such as sterol carrier protein X (SCPX), ubiquitin-conjugating enzyme, endopeptidase, receptor signalling protein kinase, ATP-dependent RNA helicase and ,-tocopherol transport. Quantitative real-time PCR verified differential expression of transcripts coding for six of the proteins observed from the proteomic analysis. BBI evidently affects expression of proteins associated with protein degradation, transport and fatty acid catabolism. We then tested the hypothesis that SCPX was critical for the Drosophila third instars' response to BBI treatment. Inhibition of SCPX caused the third instars to become more susceptible to dietary BBI. [source] WRN, the protein deficient in Werner syndrome, plays a critical structural role in optimizing DNA repairAGING CELL, Issue 4 2003Lishan Chen Summary Werner syndrome (WS) predisposes patients to cancer and premature aging, owing to mutations in WRN. The WRN protein is a RECQ-like helicase and is thought to participate in DNA double-strand break (DSB) repair by non-homologous end joining (NHEJ) or homologous recombination (HR). It has been previously shown that non-homologous DNA ends develop extensive deletions during repair in WS cells, and that this WS phenotype was complemented by wild-type (wt) WRN. WRN possesses both 3, , 5, exonuclease and 3, , 5, helicase activities. To determine the relative contributions of each of these distinct enzymatic activities to DSB repair, we examined NHEJ and HR in WS cells (WRN,/,) complemented with either wtWRN, exonuclease-defective WRN (E,), helicase-defective WRN (H,) or exonuclease/helicase-defective WRN (E,H,). The single E, and H, mutants each partially complemented the NHEJ abnormality of WRN,/, cells. Strikingly, the E,H, double mutant complemented the WS deficiency nearly as efficiently as did wtWRN. Similarly, the double mutant complemented the moderate HR deficiency of WS cells nearly as well as did wtWRN, whereas the E, and H, single mutants increased HR to levels higher than those restored by either E,H, or wtWRN. These results suggest that balanced exonuclease and helicase activities of WRN are required for optimal HR. Moreover, WRN appears to play a structural role, independent of its enzymatic activities, in optimizing HR and efficient NHEJ repair. Another human RECQ helicase, BLM, suppressed HR but had little or no effect on NHEJ, suggesting that mammalian RECQ helicases have distinct functions that can finely regulate recombination events. [source] The RNA degradosome in Bacillus subtilis: identification of CshA as the major RNA helicase in the multiprotein complexMOLECULAR MICROBIOLOGY, Issue 4 2010Martin Lehnik-Habrink Summary In most organisms, dedicated multiprotein complexes, called exosome or RNA degradosome, carry out RNA degradation and processing. In addition to varying exoribonucleases or endoribonucleases, most of these complexes contain a RNA helicase. In the Gram-positive bacterium Bacillus subtilis, a RNA degradosome has recently been described; however, no RNA helicase was identified. In this work, we tested the interaction of the four DEAD box RNA helicases encoded in the B. subtilis genome with the RNA degradosome components. One of these helicases, CshA, is able to interact with several of the degradosome proteins, i.e. RNase Y, the polynucleotide phosphorylase, and the glycolytic enzymes enolase and phosphofructokinase. The determination of in vivo protein,protein interactions revealed that CshA is indeed present in a complex with polynucleotide phosphorylase. CshA is composed of two RecA-like domains that are found in all DEAD box RNA helicases and a C-terminal domain that is present in some members of this protein family. An analysis of the contribution of the individual domains of CshA revealed that the C-terminal domain is crucial both for dimerization of CshA and for all interactions with components of the RNA degradosome, including RNase Y. A transfer of this domain to CshB allowed the resulting chimeric protein to interact with RNase Y suggesting that this domain confers interaction specificity. As a degradosome component, CshA is present in the cell in similar amounts under all conditions. Taken together, our results suggest that CshA is the functional equivalent of the RhlB helicase of the Escherichia coli RNA degradosome. [source] Staphylococcus aureus primase has higher initiation specificity, interacts with single-stranded DNA stronger, but is less stimulated by its helicase than Escherichia coli primaseMOLECULAR MICROBIOLOGY, Issue 6 2008Scott A. Koepsell Summary The study of primases from model organisms such as Escherichia coli, phage T7 and phage T4 has demonstrated the essential nature of primase function, which is to generate de novo RNA polymers to prime DNA polymerase. However, little is known about the function of primases from other eubacteria. Their overall low primary sequence homology may result in functional differences. To help understand which primase functions were conserved, primase and its replication partner helicase from the pathogenic Gram-positive bacteria Staphylococcus aureus were compared in detail with that of E. coli primase and helicase. The conserved properties were to primer initiation and elongation and included slow kinetics, low fidelity and poor sugar specificity. The significant differences included S. aureus primase having sixfold higher kinetic affinity for its template than E. coli primase under equivalent conditions. This naturally higher activity was balanced by its fourfold lower stimulation by its replication fork helicase compared with E. coli primase. The most significant difference between the two primases was that S. aureus helicase stimulation did not broaden the S. aureus primase initiation specificity, which has important biological implications. [source] Strand-specific loading of DnaB helicase by DnaA to a substrate mimicking unwound oriCMOLECULAR MICROBIOLOGY, Issue 4 2002Christoph Weigel Summary We analysed the enzymatic activity (strand dis-placement) of the Escherichia coli DnaB helicase on a mirror-image pair of oligonucleotide-based substrates mimicking the unwound replication origin oriC. Loading of the helicase complex occurred exclusively to the single-stranded ,lower strand' part of the substrates. Full helicase activity required DnaA bound to the double-stranded part of the substrates (oriC DnaA box R1) and to their single-stranded ,upper strand' part. We assume that in vivo DnaA also loads the first of two helicase complexes , required for the assembly of two replication forks , to the lower strand of oriC during initiation of bidirectional chromosome replication in E. coli. [source] Interaction of the plant glycine-rich RNA-binding protein MA16 with a novel nucleolar DEAD box RNA helicase protein from Zea maysTHE PLANT JOURNAL, Issue 6 2004Elisenda Gendra Summary The maize RNA-binding MA16 protein is a developmentally and environmentally regulated nucleolar protein that interacts with RNAs through complex association with several proteins. By using yeast two-hybrid screening, we identified a DEAD box RNA helicase protein from Zea mays that interacted with MA16, which we named Z. maysDEAD box RNA helicase 1 (ZmDRH1). The sequence of ZmDRH1 includes the eight RNA helicase motifs and two glycine-rich regions with arginine,glycine-rich (RGG) boxes at the amino (N)- and carboxy (C)-termini of the protein. Both MA16 and ZmDRH1 were located in the nucleus and nucleolus, and analysis of the sequence determinants for their cellular localization revealed that the region containing the RGG motifs in both proteins was necessary for nuclear/nucleolar localization The two domains of MA16, the RNA recognition motif (RRM) and the RGG, were tested for molecular interaction with ZmDRH1. MA16 specifically interacted with ZmDRH1 through the RRM domain. A number of plant proteins and vertebrate p68/p72 RNA helicases showed evolutionary proximity to ZmDRH1. In addition, like p68, ZmDRH1 was able to interact with fibrillarin. Our data suggest that MA16, fibrillarin, and ZmDRH1 may be part of a ribonucleoprotein complex involved in ribosomal RNA (rRNA) metabolism. [source] Characterisation of gene expression in bovine adipose tissue before and after fatteningANIMAL GENETICS, Issue 3 2000M Oishi Summary It has been reported that fattening causes bovine adipose tissue development associated with an enlargement in adipocyte cell size. As a first study to elucidate mechanisms of bovine adipose tissue development during fattening, our experiment was designed to characterise gene expression in bovine adipose tissue before and after fattening. We randomly isolated a large number of cDNA clones derived from bovine adipose tissue before and after fattening. Sequence analysis of the isolated clones showed that 3 and 10 clones from before and after fattening, respectively, correspond to genes related to adipocyte development and/or function in the adipose tissue. In addition, we isolated cDNA clones that possess negative signal by hybridising the cDNA population from the adipose tissue after fattening with that before fattening as a probe. As a result, we identified five types of transcripts observed in the adipose tissue after fattening but not before fattening. Two of the five are likely to encode bovine orthologs of phospholipase A2 and RNA helicase p68, while the other three represent unknown genes. Further functional investigation of the identified genes might lead to elucidation of mechanisms of bovine adipose tissue development during fattening. [source] RNA helicase encoded by melanoma differentiation,associated gene 5 is a major autoantigen in patients with clinically amyopathic dermatomyositis: Association with rapidly progressive interstitial lung disease,ARTHRITIS & RHEUMATISM, Issue 7 2009Shinji Sato Objective To identify the autoantigen recognized by the autoantibody that is associated with clinically amyopathic dermatomyositis (C-ADM) and rapidly progressive interstitial lung disease (ILD). Methods An anti,CADM-140 antibody,positive prototype serum sample was used to screen a HeLa cell,derived complementary DNA (cDNA) library. Selected cDNA clones were further evaluated by immunoprecipitation of their in vitro,transcribed and in vitro,translated products using anti,CADM-140 antibody,positive and anti-CADM-140 antibody,negative sera. The lysates of COS-7 cells transfected with the putative antigen were similarly tested. An enzyme-linked immunosorbent assay (ELISA) to detect the anti,CADM-140 antibody was established using a recombinant CADM-140 antigen, and its specificity and sensitivity for C-ADM and rapidly progressive ILD were assessed in 294 patients with various connective tissue diseases. Results By cDNA library screening and immunoprecipitation of in vitro,transcribed and in vitro,translated products, we obtained a cDNA clone encoding melanoma differentiation,associated gene 5 (MDA-5). The anti,CADM-140 antibodies in patients' sera specifically reacted with MDA-5 protein expressed in cells transfected with full-length MDA-5 cDNA, confirming the identity of MDA-5 as the CADM-140 autoantigen. The ELISA, using recombinant MDA-5 protein as the antigen, showed an analytical sensitivity of 85% and analytical specificity of 100%, in comparison with the "gold standard" immunoprecipitation assay, and was useful for identifying patients with C-ADM and/or rapidly progressive ILD. Conclusion Given that RNA helicase encoded by MDA-5 is a critical molecule involved in the innate immune defense against viruses, viral infection may play an important role in the pathogenesis of C-ADM and rapidly progressive ILD. Moreover, our ELISA using recombinant MDA-5 protein makes detection of the anti,CADM-140 antibody routinely available. [source] Global surface ultraviolet radiation intensity may modulate the clinical and immunologic expression of autoimmune muscle diseaseARTHRITIS & RHEUMATISM, Issue 8 2003Satoshi Okada Objective To determine if geoclimatic factors may influence the nature and frequency of dermatomyositis (DM), polymyositis, and associated autoantibodies around the world. Methods We assessed, in the first global evaluation of these conditions, the relationship between 13 geoclimatic variables that may modulate disease and the relative proportion of DM and its associated autoantibody anti,Mi-2, directed against an SNF2-superfamily helicase associated with the nucleosome remodeling and histone acetylation and deacetylation complex, in a global myositis population. Altogether, 919 consecutive patients from populations at 15 locations were studied. Results Univariate and multivariate analyses demonstrated that of the variables evaluated, surface ultraviolet (UV) radiation intensity (irradiance) most strongly contributed to the relative proportion of DM and was strongly related to the proportion of anti,Mi-2 autoantibodies (weighted r = 0.939, P < 4 × 10 -7 and weighted r = 0.69, P = 0.02, respectively). Published ethnogeographic immunogenetic allele frequencies imply that the striking differences in the proportion of DM- and DM-specific autoantibodies observed around the world are not the result of inherent global variations in known genetic risk factors. Conclusion These data suggest that UV radiation exposure may modulate the clinical and immunologic expression of an autoimmune disease in different populations around the world. [source] Crystallization and preliminary X-ray analysis of RecG, a replication-fork reversal helicase from Thermotoga maritima complexed with a three-way DNA junctionACTA CRYSTALLOGRAPHICA SECTION D, Issue 11 2001Martin R. Singleton The monomeric 3,-5, helicase RecG from the thermophilic bacterium Thermotoga maritima has been crystallized in complex with a three-way DNA junction, the preferred physiological substrate. The crystals were obtained by hanging-drop vapour diffusion. The crystals belong to space group C2, with unit-cell parameters a = 133.7, b = 144.6, c = 84.0,Å, , = 113.8°. Native data to a resolution of 3.25,Å were collected from crystals flash-cooled to 100,K. [source] Stalled replication forks: Making ends meet for recognition and stabilizationBIOESSAYS, Issue 8 2010Hisao Masai Abstract In bacteria, PriA protein, a conserved DEXH-type DNA helicase, plays a central role in replication restart at stalled replication forks. Its unique DNA-binding property allows it to recognize and stabilize stalled forks and the structures derived from them. Cells must cope with fork stalls caused by various replication stresses to complete replication of the entire genome. Failure of the stalled fork stabilization process and eventual restart could lead to various forms of genomic instability. The low viability of priA null cells indicates a frequent occurrence of fork stall during normal growth that needs to be properly processed. PriA specifically recognizes the 3,-terminus of the nascent leading strand or the invading strand in a displacement (D)-loop by the three-prime terminus binding pocket (TT-pocket) present in its unique DNA binding domain. Elucidation of the structural basis for recognition of arrested forks by PriA should provide useful insight into how stalled forks are recognized in eukaryotes. [source] Crystallization and preliminary crystallographic studies of human RIG-I in complex with double-stranded RNAACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 6 2009Hyunjin Moon Retinoic acid inducible gene-I (RIG-I) is an essential component of the innate immune system that is responsible for the detection and elimination of invading viruses. RIG-I recognizes viral RNAs inside the cell and then initiates downstream signalling to activate the IRF-3 and NF-,B genes, which results in the production of type I interferons. RIG-I is composed of an N-terminal CARD domain for signalling and C-terminal helicase and repressor domains for RNA recognition. A RIG-I,RNA binding assay was performed to investigate the in vitro RIG-I,RNA binding properties. Selenomethionine-incorporated RIG-I was expressed using Escherichia coli and purified for crystallization. X-ray data were collected from RIG-I,dsRNA complex crystals to 2.8,Å resolution using synchrotron radiation. [source] Crystallization and preliminary characterization of the Thermus thermophilus RNA helicase Hera C-terminal domainACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 3 2009Markus G. Rudolph Heat-resistant RNA-dependent ATPase (Hera) from Thermus thermophilus is a DEAD-box RNA helicase. Two constructs encompassing the second RecA-like domain and the C-terminal domain of Hera were overproduced in Escherichia coli and purified to homogeneity. Single crystals of both Hera constructs were obtained in three crystal forms. A tetragonal crystal form belonged to space group P41212, with unit-cell parameters a = 65.5, c = 153.0,Å, and contained one molecule per asymmetric unit. Two orthorhombic forms belonged to space group P212121, with unit-cell parameters a = 62.8, b = 70.9, c = 102.3,Å (form I) and a = 41.6, b = 67.6, c = 183.5,Å (form II). Both orthorhombic forms contained two molecules per asymmetric unit. All crystals diffracted X-rays to beyond 3,Å resolution, but the tetragonal data sets displayed high Wilson B values and high mean |E2, 1| values, indicating potential disorder and anisotropy. The tetragonal crystal was phased by MAD using a single selenium site. [source] Induction of mitotic cell death in cancer cells by small interference RNA suppressing the expression of RecQL1 helicaseCANCER SCIENCE, Issue 1 2008Kazunobu Futami RecQL1 DNA helicase of the human RecQ helicase family participates in DNA repair and recombination pathways during cell-cycle replication. When we examined the effect of RecQL1 suppression on cell growth, we found that RecQL1 silencing by small interference RNA efficiently prevented proliferation of a wide range of cancer cells by inducing mitotic catastrophe and mitotic cell death. In contrast, such mitotic cell death was not seen in the growing normal fibroblasts used as controls, even if RecQL1 expression was fully downregulated. Our results support the hypothesis that endogenous DNA damage that occurs during DNA replication and remains unrepaired in cancer cells due to RecQL1 silencing induces cancer cell-specific mitotic catastrophe through a less-strict checkpoint in cancer cells than in normal cells. We speculate that normal cells are exempt from such mitotic cell death, despite slow growth, because cell-cycle progression is controlled strictly by a strong checkpoint system that detects DNA damage and arrests progression of the cell cycle until DNA damage is repaired completely. These results suggest that RecQL1 helicase is an excellent molecular target for cancer chemotherapy. (Cancer Sci 2008; 99: 71,80) [source] Mutations in PHD-like domain of the ATRX gene correlate with severe psychomotor impairment and severe urogenital abnormalities in patients with ATRX syndromeCLINICAL GENETICS, Issue 1 2006C Badens Mutations in ATRX are associated with a wide and clinically heterogeneous spectrum of X-linked mental retardation syndromes. The ATRX protein, involved in chromatin remodelling, belongs to the family of SWI/SNF DNA helicases and contains a plant homeodomain (PHD)-like domain. To date, more than 60 different mutations have been reported in ATRX. One of them is recurrent and accounts for 20% of all the reported mutations, whereas all others are private. Most mutations are clustered in the two major functional domains, the helicase and the PHD-like domain. So far, no clear genotype,phenotype correlation has been established, with exception to the rare truncating mutations located at the C-terminal part of the protein, which are consistently associated with severe urogenital defects. In this study, we report the molecular analysis performed in 16 families positive for ATRX. Our findings indicate that, in addition to the previously described mutation ,hotspot' in the PHD-like domain, two other protein sections emerge as minor ,hotspots' in the helicase region encoded by exons 18,20 and 26,29, respectively, gathering 33% of all described mutations. Additionally, based on the clinical data collected for 22 patients from the 16 families, we observe that mutations in the PHD-like domain produce severe and permanent psychomotor deficiency, usually preventing patients from walking, as well as constant urogenital abnormalities, while mutations in the helicase domain lead to delayed but correct psychomotor acquisitions together with mild or absent urogenital abnormalities. In summary, mutations in the helicase domain are associated with milder phenotypes than mutations in the PHD-like domain. [source] Authentic interdomain communication in an RNA helicase reconstituted by expressed protein ligation of two helicase domainsFEBS JOURNAL, Issue 2 2007Anne R. Karow RNA helicases mediate structural rearrangements of RNA or RNA,protein complexes at the expense of ATP hydrolysis. Members of the DEAD box helicase family consist of two flexibly connected helicase domains. They share nine conserved sequence motifs that are involved in nucleotide binding and hydrolysis, RNA binding, and helicase activity. Most of these motifs line the cleft between the two helicase domains, and extensive communication between them is required for RNA unwinding. The two helicase domains of the Bacillus subtilis RNA helicase YxiN were produced separately as intein fusions, and a functional RNA helicase was generated by expressed protein ligation. The ligated helicase binds adenine nucleotides with very similar affinities to the wild-type protein. Importantly, its intrinsically low ATPase activity is stimulated by RNA, and the Michaelis,Menten parameters are similar to those of the wild-type. Finally, ligated YxiN unwinds a minimal RNA substrate to an extent comparable to that of the wild-type helicase, confirming authentic interdomain communication. [source] Dicer-related drh-3 gene functions in germ-line development by maintenance of chromosomal integrity in Caenorhabditis elegansGENES TO CELLS, Issue 9 2007Masaharu Nakamura In the course of systematic RNA interference (RNAi)-based screens with helicase-like genes in Caenorhabditis elegans, we have identified the drh-3(D2005.5) gene as a candidate gene for protection against X-ray irradiation. This gene encodes a novel RNA helicase-like protein that is similar to two nematode Dicer-related helicases (DRH). Here, we have showed the increased expression of drh-3 transcripts during maturation of larvae to adults, and characterized the phenotype of drh-3 -interferred nematodes using feeding RNAi method. RNAi-mediated depletion of the drh-3 transcripts caused embryonic lethality of F1 progeny and temperature-sensitive reproductive capacity but did not affect the nematode life span. F1 progeny from drh-3(RNAi) animals exhibited increased lethality after X-ray irradiation or exposure to camptothecin. In drh-3(RNAi) worms, aggregated chromosomes were observed in diakinesis oocyte nuclei. In developing early zygotic embryos from drh-3(RNAi) worms, abnormally segregated chromosomes were observed and embryonic development was largely arrested at the mid-stages of embryogenesis. Finally, examination of checkpoint responses in mitotic germ cells with regards to replication arrest by hydroxyurea and X-ray-induced DNA damage suggested that both checkpoints function normally under these genotoxic stress conditions. Taken together, these results indicate that the drh-3 gene is essential for the development of germ-lines by maintaining chromosomal integrity in C. elegans. [source] | ||||||||||||||||