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Full Text Available.BackgroundAbout 130 million people are infected with the hepatitis C virus (HCV) worldwide, but effective treatment options are not yet available. One of the most promising targets for antiviral therapy is nonstructural protein 3 (NS3). To identify possible changes in the structure of NS3 associated with virological sustained response or non-response of patients, a model was constructed for each helicase NS3 protein coding sequence. From this, the goal was to verify the interaction between helicases variants and their ligands.FindingsEvidence was found that the NS3 helicase portion of non-responder patients contained substitutions in its ATP and RNA binding sites. K210E substitution can cause an imbalance in the distribution of loads, leading to a decrease in the number of ligations between the essential amino acids required for the hydrolysis of ATP. W501R substitution causes an imbalance in the distribution of loads, leading and forcing the RNA to interact with the amino acid Thr269, but not preventing binding of ribavirin inhibitor.ConclusionsUseful information is provided on the genetic profiling of the HCV genotype 3, specifically the coding region of the NS3 protein, improving our understanding of the viral genome and the regions of its protein catalytic site.
Full Text Available.BackgroundHepatitis C virus (HCV) infection is a major public health problem with more than 170 million cases of chronic infections worldwide. There is no protective vaccine currently available for HCV, therefore the development of novel strategy to prevent chronic infection is important. We reported earlier that a recombinant human antibody clone blocks viral NS3 helicase activity and inhibits replication of HCV 1b virus. This study was performed further to explore the mechanism of action of this recombinant antibody and to determine whether or not this antibody inhibits replication and infectivity of a highly efficient JFH1 HCV 2a virus clone.ResultsThe antiviral effect of intracellular expressed antibody against the HCV 2a virus strain was examined using a full-length green fluorescence protein (GFP) labeled infectious cell culture system. For this purpose, a Huh-7.5 cell line stably expressing the NS3 helicase gene specific IgG1 antibody was prepared. Replication of full-length HCV-GFP chimera RNA and negative-strand RNA was strongly inhibited in Huh-7.5 cells stably expressing NS3 antibody but not in the cells expressing an unrelated control antibody. Huh-7.5 cells stably expressing NS3 helicase antibody effectively suppressed infectious virus production after natural infection and the level of HCV in the cell free supernatant remained undetectable after first passage. In contrast, Huh-7.5 cells stably expressing an control antibody against influenza virus had no effect on virus production and high-levels of infectious HCV were detected in culture supernatants over four rounds of infectivity assay. A recombinant adenovirus based expression system was used to demonstrate that Huh-7.5 replicon cell line expressing the intracellular antibody strongly inhibited the replication of HCV-GFP RNA.ConclusionRecombinant human anti-HCV NS3 antibody clone inhibits replication of HCV 2a virus and infectious virus production. Intracellular expression of this recombinant antibody offers a potential antiviral strategy to inhibit intracellular HCV replication and production.
Preliminary crystallographic characterization of an RNA helicase from Kunjin virus
2006-01-01
Kunjin virus is a member of the Flavivirus genus and is an Australian variant of West Nile virus. The C-terminal domain of the Kunjin virus NS3 protein displays helicase activity. The protein is thought to separate daughter and template RNA strands, assisting the initiation of replication by unwinding RNA secondary structure in the 3' nontranslated region. Expression, purification and preliminary crystallographic characterization of the NS3 helicase domain are reported. It is shown that Kunjin virus helicase may adopt a dimeric assembly in absence of nucleic acids, oligomerization being a means to provide the helicases with multiple nucleic acid-binding capability, facilitating translocation along the RNA strands. Kunjin virus NS3 helicase domain is an attractive model for studying the molecular mechanisms of flavivirus replication, while simultaneously providing a new basis for the rational development of anti-flaviviral compounds. Publisher: International Union of Crystallography Coverage: 2006-01-01T00:00:00Z
A model for DNA helicase mechanism based on a flashing ratchet
2007-07-19
Helicases are molecular motors that consume energy supplied by chemical reactions to unwind double-stranded nucleic acids (like DNA and RNA) and to translocate along one of the single-strands. Motivated by the recent claims, based on experimental observations on the helicase NS3 of hepatitis C virus (HCV), that monomeric helicases are governed by a Brownian ratchet mechanism, here we develope a quantitative model. Our Brownian ratchet model, which is a somewhat new reformulation of the Betterton-J\\"ulicher theory of helicases, is generic two-state model and is applicable to all helicases which follow the Brownian ratchet mechanism. We illustrate the predictive power of the model by calculating some experimentally testable motor properties of a few monomeric helicases. Speficically, we predict the speed of unwinding of the double-stranded DNA and fluctuations around the average drift of the helicase. Our predictions are in excellent quantitative agreement with the corresponding experimental data.
2009-01-01
We have developed a novel high-throughput screening assay of hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase inhibitors using the fluorescence-quenching phenomenon via photoinduced electron transfer between fluorescent dyes and guanine bases. We prepared double-stranded DNA (dsDNA) with a 5'-fluorescent-dye (BODIPY FL)-labeled strand hybridized with a complementary strand, the 3'-end of which has guanine bases. When dsDNA is unwound by helicase, the dye emits fluorescence owing to its release from the guanine bases. Our results demonstrate that this assay is suitable for quantitative assay of HCV NS3 helicase activity and useful for high-throughput screening for inhibitors. Furthermore, we applied this assay to the screening for NS3 helicase inhibitors from cell extracts of microorganisms, and found several cell extracts containing potential ...
2010-01-01
The nonstructural protein 3 helicase (NS3h) of hepatitis C virus is a 3'-to-5' superfamily 2 RNA and DNA helicase that is essential for the replication of hepatitis C virus. We have examined the kinetic mechanism of the translocation of NS3h along single-stranded nucleic acid with bases uridylate (rU), deoxyuridylate (dU), and deoxythymidylate (dT), and have found that the macroscopic rate of translocation is dependent on both the base moiety and the sugar moiety of the nucleic acid, with approximate macroscopic translocation rates of 3 nt s^-^1 (oligo(dT)), 35 nt s^-^1 (oligo(dU)), and 42 nt s^-^1 (oligo(rU)), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein for the respective substrates such...
2006-06-01
Intracellular RNA virus infection is detected by the cytoplasmic RNA helicase RIG-I that plays an essential role in signaling to the host antiviral response. Recently, the adapter molecule that links...Full Text Available
Novel 99mTc `4 + 1' peptide conjugates: Tuning the biodistribution by variation of coligands
2010-01-01
A sophisticated coligand strategy is presented for peptide-derived radioconjugates based on 99mTc `4 + 1' mixed-ligand complexes. The new pharmacologically active coligands are assessed for 99mTc-labeling of the RGD-peptide cyclo(Arg-Gly-Asp-d-Tyr-Lys) which is an established vehicle to target avb3 integrins playing a crucial part in tumor pathogenesis. Complexes of the general formula [99mTc(NS3R)X] were synthesized and evaluated, in which Tc(III) is coordinated by NS3R, a derivative of the tetradentate chelator 2,2prime,2Prime-nitrilotriethanethiol (NS3), and by X, a monodentate binding isocyanide bearing the biomolecule. The novel tetradentate chelators (NS3R = NS3crown, NS3en, NS3(COOH)3) constitute NS3 with a crown ether, an amine or a tricarboxylic acid as pharmacological modifiers. ...
2010-04-09
Full Text Available.The superfamily 2 vaccinia viral helicase nucleoside triphosphate phosphohydrolase-II (NPH-II) exhibits robust RNA helicase activity but typically displays little activity on DNA substrates. NPH-II is thus believed to make primary contacts with backbone residues of an RNA substrate. We report an unusual nucleobase bias, previously unreported in any superfamily 1 or 2 helicase, whereby purines are heavily preferred as components of both RNA and DNA tracking strands. The observed sequence bias allows NPH-II to efficiently unwind a DNA·RNA hybrid containing a purine-rich DNA track derived from the 3′-untranslated region of an early vaccinia gene. These results provide insight into potential biological functions of NPH-II and the role of sequence in targeting NPH-II to appropriate substrates. Furthermore, they demonstrate that in addition to backbone contacts, nucleotide bases play an important role in modulating the behavior of NPH-II. They also establish that processive helicase enzymes can display sequence selectivity.
2008-01-01
Hepatitis C virus (HCV) infection is frequently associated with the development of hepatocellular carcinomas and non-Hodgkin's B-cell lymphomas. Nonstructural protein 3 (NS3) of HCV possesses serine protease, nucleoside triphosphatase, and helicase activities, while NS4A functions as a cofactor for the NS3 serine protease. Here, we show that HCV NS3/4A interacts with the ATM (ataxia-telangiectasia mutated), a cellular protein essential for cellular response to irradiation. The expression of NS3/4A caused cytoplasmic translocation of either endogenous or exogenous ATM and delayed dephosphorylation of the phosphorylated ATM and gamma-H2AX following ionizing irradiation. As a result, the irradiation-induced gamma-H2AX foci persisted longer in the NS3/4A-expressing cells. Furthermore, these cells showed increased comet tail ...
Eukaryotes possess mechanisms to limit crossing over during homologous recombination, thus avoiding possible chromosomal rearrangements. We show here that budding yeast Mph1, an ortholog of human FancM helicase, utilizes its helicase activity to suppress spontaneous unequal sister chromatid exchanges and DNA double-strand break-induced chromosome crossovers. Since the efficiency and kinetics of break repair are unaffected, Mph1 appears to channel repair intermediates into a noncrossover pathway. Importantly, Mph1 works independently of two other helicases—Srs2 and Sgs1—that also attenuate crossing over. By chromatin immunoprecipitation, we find targeting of Mph1 to double-strand breaks in cells. Purified Mph1 binds D-loop structures and is particularly adept at unwinding these structures. Importantly, Mph1, but not a helicase-defective variant, dissociates Rad51-made D-loops. Overall, the results from our analyses suggest a new role of Mph1 in promoting the noncrossover repair of DNA double-strand breaks.
UvrD helicase of Plasmodium falciparum
2008-01-01
Malaria caused by the mosquito-transmitted parasite Plasmodium is the cause of enormous number of deaths every year in the tropical and subtropical areas of the world. Among four species of Plasmodium, Plasmodium falciparum causes most fatal form of malaria. With time, the parasite has developed insecticide and drug resistance. Newer strategies and advent of novel drug targets are required so as to combat the deadly form of malaria. Helicases is one such class of enzymes which has previously been suggested as potential antiviral and anticancer targets. These enzymes play an essential role in nearly all the nucleic acid metabolic processes, catalyzing the transient opening of the duplex nucleic acids in an NTP-dependent manner. DNA helicases from the PcrA/UvrD/Rep subfamily are important fo...
Hitting the Bull’s Eye: Novel Directed Cancer Therapy Through Helicase-Targeted Synthetic Lethality
Designing strategies for anti-cancer therapy have posed a significant challenge. One approach has been to inhibit specific DNA repair proteins and their respective pathways to enhance chemotherapy and radiation therapy used to treat cancer patients. Synthetic lethality represents an approach that exploits pre-existing DNA repair deficiencies in certain tumors to develop inhibitors of DNA repair pathways that compensate for the tumor-associated repair deficiency. Since helicases play critical roles in the DNA damage response and DNA repair, particularly in actively dividing and replicating cells, it is proposed that the identification and characterization of synthetic lethal relationships of DNA helicases will be of value in developing improved anti-cancer treatment strategies. In this review, we discuss this hypothesis and current evidence for synthetic lethal interactions of eukaryotic DNA helicases in model systems.
Anticipating chromosomal replication fork arrest: SSB targets repair DNA helicases to active forks
2007-10-03
Full Text Available.In bacteria, several salvage responses to DNA replication arrest culminate in reassembly of the replisome on inactivated forks to resume replication. The PriA DNA helicase is a prominent trigger of this replication restart process, preceded in many cases by a repair and/or remodeling of the arrested fork, which can be performed by many specific proteins. The mechanisms that target these rescue effectors to damaged forks in the cell are unknown. We report that the single-stranded DNA binding (SSB) protein is the key factor that links PriA to active chromosomal replication forks in vivo. This targeting mechanism determines the efficiency by which PriA reaches its specific DNA-binding site in vitro and directs replication restart in vivo. The RecG and RecQ DNA helicases, which are involved in intricate replication reactivation pathways, also associate with the chromosomal replication forks by similarly interacting with SSB. These results identify SSB as a platform for linking a ‘repair toolbox' with active replication forks, providing a first line of rescue responses to accidental arrest.
2010-01-01
Hepatitis C virus (HCV) infection is a common cause of chronic liver disease and a serious threat to human health. The HCV NS3/4A serine protease is necessary for viral replication and innate immune evasion, and represents a well-validated target for specific antiviral therapy. We previously reported the isolation of single-chain antibodies (scFvs) that inhibit NS3/4A protease activity in vitro. Expressed intracellularly (intrabodies), these scFvs blocked NS3-mediated proliferation of NS3-transfected cells. Here we show that anti-NS3 scFvs suppress HCV RNA replication when expressed intracellularly in Huh7 hepatoma cells bearing either subgenomic or genome-length HCV RNA replicons. The expression of intrabodies directed against NS3 inhibited the autonomous amplification of HCV replicons re...
Mitochondrial helicases and mitochondrial genome maintenance
2010-01-01
Helicases are essential enzymes that utilize the energy of nucleotide hydrolysis to drive unwinding of nucleic acid duplexes. Helicases play roles in all aspects of DNA metabolism including DNA repair, DNA replication and transcription. The subcellular locations and functions of several helicases have been studied in detail; however, the roles of specific helicases in mitochondrial biology remain poorly characterized. This review presents important recent advances in identifying and characterizing mitochondrial helicases, some of which also operate in the nucleus.
2008-01-01
This study describes a nanodiagnostic method using thermophilic helicase-dependent isothermal amplification (tHDA) and gold nanoparticle probes for colorimetric detection of Helicobacter pylori DNA. The primers targeting ureC gene were used for the amplification of bacterial DNA by the isothermal tHDA reaction, resulting in the accumulation of DNA amplicons. The amplicons were hybridized with specific gold nanoparticle probes. The hybrids were colorimetrically detected by the assembly of gold nanoparticles. Using this method, we detected as little as 10 CFU mL1 of H. pylori within less than 1 h. Results obtained from the gastric biopsy samples showed 92.5% and 95.4% of sensitivity and specificity, respectively, in comparison with culture results, and 100% and 98.8% of sensitivity and sp...
2009-01-01
Magnetic modulation biosensing (MMB) system is experimentally demonstrated for rapid and homogeneous detection of the Ibaraki virus NS3 cDNA. A novel fluorescent resonance energy transfer (FRET)-based probe discriminates the target DNA from the control. When detection is made, the FRET-based probe is cleaved using Taq-polymerase activity and fluorescent light is produced. The biotinylated probes are attached to streptavidin-coupled superparamagnetic beads and are maneuvered into oscillatory motion by applying an alternating magnetic field gradient through two electromagnetic poles. The beads are condensed into the detection area and their movement in and out the orthogonal laser beam produces a periodic fluorescent signal that is demodulated using synchronous detection. 1.9pM of the Ibarak...
2010-01-01
DEAD box proteins consist of a common helicase core formed by two globular RecA domains that are separated by a cleft. The helicase core acts as a nucleotide-dependent switch that alternates between open and closed conformations during the catalytic cycle of duplex separation, thereby providing basic helicase activity. Flanking domains can direct the helicase core to a specific RNA substrate by mediating high-affinity or high-specificity RNA binding. In addition, they may position RNA for the helicase core or may directly contribute to unwinding. While structures of different helicase cores have been determined previously, little is known about the orientation of flanking domains relative to the helicase core. YxiN is a DEAD box protein that consists of a helicase core and a C-terminal RNA...
Intersubunit allosteric communication mediated by a conserved loop in the MCM helicase
2009-01-27
The minichromosome maintenance (MCM) helicase is the presumptive replicative helicase in archaea and eukaryotes. The archaeal homomultimeric MCM has a two-tier structure. One tier contains the AAA+...Full Text Available
Velocity and processivity of helicase unwinding of double-stranded nucleic acids
2005-09-15
Helicases are molecular motors which unwind double-stranded nucleic acids (dsNA) in cells. Many helicases move with directional bias on single-stranded (ss) nucleic acids, and couple their directional translocation to strand separation. A model of the coupling between translocation and unwinding uses an interaction potential to represent passive and active helicase mechanisms. A passive helicase must wait for thermal fluctuations to open dsNA base pairs before it can advance and inhibit NA closing. An active helicase directly destabilizes dsNA base pairs, accelerating the opening rate. Here we extend this model to include helicase unbinding from the nucleic-acid strand. The helicase processivity depends on the form of the interaction potential. A passive helicase has a mean attachment time which does not change between ss translocation and ds unwinding, while an active helicase in general shows a decrease in attachment time during unwinding relative to ss translocation. In addition, we describe how helicase unwinding velocity and processivity vary if the base-pair binding free energy is changed.
2008-01-01
Human cytomegalovirus (HCMV) is a ubiquitous virus that causes significant morbidity and mortality in immunocompromised individuals. Although there are prophylactic treatments available, all current antiviral drugs ultimately target the DNA polymerase, resulting in the increasing emergence of antiviral resistant strains in the clinical setting. There is a fundamental need for understanding the role of other essential genes in DNA replication as a foundation for developing new antiviral treatments that are safe and which utilize a mechanism of action different to existing therapies. In this study we looked at six HCMV replication genes encoding for the DNA polymerase accessory protein (UL44), single stranded DNA binding protein (UL57), primase (UL70), helicase (UL105), primase-helicase associated protein (UL102), and the putative initiator protein (UL84) in order to increase our understanding of their role in DNA replication. The aim of this project was to identify variation within these genes as well as to predict putative domains and motifs in order to ultimately express and study the functional properties of the HCMV primase (UL70) through the use of recombinant mutants. Sequencing of these genes revealed a high degree of conservation between the isolates with amino acid sequence identity of >97% for all genes. Using ScanProsite software from the Expert Protein Analysis System (ExPASy) proteomics server, we have mapped putative motifs throughout these HCMV replication genes. In particular, highly conserved putative Nlinked glycosylation sites were identified in UL105 that were also conserved across 33 homologues as well as several unique motifs including casein kinase II phosphorylation sites (CKII) in UL105 and UL84, a microbodies signal motif in UL57 and an integrin binding site in the UL102 helicase-primase associated protein. Our investigations have also elucidated motif-rich regions of the UL44 DNA polymerase accessory protein, mapped functionally important domains of the UL105 helicase and identified cysteine motifs that have implications for folding of the UL70 primase. Taken together, these findings provide insights to regions of these HCMV replication proteins that are important for post-translation modification, activation and overall function, and this information can be utilized to target further research into these proteins and advance the development of novel antiviral agents that target these processes. Publisher: Publisher:University of New South Wales. Medical Sciences Language: EN Rights: http://unsworks.unsw.edu.au/copyright
2010-01-01
DEAD-box RNA helicases of the bacterial DbpA subfamily are localized to their biological substrate when a carboxy-terminal RNA recognition motif domain binds tightly and specifically to a segment of 23S ribosomal RNA (rRNA) that includes hairpin 92 of the peptidyl transferase center. A complex between a fragment of 23S rRNA and the RNA binding domain (RBD) of the Bacillus subtilis DbpA protein YxiN was crystallized and its structure was determined to 2.9 A resolution, revealing an RNA recognition mode that differs from those observed with other RNA recognition motifs. The RBD is bound between two RNA strands at a three-way junction. Multiple phosphates of the RNA backbone interact with an electropositive band generated by lysines of the RBD. Nucleotides of the single-stranded loop of hairp...
2010-01-01
The hepatitis C virus (HCV) serine protease (NS3/4A) processes the NS3-NS5B segment of the viral polyprotein and also cleaves host proteins involved in interferon signaling, making it an important target for antiviral drug discovery and suggesting a wide breadth of substrate specificity. We compared substrate specificities of the HCV protease with that of the GB virus B (GBV-B), a distantly related nonhuman primate hepacivirus, by exchanging amino acid sequences at the NS4B/5A and/or NS5A/5B cleavage junctions between these viruses within the backbone of subgenomic replicons. This mutagenesis study demonstrated that the GBV-B protease had a broader substrate tolerance, a feature corroborated by structural homology modeling. However, despite efficient polyprotein processing, GBV-B RNAs cont...
Prp43p contains a processive helicase structural architecture with a specific regulatory domain
2010-01-01
The DEAH/RNA helicase A (RHA) helicase family comprises proteins involved in splicing, ribosome biogenesis and transcription regulation. We report the structure of yeast Prp43p, a DEAH/RHA helicase remarkable in that it functions in both splicing and ribosome biogenesis. Prp43p displays a novel structural architecture with an unforeseen homology with the Ski2-like Hel308 DNA helicase. Together with the presence of a β-hairpin in the second RecA-like domain, Prp43p contains all the structural elements of a processive helicase. Moreover, our structure reveals that the C-terminal domain contains an oligonucleotide/oligosaccharide-binding (OB)-fold placed at the entrance of the putative nucleic acid cavity. Deletion or mutations of this domain decrease the affinity of Prp43p for RNA and s...
Full Text Available.Werner syndrome is a rare disorder that manifests as premature aging and age-related diseases. WRN is the gene mutated in WS, and is one of five human RecQ helicase family members. WS cells exhibit genomic instability and altered proliferation, and in vitro studies suggest that WRN has a role in suppressing homologous recombination. However, more recent studies propose that other RecQ helicases (including WRN) promote early events of homologous recombination. To study the role of WRN helicase on spontaneous homologous recombination, we obtained a mouse with a deleted WRN helicase domain and combined it with the in vivo pink-eyed unstable homologous recombination system. In this paper, we demonstrate that WRN helicase is not necessary for suppressing homologous recombination in vivo contrary to previous reports using a similar mouse model.
Full Text Available.BackgroundThe dengue virus two-component protease NS2B/NS3 mediates processing of the viral polyprotein precursor and is therefore an important determinant of virus replication. The enzyme is now intensively studied with a view to the structure-based development of antiviral inhibitors. Although 3-dimensional structures have now been elucidated for a number of flaviviral proteases, enzyme-substrate interactions are characterized only to a limited extend. The high selectivity of the dengue virus protease for the polyprotein precursor offers the distinct advantage of designing inhibitors with exquisite specificity for the viral enzyme. To identify important determinants of substrate binding and catalysis in the active site of the dengue virus NS3 protease, nine residues, L115, D129, G133, T134, Y150, G151, N152, S163 and I165, located within the S1 and S2 pockets of the enzyme were targeted by alanine substitution mutagenesis and effects on enzyme activity were fluorometrically assayed.MethodsAlanine substitutions were introduced by site-directed mutagenesis at residues L115, D129, G133, T134, Y150, G151, N152, S163 and I165 and recombinant proteins were purified from overexpressing E. coli. Effects of these substitutions on enzymatic activity of the NS3 protease were assayed by fluorescence release from the synthetic model substrate GRR-amc and kinetic parameters K
2005-01-01
The flavivirus West Nile virus (WNV) has spread rapidly throughout the world in recent years causing fever, meningitis, encephalitis, and fatalities. Because the viral protease NS2B/NS3 is essential for replication, it is attracting attention as a potential therapeutic target, although there are currently no antiviral inhibitors for any flavivirus. This paper focuses on elucidating interactions between a hexapeptide substrate (Ae-KPGLKR-p-nitroanilide) and residues at S1 and S2 in the active site of WNV protease by comparing the catalytic activities of selected mutant recombinant proteases in vitro. Homology modeling enabled the predictions of key mutations in VWNV NS3 protease at S1 (V115A/F, D129A/ E/N, S135A, Y150A/F, S160A, and S163A) and S2 (N152A) that might influence substrate recognition and catalytic efficiency. Key conclusions are that the substrate P1 Arg strongly interacts with S1 residues Asp-129, Tyr-150, and Ser-163 and, to a lesser extent, Ser-160, and P2 Lys makes an essential interaction with Asn-152 at S2. The inferred substrate-enzyme interactions provide a basis for rational protease inhibitor design and optimization. High sequence conservation within flavivirus proteases means that this study may also be relevant to design of protease inhibitors for other flavivirus proteases. Publisher: The American Society for Biochemistry and Molecular Biology . Contributor: Herbert, T. Coverage: 2005-01-28T00:00:00Z
An automated multidimensional protein identification technology, which combines biphasic liquid chromatography with electrospray ionisation tandem mass spectrometry (MS/MS), was employed to analyse tryptic peptides from Escherichia coli cells treated with the antiproliferation agent [(eta(6)-p-cymene)RuCl(2)(DMSO)], where DMSO is dimethyl sulfoxide. MS/MS spectra were recorded for molecular ions generated by neutral loss of p-cymene from intensive peptide ions coordinated by the (eta(6)-p-cymene)Ru(II) fragment. Matching of the MS/MS spectra of the ruthenated peptides to spectra of proteins in the E. coli database enabled the identification of five protein targets for [(eta(6)-p-cymene)RuCl(2)(DMSO)]. One of these is the constitutive cold-shock protein cspC, which regulates the expression of genes encoding stress-response proteins, and three of the other targets, ppiD, osmY and sucC, are proteins of the latter type. The DNA damage-inducible helicase dinG was likewise established as a protein target. Aspartate carboxylate functions were identified as the probable Ru binding sites in cspC, ppiD and dinG, and threonine and lysine side chains in osmY and sucC, respectively.
2010-03-01
In trypanosomes, the predominant mechanisms of regulation of gene expression are post-transcriptional. The DEAD-box RNA helicase DHH1 was identified in a screen for gene products that are necessary...Full Text Available
Senescence delay and repression of p16
2009-08-01
Lymphoid specific helicase (Lsh) belongs to the family of SNF2/helicases. Disruption of Lsh leads to developmental growth retardation and premature aging in mice. However, the specific effect of Lsh...Full Text Available
RecQ helicases: lessons from model organisms
2006-09-01
RecQ DNA helicases function during DNA replication and are essential for the maintenance of genome stability. There is increasing evidence that spontaneous genomic instability occurs primarily during...Full Text Available
Phosphate release contributes to the rate-limiting step for unwinding by an RNA helicase
2010-03-01
RNA helicases function in numerous aspects of RNA biology. These enzymes are RNA-stimulated ATPases that translocate on RNA and unwind or remodel structured RNA in an ATP-dependent fashion. How ATP...Full Text Available
Opening of DNA double strands by helicases. Active versus passive opening
2002-12-10
Helicase opening of double-stranded nucleic acids may be "active" (the helicase directly destabilizes the dsNA to promote opening) or "passive" (the helicase binds ssNA available due to a thermal fluctuation which opens part of the dsNA). We describe helicase opening of dsNA, based on helicases which bind single NA strands and move towards the double-stranded region, using a discrete ``hopping'' model. The interaction between the helicase and the junction where the double strand opens is characterized by an interaction potential. The form of the potential determines whether the opening is active or passive. We calculate the rate of passive opening for the helicase PcrA, and show that the rate increases when the opening is active. Finally, we examine how to choose the interaction potential to optimize the rate of strand separation. One important result is our finding that active opening can increase the unwinding rate by 7 fold compared to passive opening.
Double-stranded RNA-dependent ATPase DRH-3
2010-08-13
RNA helicases are proteins essential to almost every facet of RNA metabolism, including the gene-silencing pathways that employ small RNAs. A phylogenetically related group of helicases is required...Full Text Available
Comparative Structural Analysis of Human DEAD-Box RNA Helicases
DEAD-box RNA helicases play various, often critical, roles in all processes where RNAs are involved. Members of this family of proteins are linked to human disease, including cancer and viral infections....Full Text Available
BLM helicase stimulates the ATPase and chromatin-remodeling activities of RAD54
2009-09-01
SummaryMutation of BLM helicase results in the autosomal recessive disorder Bloom syndrome (BS). Patients with BS exhibit hyper-recombination and are prone to almost all forms of cancer....Full Text Available
RecQ helicase, in concert with RecA and SSB proteins, initiates and disrupts DNA recombination
1998-04-15
RecQ helicase is important to homologous recombination and DNA repair in Escherichia coli. We demonstrate that RecQ helicase, in conjunction with RecA and SSB proteins, can initiate...Full Text Available
P68 (DDX5) and p72 (DDX17) are members of the DEAD-box RNA helicase family. They can unwind double-stranded RNA and also contribute to the remodeling of ribonucleoprotein complexes. These activities...Full Text Available
2010-05-01
Full Text Available.The formation of single-stranded DNA (ssDNA) at double-strand break (DSB) ends is essential in repair by homologous recombination and is mediated by DNA helicases and nucleases. Here we estimated the length of ssDNA generated during DSB repair and analyzed the consequences of elimination of processive resection pathways mediated by Sgs1 helicase and Exo1 nuclease on DSB repair fidelity. In wild-type cells during allelic gene conversion, an average of 2–4 kb of ssDNA accumulates at each side of the break. Longer ssDNA is formed during ectopic recombination or break-induced replication (BIR), reflecting much slower repair kinetics. This relatively extensive resection may help determine sequences involved in homology search and prevent recombination within short DNA repeats next to the break. In sgs1Δ exo1Δ mutants that form only very short ssDNA, allelic gene conversion decreases 5-fold and DSBs are repaired by BIR or de novo telomere formation resulting in loss of heterozygosity. The absence of the telomerase inhibitor, PIF1, increases de novo telomere pathway usage to about 50%. Accumulation of Cdc13, a protein recruiting telomerase, at the break site increases in sgs1Δ exo1Δ, and the requirement of the Ku complex for new telomere formation is partially bypassed. In contrast to this decreased and alternative DSB repair, the efficiency and accuracy of gene targeting increases dramatically in sgs1Δ exo1Δ cells, suggesting that transformed DNA is very stable in these mutants. Altogether these data establish a new role for processive resection in the fidelity of DSB repair.
2010-03-01
Full Text Available.The human Werner and Bloom syndromes (WS and BS) are caused by deficiencies in the WRN and BLM RecQ helicases, respectively. WRN, BLM and their Saccharomyces cerevisiae homologue Sgs1, are particularly active in vitro in unwinding G-quadruplex DNA (G4-DNA), a family of non-canonical nucleic acid structures formed by certain G-rich sequences. Recently, mRNA levels from loci containing potential G-quadruplex-forming sequences (PQS) were found to be preferentially altered in sgs1Δ mutants, suggesting that G4-DNA targeting by Sgs1 directly affects gene expression. Here, we extend these findings to human cells. Using microarrays to measure mRNAs obtained from human fibroblasts deficient for various RecQ family helicases, we observe significant associations between loci that are upregulated in WS or BS cells and loci that have PQS. No such PQS associations were observed for control expression datasets, however. Furthermore, upregulated genes in WS and BS showed no or dramatically reduced associations with sequences similar to PQS but that have considerably reduced potential to form intramolecular G4-DNA. These findings indicate that, like Sgs1, WRN and BLM can regulate transcription globally by targeting G4-DNA.
2006-01-01
We derive, using the pure-spinor formalism, the complete-including the fermions-4-point effective action of both type II superstrings to all orders in alpha', at tree level in string loops. We find that, in the quartic-field approximation, the supergravity Lagrangian can be thought of as the tensor product, in a suitable sense, of two copies of the super-Yang-Mills Lagrangian in ten dimensions. The NS-NS 3-form enters the supergravity Lagrangian through a modified connection with torsion. As a byproduct, we derive the complete, i.e. to all orders in the theta-expansion, closed-string vertex operator in a flat target-space background
Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.
2008-06-02
Mutations in XPD helicase, required for nucleotide excision repair (NER) as part of the transcription/repair complex TFIIH, cause three distinct phenotypes: cancer-prone xeroderma pigmentosum (XP), or aging disorders Cockayne syndrome (CS), and trichothiodystrophy (TTD). To clarify molecular differences underlying these diseases, we determined crystal structures of the XPD catalytic core from Sulfolobus acidocaldarius and measured mutant enzyme activities. Substrate-binding grooves separate adjacent Rad51/RecA-like helicase domains (HD1, HD2) and an arch formed by 4FeS and Arch domains. XP mutations map along the HD1 ATP-binding edge and HD2 DNA-binding channel and impair helicase activity essential for NER. XP/CS mutations both impair helicase activity and likely affect HD2 functional movement. TTD mutants lose or retain helicase activity but map to sites in all four domains expected to cause framework defects impacting TFIIH integrity. These results provide a foundation for understanding disease consequences of mutations in XPD and related 4Fe-4S helicases including FancJ.
Full Text Available.Alzheimer's disease (AD) is the main cause of dementia in our increasingly aging population. The debilitating cognitive and behavioral symptoms characteristic of AD make it an extremely distressing illness for patients and carers. Although drugs have been developed to treat AD symptoms and to slow disease progression, there is currently no cure. The incidence of AD is predicted to increase to over one hundred million by 2050, placing a heavy burden on communities and economies, and making the development of effective therapies an urgent priority. Two proteins are thought to have major contributory roles in AD: the microtubule associated protein tau, also known as MAPT; and the amyloid-beta peptide (A-beta), a cleavage product of amyloid precursor protein (APP). Oxidative stress is also implicated in AD pathology from an early stage. By targeting eIF4A, an RNA helicase involved in translation initiation, the synthesis of APP and tau, but not neuroprotective proteins, can be simultaneously and specifically reduced, representing a novel avenue for AD intervention. We also show that protection from oxidative stress is increased upon eIF4A inhibition. We demonstrate that the reduction of these proteins is not due to changes in mRNA levels or increased protein degradation, but is a consequence of translational repression conferred by inhibition of the helicase activity of eIF4A. Inhibition of eIF4A selectively and simultaneously modulates the synthesis of proteins involved in Alzheimer's disease: reducing A-beta and tau synthesis, while increasing proteins predicted to be neuroprotective.
Recombinational DNA repair and human disease
2002-11-30
We review the genes and proteins related to the homologous recombinational repair (HRR) pathway that are implicated in cancer through either genetic disorders that predispose to cancer through chromosome instability or the occurrence of somatic mutations that contribute to carcinogenesis. Ataxia telangiectasia (AT), Nijmegen breakage syndrome (NBS), and an ataxia-like disorder (ATLD), are chromosome instability disorders that are defective in the ataxia telangiectasia mutated (ATM), NBS, and Mre11 genes, respectively. These genes are critical in maintaining cellular resistance to ionizing radiation (IR), which kills largely by the production of double-strand breaks (DSBs). Bloom syndrome involves a defect in the BLM helicase, which seems to play a role in restarting DNA replication forks that are blocked at lesions, thereby promoting chromosome stability. The Werner syndrome gene (WRN) helicase, another member of the RecQ family like BLM, has very recently been found to help mediate homologous recombination. Fanconi anemia (FA) is a genetically complex chromosomal instability disorder involving seven or more genes, one of which is BRCA2. FA may be at least partially caused by the aberrant production of reactive oxidative species. The breast cancer-associated BRCA1 and BRCA2 proteins are strongly implicated in HRR; BRCA2 associates with Rad51 and appears to regulate its activity. We discuss in detail the phenotypes of the various mutant cell lines and the signaling pathways mediated by the ATM kinase. ATM's phosphorylation targets can be grouped into oxidative stress-mediated transcriptional changes, cell cycle checkpoints, and recombinational repair. We present the DNA damage response pathways by using the DSB as the prototype lesion, whose incorrect repair can initiate and augment karyotypic abnormalities.
2010-01-01
Summary We recently showed that upregulation of a key oncogene FOXM1 precedes head and neck squamous cell carcinoma (HNSCC) malignancy. Furthermore, we also identified a centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, which are both downstream targets of FOXM1. In this study, we have investigated the expression profiles of CEP55 and HELLS using immunohistochemistry and quantified by digital densitometry in a tissue panel (20 samples) consisting of normal oral mucosa, dysplasias, HNSCC and lymph node metastasis (LnMet) samples. Furthermore, we corroborated our findings using absolute real-time PCR (qPCR) on a panel of 12 primary normal human oral keratinocytes, five dysplasia and 10 HNSCC cell lines. Finally, we validated our study using bioinformatics microarr...
2008-01-01
To investigate involvement of miRNAs in radiation responses we used microRNAome profiling to analyze the sex-specific response of radiation sensitive hematopoietic lymphoid tissues. We show that radiation exposure resulted in a significant and sex-specific deregulation of microRNA expression in murine spleen and thymus tissues. Among the regulated miRNAs, we found that changes in expression of miR-34a and miR-7 may be involved in important protective mechanisms counteracting radiation cytotoxicity. We observed a significant increase in the expression of tumor-suppressor miR-34a, paralleled by a decrease in the expression of its target oncogenes NOTCH1, MYC, E2F3 and cyclin D1. Additionally, we show that miR-7 targets the lymphoid-specific helicase LSH, a pivotal regulator of DNA methylation and genome stability. While miR-7 was significantly down-regulated ...
A novel vascular targeting strategy for brain-derived endothelial cells using a TCR mimic antibody
2010-01-01
Organ-specific vascular targeting, for example, to the blood-brain barrier, requires the identification of unique molecular addresses on a subset of endothelial cells. The present study describes a crucial step towards tapping the exquisite specificity of the peptide/HLA class I system for this goal. We utilized a novel T-cell receptor (TCR) mimic antibody of high affinity and specificity, which is restricted by HLA-A2 and has been generated to recognize a peptide epitope derived from p68 RNA helicase (YLLPAIVHI). The parent protein is highly expressed by brain endothelial cells. Flow cytometry and confocal imaging showed that the antibody binds to HLA-A2-positive human brain-derived endothelial cells, both immortalized hCMEC/D3 cells and primary cells. The TCR mimic antibody undergoes int...
Substrate Inhibition Kinetic Model for West Nile Virus NS2B-NS3 Protease †
2008-11-11
Full Text Available.West Nile virus (WNV) has recently emerged in North America as a significant disease threat to humans and animals. Unfortunately, no approved antiviral drugs exist to combat WNV or other members of the genus Flavivirus in humans. The WNV NS2B-NS3 protease has been one of the primary targets for anti-WNV drug discovery and design since it is required for virus replication. As part of our efforts to develop effective WNV inhibitors, we reexamined the reaction kinetics of the NS2B-NS3 protease and the inhibition mechanisms of newly discovered inhibitors. The WNV protease showed substrate inhibition in assays utilizing fluorophore-linked peptide substrates GRR, GKR, and DFASGKR. Moreover, a substrate inhibition reaction step was required to accurately model kinetic data generated from protease assays with a peptide inhibitor. The substrate inhibition model suggested peptide substrates could bind to two binding sites on the protease. Reaction product analogs also showed inhibition of the protease, demonstrating product inhibition in addition to, and distinct from, substrate inhibition. We propose that small peptide substrates and inhibitors may interact with protease residues that form either the P3-P1 binding surface (i.e., the S3-S1 sites) or the P1′-P3′ interaction surface (i.e., the S1′-S3′ sites). Optimization of substrate analog inhibitors that target these two independent sites may lead to novel anti-WNV drugs.
Active and passive mechanisms of helicases
2010-09-01
Full Text Available.In this work, we discuss the active or passive character of helicases. In the past years, several studies have used the theoretical framework proposed by Betterton and Julicher [Betterton, M.D. and Julicher, F. (2005) Opening of nucleic-acid double strands by helicases: active versus passive opening. Phys. Rev. E, 71, 11904–11911.] to analyse the unwinding data and assess the mechanism of the helicase under study (active versus passive). However, this procedure has given rise to apparently contradictory interpretations: helicases exhibiting similar behaviour have been classified as both active and passive enzymes [Johnson, D.S., Bai, L. Smith, B.Y., Patel, S.S. and Wang, M.D. (2007) Single-molecule studies reveal dynamics of DNA unwinding by the ring-shaped T7 helicase. Cell, 129, 1299–1309; Lionnet, T., Spiering, M.M., Benkovic, S.J., Bensimon, D. and Croquette, V. (2007) Real-time observation of bacteriophage T4 gp41 helicase reveals an unwinding mechanism Proc. Natl Acid. Sci., 104, 19790–19795]. In this work, we show that when the helicase under study has not been previously well characterized (namely, if its step size and rate of slippage are unknown) a multi-parameter fit to the afore-mentioned model can indeed lead to contradictory interpretations. We thus propose to differentiate between active and passive helicases on the basis of the comparison between their observed translocation velocity on single-stranded nucleic acid and their unwinding rate of double-stranded nucleic acid (with various GC content and under different tensions). A threshold separating active from passive behaviour is proposed following an analysis of the reported activities of different helicases. We study and contrast the mechanism of two helicases that exemplify these two behaviours: active for the RecQ helicase and passive for the gp41 helicase.
Bombesin (BBN) peptide exhibits high selectivity and affinity for the gastrin-releasing peptide receptor (GRPr). The GRPr is overexpressed on many human cancer cell types, thus making BBN a potent delivery vehicle for radionuclide targeting. In this study, the biologically active minimal sequence BBN(7-14) was labeled using the novel Tc '4 + 1' mixed-ligand system, [Tc(NS3)(CN-R)], in which Tc(III) is coordinated by a monodentate isocyanide linker bearing the peptide and the tetradentate, tripodal chelator, 2,2',2''-nitrilotriethanethiol (NS3). BBN(7-14) was N-terminally modified with Gly-Gly-Gly, betaAla, and Ser-Ser-Ser spacer groups (X) and functionalized with 4-(isocyanomethyl)benzoic acid (L1) or 4-isocyanobutanoic acid (L2), resulting in a series of [M(NS3)(L-X-BBN(7-14))] conjugates (M = 99mTc, Re). The isocyanide ligand frameworks were introduced using novel bifunctional coupling agents. The spacer groups (X), the monodentate isocyanide units, and a tetradentate NS3 chelator bearing a pendant carboxylic acid (NS3COOH) were proposed as pharmacological modifiers. 99mTc-labeling was performed in a two-step procedure by first preparing 99mTc-EDTA/mannitol followed by reactions with the isocyanides and NS3 or NS3COOH ligand frameworks. The 99mTc complexes were obtained with a radiochemical yield of 30-80% depending on the amount of the isocyanide (20-100 nmol) used. These new conjugates were purified by reversed-phased high-performance liquid chromatography (RP-HPLC) to give a radiochemical purity of >or=95%. The 99mTc conjugates exhibited high in vitro stability (>90%, 24 h). Analogous nonradioactive Re conjugates were synthesized and characterized by electrospray ionization mass spectrometry (ESI-MS). RP-HPLC analyses of the Re conjugates indicated that they exhibited identical retention times to the corresponding 99mTc conjugates under identical HPLC conditions, demonstrating structural similarity between the two metalated species. The [Re(NS3)(L-X-BBN(7-14))] conjugates exhibited GRPr affinity in the nanomolar range as demonstrated by in vitro competitive binding assays using PC-3 human prostate cancer cells. In vitro internalization/externalization assays indicated that approximately 65% of [99mTc(NS3)(L2-betaAla-BBN(7-14))] conjugate was either surface-bound or internalized in PC-3 cells. Cell-associated activity for all other 99mTc conjugates was below 20%. Biodistribution studies of [99mTc(NS3)(L-betaAla-BBN(7-14))], L = L1 or L2, in normal, CF-1 mice showed minimal accumulation in normal pancreas (a tissue expressing the GRPr in high density in rodent models) and rapid hepatobiliary elimination. Introduction of a carboxyl group onto the NS3 ligand framework had only minimal effects to increase renal excretion. Activity distribution and accumulation was highly dominated by the relatively lipophilic '4 + 1' complex unit.
The Crystal Structure of the Thermus Aquaticus DnaB Helicase Monomer
2007-01-01
The ring-shaped hexameric DnaB helicase unwinds duplex DNA at the replication fork of eubacteria. We have solved the crystal structure of the full-length Thermus aquaticus DnaB monomer, or possibly dimer, at 2.9 {angstrom} resolution. DnaB is a highly flexible two domain protein. The C-terminal domain exhibits a RecA-like core fold and contains all the conserved sequence motifs that are characteristic of the DnaB helicase family. The N-terminal domain contains an additional helical hairpin that makes it larger than previously appreciated. Several DnaB mutations that modulate its interaction with primase are found in this hairpin. The similarity in the fold of the DnaB N-terminal domain with that of the C-terminal helicase-binding domain (HBD) of the DnaG primase also includes this hairpin. Comparison of hexameric homology models of DnaB with the structure of the papillomavirus E1 helicase suggests the two helicases may function through different mechanisms despite their sharing a common ancestor.
Roles of RECQ helicases in recombination based DNA repair, genomic stability and aging
The maintenance of the stability of genetic material is an essential feature of every living organism. Organisms across all kingdoms have evolved diverse and highly efficient repair mechanisms to protect the genome from deleterious consequences of various genotoxic factors that might tend to destabilize the integrity of the genome in each generation. One such group of proteins that is actively involved in genome surveillance is the RecQ helicase family. These proteins are highly conserved DNA helicases, which have diverse roles in multiple DNA metabolic processes such as DNA replication, recombination and DNA repair. In humans, five RecQ helicases have been identified and three of them namely, WRN, BLM and RecQL4 have been linked to genetic diseases characterized by genome instability, premature aging and cancer predisposition. This helicase family plays important roles in various DNA repair pathways including protecting the genome from illegitimate recombination during chromosome segregation in mitosis and assuring genome stability. This review mainly focuses on various roles of human RecQ helicases in the process of recombination-based DNA repair to maintain genome stability and physiological consequences of their defects in the development of cancer and premature aging.
2010-01-01
Many quantitative approaches for analysis of helicase-nucleic acid interactions require a robust and specific signal, which reports on the presence of the helicase and its position on a nucleic acid lattice. Since 2006, iron-sulfur (FeS) clusters have been found in a number of helicases. They serve as endogenous quenchers of Cy3 and Cy5 fluorescence which can be exploited to characterize FeS cluster containing helicases both in ensemble-based assays and at the single-molecule level. Synthetic oligonucleotides site-specifically labeled with either Cy3 or Cy5 can be used to create a variety of DNA substrates that can be used to characterized DNA binding, as well as helicase translocation and unwinding. Equilibrium binding affinities for ssDNA, duplex and branched DNA substrates can be determ...
2007-01-01
West Nile virus is a medically significant emerging pathogen for which there is no effective antiviral therapy. The viral protease encoded by NS2B and NS3 is an attractive target for development of an inhibitor and has been the focus of numerous studies. Most have employed recombinant proteases based on an expression strategy we developed which links the essential hydrophilic cofactor domain within NS2B to the NS3 protease domain by a flexible glycine linker. However, autoproteolysis has been a significant problem associated with this construct. The recently resolved crystal structure of the cofactor bound WNV NS3 protease for example, was found to be truncated by 18 residues at its N-terminus. In this study, the autocatalytic cleavage site was identified and removed along with nonessential regions of the glycine linker and cofactor domain. In addition, the optimal size of the NS3 protease was defined. Based on this optimized construct, a recombinant protease incorporating the full length of NS3 was also successfully expressed and purified. Somewhat surprisingly, comparative analysis of the proteolytic activity of this recombinant with that of the protease domain alone revealed little influence of the C-terminal two thirds of NS3 on substrate binding. These modifications have yielded highly stable and constrained recombinant proteases, which are more suitable than existing constructs for both activity and structural studies. Publisher: Academic Press Coverage: 2007-05-01T00:00:00Z
Unraveling the Fanconi anaemia-DNA repair connection through DNA helicase and translocase activities
How the Fanconi anaemia (FA) chromosome stability pathway functions to cope with interstrand crosslinks and other DNA lesions has been elusive, even after FANCD1 proved to be BRCA2, a partner of Rad51 in homologous recombination. The identification and characterization of two new Fanconi proteins having helicase motifs, FANCM and FANCJ/BRIP1/BACH1, implicates the FANC nuclear core complex as a participant in recognizing or processing damaged DNA, and the BRIP1 helicase as acting independently of this complex.
Unraveling the Fanconi anaemia-DNA repair connection through DNA helicase and translocase activities
2005-08-16
How the Fanconi anaemia (FA) chromosome stability pathway functions to cope with interstrand crosslinks and other DNA lesions has been elusive, even after FANCD1 proved to be BRCA2, a partner of Rad51 in homologous recombination. The identification and characterization of two new Fanconi proteins having helicase motifs, FANCM and FANCJ/BRIP1/BACH1, implicates the FANC nuclear core complex as a participant in recognizing or processing damaged DNA, and the BRIP1 helicase as acting independently of this complex.
2009-01-01
Aryl diketoacids have been identified as the first SARS-CoV NTPase/helicase inhibitors with a distinct pharmacophore featuring an arylmethyl group attached to a diketoacid. In order to search for the pharmacophore space around the diketoacid core, three classes of dihydroxychromone derivatives were prepared. Based on SAR study, an extended feature of the pharmacophore model of SARS-CoV NTPase/helicase was proposed which is constituted of a diketoacid core, a hydrophobic arylmethyl substituent, and a free catechol unit.
Substrate specific stimulation of NEIL1 by WRN but not the other human RecQ helicases
2010-01-01
NEIL1, the mammalian homolog of Escherichia coli endonuclease VIII, is a DNA glycosylase that repairs ring-fragmented purines, saturated pyrimidines and several oxidative lesions like 5-hydroxyuracil, 5-hydroxycytosine, etc. Previous studies from our laboratory have shown that Werner Syndrome protein (WRN), one of the five human RecQ helicases, stimulates NEIL1 DNA glycosylase activity on oxidative DNA lesions. The goal of this study was to extend this observation and analyze the interaction between NEIL1 and all five human RecQ helicases. The DNA substrate specificity of the interaction between WRN and NEIL1 was also analyzed. The results indicate that WRN is the only human RecQ helicase that stimulates NEIL1 DNA glycosylase activity, and that this stimulation requires a double-stranded D...
2008-01-01
Summary The loading of oligomeric helicases onto replication origins marks an essential step in replisome assembly. In cells, dedicated AAA+ ATPases regulate loading, however, the mechanism by which these factors recruit and deposit helicases has remained unclear. To better understand this process, we determined the structure of the ATPase region of the bacterial helicase loader DnaC from Aquifex aeolicus to 2.7 A resolution. The structure shows that DnaC is a close paralog of the bacterial replication initiator, DnaA, and unexpectedly shares an ability to form a helical assembly similar to that of ATP-bound DnaA. Complementation and ssDNA-binding assays validate the importance of homomeric DnaC interactions, while pull-down experiments show that the DnaC and DnaA AAA+ domains interact in ...
Delineation of WRN helicase function with EXO1 in the replicational stress response
2010-01-01
The WRN gene defective in the premature aging disorder Werner syndrome encodes a helicase/exonuclease. We examined the ability of WRN to rescue DNA damage sensitivity of a yeast mutant defective in the Rad50 subunit of Mre11-Rad50-Xrs2 nuclease complex implicated in homologous recombination repair. Genetic studies revealed WRN operates in a yEXO1-dependent pathway to rescue rad50 sensitivity to methylmethane sulfonate (MMS). WRN helicase, but not exonuclease, is required for MMS resistance. WRN missense mutations in helicase or RecQ C-terminal domains interfered with the ability of WRN to rescue rad50 MMS sensitivity. WRN does not rescue rad50 ionizing radiation (IR) sensitivity, suggesting that WRN, in collaboration with yEXO1, is tailored to relieve replicational stress imposed by alkyla...
DNA Helicase Activity in Purified Human RECQL4 Protein
2009-01-01
Human RECQL4 protein was expressed in insect cells using a baculovirus protein expression system and it was purified to near homogeneity. The protein sedimented at a position between catalase (230 kDa) and ferritin (440 kDa) in glycerol gradient centrifugation, suggesting that it forms homo-multimers. Activity to displace annealed 17-mer oligonucleotide in the presence of ATP was co-sedimented with hRECQL4 protein. In ion-exchange chromatography, both DNA helicase activity and single-stranded DNA-dependent ATPase activity were co-eluted with hRECQL4 protein. The requirements of ATP and Mg for the helicase activity were different from those for the ATPase activity. The data suggest that the helicase migrates on single-stranded DNA in a 3prime-5prime direction. These results suggest that the...
A study of phase explosion of metal using high power Nd:YAG laser ablation
The interaction of high-power pulsed-laser beam with metal targets in air from 1.06 {mu}m, 5 ns, 3 J/pulse max, Nd:YAG pulsed laser is investigated together with hydrodynamic theories of laser-supported detonation (LSD) wave and multi-material reactive Euler equations. The high speed blast wave generated by the laser ablation of metal reaches maximum velocity of several thousand meters per second. The apparently similar flow conditions to those of reactive shock wave allow one to apply the equations of motion for energetic materials and to understand the explosive behavior of metal vaporization upon laser ablation. The characteristic time at which planar to spherical wave transition occurs is confirmed at low (20 mJ/pulse) to higher (200 mJ/pulse) beam intensities. The flow structure behind the leading shock wave during the early planar shock state is confirmed by the high-resolution multi-material hydrocode originally developed for shock compression of condensed matter.
A study of phase explosion of metal using high power Nd:YAG laser ablation
2007-01-01
The interaction of high-power pulsed-laser beam with metal targets in air from 1.06 mum, 5 ns, 3 J/pulse max, Nd:YAG pulsed laser is investigated together with hydrodynamic theories of laser-supported detonation (LSD) wave and multi-material reactive Euler equations. The high speed blast wave generated by the laser ablation of metal reaches maximum velocity of several thousand meters per second. The apparently similar flow conditions to those of reactive shock wave allow one to apply the equations of motion for energetic materials and to understand the explosive behavior of metal vaporization upon laser ablation. The characteristic time at which planar to spherical wave transition occurs is confirmed at low (20 mJ/pulse) to higher (200 mJ/pulse) beam intensities. The flow structure behind the leading shock wave during the early planar shock state is ...
2009-09-01
Mrc1 plays a role in mediating the DNA replication checkpoint. We surveyed replication elongation proteins that interact directly with Mrc1 and identified a replicative helicase, Mcm6, as a specific...Full Text Available
Visualizing helicases unwinding DNA at the single molecule level
2010-07-01
Full Text Available.DNA helicases are motor proteins that catalyze the unwinding of double-stranded DNA into single-stranded DNA using the free energy from ATP hydrolysis. Single molecule approaches enable us to address detailed mechanistic questions about how such enzymes move processively along DNA. Here, an optical method has been developed to follow the unwinding of multiple DNA molecules simultaneously in real time. This was achieved by measuring the accumulation of fluorescent single-stranded DNA-binding protein on the single-stranded DNA product of the helicase, using total internal reflection fluorescence microscopy. By immobilizing either the DNA or helicase, localized increase in fluorescence provides information about the rate of unwinding and the processivity of individual enzymes. In addition, it reveals details of the unwinding process, such as pauses and bursts of activity. The generic and versatile nature of the assay makes it applicable to a variety of DNA helicases and DNA templates. The method is an important addition to the single-molecule toolbox available for studying DNA processing enzymes.
2010-01-01
The Escherichia coli PriA helicase complex with the double-stranded DNA (dsDNA), the location of the strong DNA-binding subsite, and the effect of the nucleotide cofactors, bound to the strong and weak nucleotide-binding site of the enzyme on the dsDNA affinity, have been analyzed using the fluorescence titration, analytical ultracentrifugation, and photo-cross-linking techniques. The total site size of the PriA-dsDNA complex is only 5+/-1 bp, that is, dramatically lower than 20+/-3 nucleotides occluded in the enzyme-single-stranded DNA (ssDNA) complex. The helicase associates with the dsDNA using its strong ssDNA-binding subsite in an orientation very different from the complex with the ssDNA. The strong DNA-binding subsite of the enzyme is located on the helicase domain of the PriA prote...
2001-04-24
Full Text Available.The Escherichia coli protein DbpA is unique in its subclass of DEAD box RNA helicases, because it possesses ATPase-specific activity toward the peptidyl transferase center in 23S rRNA. Although its remarkable ATPase activity had been well defined toward various substrates, its RNA helicase activity remained to be characterized. Herein, we show by using biochemical assays and atomic force microscopy that DbpA exhibits ATP-stimulated unwinding activity of RNA duplex regardless of its primary sequence. This work presents an attempt to investigate the action of DEAD box proteins by a single-molecule visualization methodology. Our atomic force microscopy images enabled us to observe directly the unwinding reaction of a DEAD box helicase on long stretches of double-stranded RNA. Specifically, we could differentiate between the binding of DbpA to RNA in the absence of ATP and the formation of a Y-shaped intermediate after its progression through double-stranded RNA in the presence of ATP. Recent studies have questioned the designation of DbpA, in particular, and DEAD box proteins in general as RNA helicases. However, accumulated evidence and the results reported herein suggest that these proteins are indeed helicases that resemble in many aspects the DNA helicases.
Full Text Available.BackgroundThe mini-chromosome maintenance protein (MCM) complex is an essential replicative helicase for DNA replication in Archaea and Eukaryotes. While the eukaryotic complex consists of six homologous proteins (MCM2-7), the archaeon Sulfolobus solfataricus has only one MCM protein (ssoMCM), six subunits of which form a homohexamer. We have recently reported a 4.35Å crystal structure of the near full-length ssoMCM. The structure reveals a total of four β-hairpins per subunit, three of which are located within the main channel or side channels of the ssoMCM hexamer model generated based on the symmetry of the N-terminal Methanothermobacter thermautotrophicus (mtMCM) structure. The fourth β-hairpin, however, is located on the exterior of the hexamer, near the exit of the putative side channels and next to the ATP binding pocket.ResultsIn order to better understand this hairpin's role in DNA binding and helicase activity, we performed a detailed mutational and biochemical analysis of nine residues on this exterior β-hairpin (EXT-hp). We examined the activities of the mutants related to their helicase function, including hexamerization, ATPase, DNA binding and helicase activities. The assays showed that some of the residues on this EXT-hp play a role for DNA binding as well as for helicase activity.ConclusionsThese results implicate several current theories regarding helicase activity by this critical hexameric enzyme. As the data suggest that EXT-hp is involved in DNA binding, the results reported here imply that the EXT-hp located near the exterior exit of the side channels may play a role in contacting DNA substrate in a manner that affects DNA unwinding.
Delineation of WRN helicase function with EXO1 in the replicational stress response.
The WRN gene defective in the premature aging disorder Werner syndrome encodes a helicase/exonuclease. We examined the ability of WRN to rescue DNA damage sensitivity of a yeast mutant defective in the Rad50 subunit of Mre11-Rad50-Xrs2 nuclease complex implicated in homologous recombination repair. Genetic studies revealed WRN operates in a yEXO1-dependent pathway to rescue rad50 sensitivity to methylmethane sulfonate (MMS). WRN helicase, but not exonuclease, is required for MMS resistance. WRN missense mutations in helicase or RecQ C-terminal domains interfered with the ability of WRN to rescue rad50 MMS sensitivity. WRN does not rescue rad50 ionizing radiation (IR) sensitivity, suggesting that WRN, in collaboration with yEXO1, is tailored to relieve replicational stress imposed by alkylated base damage. WRN and yEXO1 are associated with each other in vivo. Purified WRN stimulates hEXO1 nuclease activity on DNA substrates associated with a stalled or regressed replication fork. We propose WRN helicase operates in an EXO1-dependent pathway to help cells survive replicational stress. In contrast to WRN, BLM helicase defective in Bloom's syndrome failed to rescue rad50 MMS sensitivity, but partially restored IR resistance, suggesting a delineation of function by the human RecQ helicases.
A Human PMS2 Homologue from Aquifex aeolicus Stimulates an ATP-dependent DNA Helicase
2010-04-09
Full Text Available.Mismatch repair in Escherichia coli involves a number of proteins including MutL and UvrD. Eukaryotes also possess MutL homologues; however, no UvrD helicase homologues have been identified. The hyperthermophilic bacterium Aquifex aeolicus has a MutL protein (Aae MutL) that possesses a latent endonuclease activity similar to eukaryotic, but different from E. coli, MutL proteins. By sequence homology Aq793 is a member of the PcrA/UvrD/Rep helicase subfamily. We expressed Aae MutL and the putative A. aeolicus DNA helicase (Aq793) proteins in E. coli. Using synthetic oligonucleotide substrates, we observed that lower concentrations of Aq793 were required to unwind double-stranded DNA that had a 3′-poly(dT) overhang as compared with double-stranded DNA with a 5′-poly(dT) or lacking a poly(dT) tail. This unwinding activity was stimulated by adding Aae MutL with maximal stimulation observed at an ∼1.5:1 (MutL:Aq793) stoichiometric ratio. The enhancement of Aq793 helicase activity did not require the Aae MutL protein to retain endonuclease activity. Furthermore, the C-terminal 123 amino acid residues of Aae MutL were sufficient to stimulate Aq793 helicase activity, albeit at a significantly reduced efficacy. To the best of our knowledge this is the first time a human PMS2 homologue has been demonstrated to stimulate a PcrA/UvrD/Rep subfamily helicase, and this finding may further our understanding of the evolution of the mismatch repair pathway.
Peptide inhibitors of dengue virus NS3 protease. Part 1: Warhead
2006-01-01
Substrate-based tetrapeptide inhibitors with various warheads were designed, synthesized, and evaluated against the Dengue virus NS3 protease. Effective inhibition was achieved by peptide inhibitors with electrophilic warheads such as aldehyde, trifluoromethyl ketone, and boronic acid. A boronic acid has the highest affinity, exhibiting a Ki of 43nM.
2010-02-12
Full Text Available.The iron-sulfur-containing DNA helicases XPD, FANCJ, DDX11, and RTEL represent a small subclass of superfamily 2 helicases. XPD and FANCJ have been connected to the genetic instability syndromes xeroderma pigmentosum and Fanconi anemia. Here, we report a human individual with biallelic mutations in DDX11. Defective DDX11 is associated with a unique cellular phenotype in which features of Fanconi anemia (drug-induced chromosomal breakage) and Roberts syndrome (sister chromatid cohesion defects) coexist. The DDX11-deficient patient represents another cohesinopathy, besides Cornelia de Lange syndrome and Roberts syndrome, and shows that DDX11 functions at the interface between DNA repair and sister chromatid cohesion.
The nuclear import of RNA helicase A is mediated by importin-alpha3
2006-01-01
RNA helicase A (RHA), an ATPase/helicase, regulates the gene expression at various steps including transcriptional activation and RNA processing. RHA is known to shuttle between the nucleus and cytoplasm. We identified the nuclear localization signal (NLS) of RHA and analyzed the nuclear import mechanisms. The NLS of RHA (RHA-NLS) consisting of 19 amino acid residues is highly conserved through species and does not have the consensus classical NLS. In vitro nuclear import assays revealed that the nuclear import of RHA was Ran-dependent and mediated with the classical importin-alpha/beta-dependent pathway. The binding assay indicated that the basic residues in RHA-NLS were used for interaction with importin-alpha. Furthermore, the nuclear import of RHA-NLS was supported by importin-alpha1 and preferentially ...
The effect of 2-deoxy-d-glucose on Werner syndrome RecQ helicase gene
2009-01-01
Caloric restriction (CR) is known to effectively elongate mammalian life-spans. The compound 2-deoxy-d-glucose (2DG), which is often used as an inhibitor of glucose utilization, is a mimetic agent of CR. In this study, we examined the changes of telomerase and Werner's syndrome RecQ (WRN) helicase after treatment with 2DG, because of the involvement of recQ helicase in the regulation of telomeres. Interestingly, 2DG treatment increased the expression of WRN protein in accordance with induction of its promoter activity and gene expression. Furthermore, the activation of telomerase was observed after 2DG treatment, whereas it resulted in the reduction of cell proliferation. These results suggest that 2DG could up-regulate telomere maintenance factors accompanied with suppression of prolifera...
Telomeres do the (un)twist: helicase actions at chromosome termini
SUMMARYTelomeres play critical roles in protecting genome stability, and their dysfunction contributes to cancer and age-related degenerative diseases. The precise architecture of telomeres, including their single-stranded 3′ overhangs, bound proteins, and ability to form unusual secondary structures such as t-loops, is central to their function and thus requires careful processing by diverse factors. Furthermore, telomeres provide unique challenges to the DNA replication and recombination machinery, and are particularly suited for extension by the telomerase reverse transcriptase. Helicases use the energy from NTP hydrolysis to track along DNA and disrupt base pairing. Here we review current findings concerning how helicases modulate several aspects of telomere form and function.
Sumoylation of the BLM ortholog, Sgs1, promotes telomere-telomere recombination in budding yeast
2010-01-01
BLM and WRN are members of the RecQ family of DNA helicases, and in humans their loss is associated with syndromes characterized by genome instability and cancer predisposition. As the only RecQ DNA helicase in the yeast Saccharomyces cerevisiae, Sgs1 is known to safeguard genome integrity through its role in DNA recombination. Interestingly, WRN, BLM and Sgs1 are all known to be modified by the small ubiquitin-related modifier (SUMO), although the significance of this posttranslational modification remains elusive. Here, we demonstrate that Sgs1 is specifically sumoylated under the stress of DNA double strand breaks. The major SUMO attachment site in Sgs1 is lysine 621, which lies between the Top3 binding domain and the DNA helicase domain. Surprisingly, sumoylation of K621 was found to b...
2010-01-01
Abstract RNA helicases are adenosine tri-phosphatases that unwind the secondary structures of RNAs and are required in almost any aspect of RNA metabolism. They are highly conserved from prokaryotic to eukaryotic organisms. However, their precise roles in plant physiology and development remain to be clarified. Here we report that the mutation in the gene SLOW WALKER3 (SWA3) results in the slow and retarded progression of mitosis during megagametogenesis in Arabidopsis. SWA3 is a putative RNA helicase of the DEAD-box subfamily. Mutant megagametophyte development is arrested at four- or eight-nucleate stages, furthermore, one of the synergids in about half of the mutant embryo sacs displays abnormal polarity, with its nucleus locating at the chalazal end, instead of the micropylar end in th...
Roles of Werner syndrome protein in protection of genome integrity
2010-01-01
Werner syndrome protein (WRN) is one of a family of five human RecQ helicases implicated in the maintenance of genome stability. The conserved RecQ family also includes RecQ1, Bloom syndrome protein (BLM), RecQ4, and RecQ5 in humans, as well as Sgs1 in Saccharomyces cerevisiae, Rqh1 in Schizosaccharomyces pombe, and homologs in Caenorhabditis elegans, Xenopus laevis, and Drosophila melanogaster. Defects in three of the RecQ helicases, RecQ4, BLM, and WRN, cause human pathologies linked with cancer predisposition and premature aging. Mutations in the WRN gene are the causative factor of Werner syndrome (WS). WRN is one of the best characterized of the RecQ helicases and is known to have roles in DNA replication and repair, transcription, and telomere maintenance. Studies both in vitro and i...
2007-01-01
Concerted, stochastic and sequential mechanisms of action have been proposed for different hexameric AAA+ molecular motors. Here we report the crystal structure of the E1 helicase from bovine papillomavirus, where asymmetric assembly is for the first time observed in the absence of nucleotide cofactors and DNA. Surprisingly, the ATP-binding sites adopt specific conformations linked to positional changes in the DNA-binding hairpins, which follow a wave-like trajectory, as observed previously in the E1/DNA/ADP complex. The protein's assembly thus maintains such an asymmetric state in the absence of DNA and nucleotide cofactors, allowing consideration of the E1 helicase action as the propagation of a conformational wave around the protein ring. The data imply that the wave�...
2005-01-01
Werner syndrome (WS) is an autosomal recessive disease characterized by multiple progeroid features. The gene responsible for WS, WRN, is a member of the human RecQ helicase family. WRN is unique among this family, associated with an exonuclease activity. In the present study, we established the human 293-derived cell lines, which expressed exogenously truncated WRN protein, lacking the N-terminal exonuclease domain but having normal helicase activity, and found that they were slightly, but nonetheless significantly, radiosensitive than control cell lines, into which the empty vector had been introduced. The truncated WRN-expressing cells also exhibited increased numbers of micronuclei, chromosome aberrations, and the foci of phosphorylated histone H2AX with X-rays. These results suggested a function of WRN exonuclease activity that is separable from helicase ...
DnaB Helicase Activity Is Modulated by DNA Geometry and Force
2010-01-01
The replicative helicase for Escherichia coli is DnaB, a hexameric, ring-shaped motor protein that encircles and translocates along ssDNA, unwinding dsDNA in advance of its motion. The microscopic mechanisms of DnaB are unknown; further, prior work has found that DnaB's activity is modified by other replication proteins, indicating some mechanistic flexibility. To investigate these issues, we quantified translocation and unwinding by single DnaB molecules in three tethered DNA geometries held under tension. Our data support the following conclusions: 1), Unwinding by DnaB is enhanced by force-induced destabilization of dsDNA. 2), The magnitude of this stimulation varies with the geometry of the tension applied to the DNA substrate, possibly due to interactions between the helicase and the ...
A novel DEAD box helicase Has1p from Plasmodium falciparum: N-terminal is essential for activity
2010-01-01
Helicases catalyze the opening of nucleic acid duplexes and are implicated in many nucleic acid metabolic cellular processes that require single stranded DNA or reorganization of RNA structure. Previously we have reported that Plasmodium falciparum genome contains a number of DEAD box helicases. In the present study we report the cloning, expression and characterization of one of the novel members of DEAD box family from P. falciparum. Our results indicate that it is a homologue of Has1p from yeast and it contains DNA and RNA unwinding, nucleic acid-dependent ATPase and RNA binding activities. This enzyme can utilize all the nucleosidetriphosphates (NTPs) and deoxy nucleosidetriphosphates (dNTPs) for its unwinding activity. Using a truncated derivative of this protein we further report tha...
2006-01-01
The West Nile Virus (WNV) non-structural proteins 2B and 3 (NS2B-NS3) constitute the proteolytic complex that mediates the cleavage and processing of the viral polyprotein. NS3 recruits NS2B and NS5 proteins to direct protease and replication activities. In an effort to investigate the biology of the viral protease, we cloned cDNA encoding the NS2B-NS3 proteolytic complex from brain tissue of a WNV-infected dead crow, collected from the Lower Merion area (Merion strain). Expression of the NS2B-NS3 gene cassette induced apoptosis within 48 h of transfection. Electron microscopic analysis of NS2B-NS3-transfected cells revealed ultra-structural changes that are typical of apoptotic cells including membrane blebbing, nuclear disintegration and cytoplasmic vacuolations. The role of NS3 or NS2B in contributing to host cell apoptosis was examined. NS3 alone triggers the ...
2009-01-01
Vaccination programs for the control of bluetongue (BT) in ruminants have limitations due to difficulties in differentiating infected from vaccinated animals (DIVA). To overcome this problem a DIVA test that looks at a differential immune response to bluetongue virus (BTV) non-structural protein 3 (NS3) was developed. The NS3 encoding gene of strain BTV4/22045/PT04 was inserted into expression vector pET-28a and expressed in Escherichia coli strain JM109. Recombinant NS3 protein was used as an antigen in an indirect ELISA (NS3-ELISA) to measure the serologic response to NS3 protein in cattle and sheep. Following a cattle vaccination/challenge experiment with a bivalent inactivated BTV 2-4 vaccine, NS3 antibodies were detected at approximately 15 days post-infection in control unvaccinated ...
Antisense RNA-mediated gene silencing in fission yeast
2001-01-01
The major aims of this thesis were to investigate the influence of i) antisense gene location relative to the target gene locus (?????location effect?????), ii) double-stranded RNA (dsRNA) formation, and iii) over-expression of host-encoded proteins on antisense RNA-mediated gene regulation. To test the location effect hypothesis, strains were generated which contained the target lacZ gene at a fixed location and the antisense lacZ gene at various genomic locations including all arms of the three fission yeast chomosomes and in close proximity to the target gene locus. A long inverse-PCR protocol was developed to rapidly identify the precise site of antisense gene integration in the fission yeast transformants. No significant difference in lacZ suppression was observed when the antisense gene was integrated in close proximity to the target gene locus, compared with other genomic locations, indicating that target and antisense gene co-localisation is not a critical factor for efficient antisense RNA-mediated gene suppression in vivo. Instead, increased lacZ down-regulation correlated with an increase in the steady-state level of antisense RNA, which was dependent on genomic position effects and transgene copy number. In contrast, convergent transcription of an overlapping antisense lacZ gene was found to be very effective at inhibiting lacZ gene expression. DsRNA was also found to be a central component of antisense RNA-mediated gene silencing in fission yeast. It was shown that gene suppression could be enhanced by increasing the intracellular concentration of non-coding lacZ RNA, while expression of a lacZ panhandle RNA also inhibited beta-galactosidase activity. In addition, over-expression of the ATP-dependent RNA-helicase, ded1, was found to specifically enhance antisense RNA-mediated gene silencing. Through a unique overexpression screen, four novel factors were identified which specifically enhanced antisense RNA-mediated gene silencing by up to an additional 50%. The products of these antisense enhancing sequences (aes factors), all have natural associations with nucleic acids which is consistent with other proteins which have previously been identified to be involved in posttranscriptional gene silencing. Publisher: Awarded by:University of New South Wales. Biochemistry and Molecular Genetics Language: EN Rights: Copyright Mitch Raponi; http://unsworks.unsw.edu.au/copyright
Vertex Pharmaceuticals Researchers Report Three-Dimensional Structure Of Hepatitis C ...
1998-01-15
There are 4 million people in the US, some as prominent as Naomi Judd, with HCV. Vertex will report the 3-D map of HCV helicase in the January 15 issue of Structure. The information may be a valuable tool for designing ...
The Werner Syndrome Protein Functions in Repair of Cr(VI)-Induced Replication-Associated DNA Damage
2009-08-01
Werner syndrome is a premature aging disorder characterized by cancer predisposition that is caused by loss of the Werner syndrome protein (WRN) helicase/exonuclease DNA repair protein. Hexavalent chromium...Full Text Available
Researchers advance understanding of enzyme that regulates DNA
2010-08-20
Thanks to a single-molecule imaging technique developed by a University of Illinois professor, researchers have revealed the mechanisms of PcrA helicase, an important DNA-regulating enzyme. To prevent unwanted ...
Reduced Expression of IFIH1 Is Protective for Type 1 Diabetes
IFIH1 (interferon induced with helicase C domain 1), also known as MDA5 (melanoma differentiation-associated protein 5), is one of a family of intracellular proteins known to recognise viral RNA and...Full Text Available
Recql5 and Blm RecQ DNA Helicases Have Nonredundant Roles in Suppressing Crossovers
2005-05-01
In eukaryotes, crossovers in mitotic cells can have deleterious consequences and therefore must be suppressed. Mutations in BLM give rise to Bloom syndrome, a disease that is characterized...Full Text Available
NUCLEIC ACID BINDING ACTIVITIES OF HUMAN COCKAYNE SYNDROME B PROTEIN AND IDENTIFICATION OF Ca
2009-09-04
SummaryCockayne syndrome group B protein (CSB) is a member of the SWI/SNF2 subgroup of Superfamily 2 ATPases/nucleic acid translocases/helicases and is defective in the autosomal...Full Text Available
1997-02-01
Werner syndrome (WS) is an autosomal recessive disease with a complex phenotype that is suggestive of accelerated aging. WS is caused by mutations in a gene, WRN, that encodes a predicted 1,432-amino-acid...Full Text Available
Mammalian BLM helicase is critical for integrating multiple pathways of meiotic recombination
2010-03-22
Bloom’s syndrome (BS) is an autosomal recessive disorder characterized by growth retardation, cancer predisposition, and sterility. BS mutated (Blm), the gene mutated in BS...Full Text Available
2009-05-01
Fanconi anemia (FA) is an autosomal recessive disorder characterized by multiple congenital anomalies, progressive bone marrow failure, and high cancer risk. Cells from FA patients exhibit spontaneous...Full Text Available
Escherichia coli RecQ protein is a DNA helicase.
1990-07-01
The Escherichia coli recQ gene, a member of the RecF recombination gene family, was set in an overexpression plasmid, and its product was purified to near-homogeneity. The purified RecQ protein exhibited...Full Text Available
2006-09-01
Full Text Available.The DExD/H box family of proteins includes a large number of proteins that play important roles in RNA metabolism. Members of this family have been shown to act as RNA helicases or unwindases, using the energy from ATP hydrolysis to unwind RNA structures or dissociate RNA–protein complexes in cellular processes that require modulation of RNA structures. However, it is clear that several members of this family are multifunctional and, in addition to acting as RNA helicases in processes such as pre-mRNA processing, play important roles in transcriptional regulation. In this review I shall concentrate on RNA helicase A (Dhx9), DP103 (Ddx20), p68 (Ddx5) and p72 (Ddx17), proteins for which there is a strong body of evidence showing that they play important roles in transcription, often as coactivators or corepressors through their interaction with key components of the transcriptional machinery, such as CREB-binding protein, p300, RNA polymerase II and histone deacetylases.
2006-01-01
E1 and T-antigen of the tumour viruses bovine papillomavirus (BPV-1) and Simian virus 40 (SV40) are the initiator proteins that recognize and melt their respective origins of replication in the initial phase of DNA replication. These proteins then assemble into processive hexameric helicases upon the single-stranded DNA that they create. In T-antigen, a characteristic loop and hairpin structure (the pre-sensor 1b hairpin, PS1bH) project into a central cavity generated by protein hexamerization. This channel undergoes large ATP-dependent conformational changes, and the loop/PS1bH is proposed to form a DNA binding site critical for helicase activity. Here, we show that conserved residues in BPV E1 that probably form a similar loop/hairpin structure are required for helicase activity and also...
2010-08-01
Full Text Available.Mutations in the Werner gene promote the segmental progeroid Werner syndrome (WS) with increased genomic instability and cancer. The Werner gene encodes a DNA helicase (WRN) that can engage in direct protein–protein interactions with DHX9, also known as RNA helicase A or nuclear DNA helicase II, which represents an essential enzyme involved in transcription and DNA repair. By using several synthetic nucleic acid substrates we demonstrate that WRN preferably unwinds RNA-containing Okazaki fragment-like substrates suggesting a role in lagging strand maturation of DNA replication. In contrast, DHX9 preferably unwinds RNA–RNA and RNA–DNA substrates, but fails to unwind Okazaki fragment-like hybrids. We further show that the preferential unwinding of RNA-containing substrates by WRN is stimulated by DHX9 in vitro, both on Okazaki fragment-like hybrids and on RNA-containing ‘chicken-foot’ structures. Collectively, our results suggest that WRN and DHX9 may also cooperate in vivo, e.g. at ongoing and stalled replication forks. In the latter case, the cooperation between both helicases may serve to form and to dissolve Holliday junction-like intermediates of regressed replication forks.
2006-10-01
Full Text Available.RecQ DNA helicases and Topo III topoisomerases have conserved genetic, physical, and functional interactions that are consistent with a model in which RecQ creates a recombination-dependent substrate that is resolved by Topo III. The phenotype associated with Topo III loss suggests that accumulation of a RecQ-created substrate is detrimental. In yeast, mutation of the TOP3 gene encoding Topo III causes pleiotropic defects that are suppressed by deletion of the RecQ homolog Sgs1. We searched for gene dosage suppressors of top3 and identified Pif1, a DNA helicase that acts with polarity opposite to that of Sgs1. Pif1 overexpression suppresses multiple top3 defects, but exacerbates sgs1 and sgs1 top3 defects. Furthermore, Pif1 helicase activity is essential in the absence of Top3 in an Sgs1-dependent manner. These data clearly demonstrate that Pif1 helicase activity is required to counteract Sgs1 helicase activity that has become uncoupled from Top3. Pif1 genetic interactions with the Sgs1–Top3 pathway are dependent upon homologous recombination. We also find that Pif1 is recruited to DNA repair foci and that the frequency of these foci is significantly increased in top3 mutants. Our results support a model in which Pif1 has a direct role in the prevention or repair of Sgs1-induced DNA damage that accumulates in top3 mutants.
Werner syndrome (WS) is an autosomal recessive disease with a complex phenotype that is suggestive of accelerated aging. WS is caused by mutations in a gene, WRN, that encodes a predicted 1,432-amino-acid protein with homology to DNA and RNA helicases. Previous work identified four WS mutations in the 3' end of the gene, which resulted in predicted truncated protein products of 1,060-1,247 amino acids but did not disrupt the helicase domain region (amino acids 569-859). Here, additional WS subjects were screened for mutations, and the intron-exon structure of the gene was determined. A total of 35 exons were defined, with the coding sequences beginning in the second exon. Five new WS mutations were identified: two nonsense mutations at codons 369 and 889; a mutation at a splice-junction site, resulting in a predicted truncated protein of 760 amino acids; a 1-bp deletion causing a frameshift; and a predicted truncated protein of 391 amino acids. Another deletion is >15 kb of genomic DNA, including exons 19-23; the predicted protein is 1,186 amino acids long. Four of these new mutations either partially disrupt the helicase domain region or result in predicted protein products completely missing the helicase region. These results confirm that mutations in the WRN gene are responsible for WS. Also, the location of the mutations indicates that the presence or absence of the helicase domain does not influence the WS phenotype and suggests that WS is the result of complete loss of function of the WRN gene product.
Mutations in the consensus helicase domains of the Werner syndrome gene
Werner syndrome (WS) is an autosomal recessive disease with a complex phenotype that is suggestive of accelerated aging. WS is caused by mutations in a gene, WRN, that encodes a predicted 1,432-amino-acid protein with homology to DNA and RNA helicases. Previous work identified four WS mutations in the 3{prime} end of the gene, which resulted in predicted truncated protein products of 1,060-1,247 amino acids but did not disrupt the helicase domain region (amino acids 569-859). Here, additional WS subjects were screened for mutations, and the intron-exon structure of the gene was determined. A total of 35 exons were defined, with the coding sequences beginning in the second exon. Five new WS mutations were identified: two nonsense mutations at codons 369 and 889; a mutation at a splice-junction site, resulting in a predicted truncated protein of 760 amino acids; a 1-bp deletion causing a frameshift; and a predicted truncated protein of 391 amino acids. Another deletion is >15 kb of genomic DNA, including exons 19-23; the predicted protein is 1,186 amino acids long. Four of these new mutations either partially disrupt the helicase domain region or result in predicted protein products completely missing the helicase region. These results confirm that mutations in the WRN gene are responsible for WS. Also, the location of the mutations indicates that the presence or absence of the helicase domain does not influence the WS phenotype and suggests that WS is the result of complete loss of function of the WRN gene product. 63 refs., 1 fig., 5 tabs.
Mutations in the consensus helicase domains of the Werner syndrome gene
1997-02-01
Werner syndrome (WS) is an autosomal recessive disease with a complex phenotype that is suggestive of accelerated aging. WS is caused by mutations in a gene, WRN, that encodes a predicted 1,432-amino-acid protein with homology to DNA and RNA helicases. Previous work identified four WS mutations in the 3{prime} end of the gene, which resulted in predicted truncated protein products of 1,060-1,247 amino acids but did not disrupt the helicase domain region (amino acids 569-859). Here, additional WS subjects were screened for mutations, and the intron-exon structure of the gene was determined. A total of 35 exons were defined, with the coding sequences beginning in the second exon. Five new WS mutations were identified: two nonsense mutations at codons 369 and 889; a mutation at a splice-junction site, resulting in a predicted truncated protein of 760 amino acids; a 1-bp deletion causing a frameshift; and a predicted truncated protein of 391 amino acids. Another deletion is >15 kb of genomic DNA, including exons 19-23; the predicted protein is 1,186 amino acids long. Four of these new mutations either partially disrupt the helicase domain region or result in predicted protein products completely missing the helicase region. These results confirm that mutations in the WRN gene are responsible for WS. Also, the location of the mutations indicates that the presence or absence of the helicase domain does not influence the WS phenotype and suggests that WS is the result of complete loss of function of the WRN gene product. 63 refs., 1 fig., 5 tabs.
Ablation-induced explosion of metal using a high-power Nd:YAG laser
The interaction of a high-power pulsed-laser beam with metal targets in air from a 1.06 ?m, 5 ns, 3 J/pulse, Nd:YAG pulsed laser is investigated together with hydrodynamic theories of laser-supported blast wave and multimaterial reactive Euler equations. The high-speed blast wave generated by the laser ablation of metal reaches a maximum velocity of several thousand meters per second. The apparently similar flow conditions to those of reactive shock wave allow one to apply the equations of motion for energetic materials and to understand the explosive behavior of metal vaporization upon laser ablation. The characteristic time at which the planar to spherical wave transition occurs is investigated at low (20 mJ/pulse) to high (200 mJ/pulse) beam intensities. The flow structure behind the leading shock wave during the early planar shock state is confirmed by the high-resolution multimaterial hydrocode originally developed for shock compression of condensed matter. A repeatable lab-scale blast wave experiment is conducted at various energy levels with three different ablative targets, and both theoretical and computational analyses are used to verify the flow structures behind the leading shock front that remains spherically symmetric until all the momentum transferred from the absorbed intensity dissipates into open air a few microseconds later.
2009-03-15
Full Text Available.Recombination is important for DNA repair, but it can also contribute to genome rearrangements. RecQ helicases, including yeast Sgs1 and human BLM, safeguard genome integrity through their functions in DNA recombination. Sgs1 prevents the accumulation of Rad51-dependent sister chromatid junctions at damaged replication forks, and its functionality seems to be regulated by Ubc9- and Mms21-dependent sumoylation. We show that mutations in Smc5-6 and Esc2 also lead to an accumulation of recombinogenic structures at damaged replication forks. Because Smc5-6 is sumoylated in an Mms21-dependent manner, this finding suggests that Smc5-6 may be a crucial target of Mms21 implicated in this process. Our data reveal that Smc5-6 and Esc2 are required to tolerate DNA damage and that their functionality is critical in genotoxic conditions in the absence of Sgs1. As reported previously for Sgs1 and Smc5-6, we find that Esc2 physically interacts with Ubc9 and SUMO. This interaction is correlated with the ability of Esc2 to promote DNA damage tolerance. Collectively, these data suggest that Esc2 and Smc5-6 act in concert with Sgs1 to prevent the accumulation of recombinogenic structures at damaged replication forks, likely by integrating sumoylation activities to regulate the repair pathways in response to damaged DNA.
Recombination is important for DNA repair, but it can also contribute to genome rearrangements. RecQ helicases, including yeast Sgs1 and human BLM, safeguard genome integrity through their functions in DNA recombination. Sgs1 prevents the accumulation of Rad51-dependent sister chromatid junctions at damaged replication forks, and its functionality seems to be regulated by Ubc9- and Mms21-dependent sumoylation. We show that mutations in Smc5-6 and Esc2 also lead to an accumulation of recombinogenic structures at damaged replication forks. Because Smc5-6 is sumoylated in an Mms21-dependent manner, this finding suggests that Smc5-6 may be a crucial target of Mms21 implicated in this process. Our data reveal that Smc5-6 and Esc2 are required to tolerate DNA damage and that their functionality is critical in genotoxic conditions in the absence of Sgs1. As reported previously for Sgs1 and Smc5-6, we find that Esc2 physically interacts with Ubc9 and SUMO. This interaction is correlated with the ability of Esc2 to promote DNA damage tolerance. Collectively, these data suggest that Esc2 and Smc5-6 act in concert with Sgs1 to prevent the accumulation of recombinogenic structures at damaged replication forks, likely by integrating sumoylation activities to regulate the repair pathways in response to damaged DNA.
Molecular dissection of prethymic progenitor entry into the T lymphocyte developmental pathway
Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-I (TCF-1) (gene name Tcli). To identify additional regulators of T cell specification, a cDNA libnlrY from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expression of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin{sup -}Sca-1{sup +}Kit{sup +}CD27{sup -}) and multipotent progenitors (Lin{sup -}Sca-l{sup +}Kit{sup +}CD27{sup +}), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bclllb, TCF-I (Tcli), and HEBalt, Notch target Deltexl, Deltex3L, Fkbp5, Eval, and Tmem13l. Like GATA3 and Deltexl, Bclllb, Fkbp5, and Eval were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only BcIlI band HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative I and double negative 2) corresponding to T lineage specification. Bclllb was uniquely T lineage restricted and induced by NotchlDelta signaling specifically upon entry into the T lineage differentiation pathway.
Molecular dissection of prethymic progenitor entry into the T lymphocyte developmental pathway
Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-I (TCF-1) (gene name Tcli). To identify additional regulators of T cell specification, a cDNA libnlrY from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expression of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin{sup -}Sca-1{sup +}Kit{sup +}CD27{sup -}) and multipotent progenitors (Lin{sup -}Sca-l{sup +}Kit{sup +}CD27{sup +}), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bclllb, TCF-I (Tcli), and HEBalt, Notch target Deltexl, Deltex3L, Fkbp5, Eval, and Tmem13l. Like GATA3 and Deltexl, Bclllb, Fkbp5, and Eval were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only BcIlI band HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative I and double negative 2) corresponding to T lineage specification. Bclllb was uniquely T lineage restricted and induced by NotchlDelta signaling specifically upon entry into the T lineage differentiation pathway.
Molecular dissection of prethymic progenitor entry into the T lymphocyte developmental pathway
2008-01-01
Notch signaling activates T lineage differentiation from hemopoietic progenitors, but relatively few regulators that initiate this program have been identified, e.g., GATA3 and T cell factor-I (TCF-1) (gene name Tcli). To identify additional regulators of T cell specification, a cDNA libnlrY from mouse Pro-T cells was screened for genes that are specifically up-regulated in intrathymic T cell precursors as compared with myeloid progenitors. Over 90 genes of interest were identified, and 35 of 44 tested were confirmed to be more highly expressed in T lineage precursors relative to precursors of B and/or myeloid lineage. To a remarkable extent, however, expression of these T lineage-enriched genes, including zinc finger transcription factor, helicase, and signaling adaptor genes, was also shared by stem cells (Lin{sup -}Sca-1{sup +}Kit{sup +}CD27{sup -}) and multipotent progenitors (Lin{sup -}Sca-l{sup +}Kit{sup +}CD27{sup +}), although down-regulated in other lineages. Thus, a major fraction of these early T lineage genes are a regulatory legacy from stem cells. The few genes sharply up-regulated between multipotent progenitors and Pro-T cell stages included those encoding transcription factors Bclllb, TCF-I (Tcli), and HEBalt, Notch target Deltexl, Deltex3L, Fkbp5, Eval, and Tmem13l. Like GATA3 and Deltexl, Bclllb, Fkbp5, and Eval were dependent on Notch/Delta signaling for induction in fetal liver precursors, but only BcIlI band HEBalt were up-regulated between the first two stages of intrathymic T cell development (double negative I and double negative 2) corresponding to T lineage specification. Bclllb was uniquely T lineage restricted and induced by NotchlDelta signaling specifically upon entry into the T lineage differentiation pathway.
Cigarette Smoke Induces Cellular Senescence via Werner's Syndrome Protein Down-regulation
Rationale: Werner's syndrome is a genetic disorder that causes premature aging due to loss-of-function mutations in a gene encoding a member of the RecQ helicase family. Both Werner's syndrome and cigarette smoking accelerate aging. No studies have examined the effect of cigarette smoke on Werner's syndrome protein.Objectives: To investigate the role of Werner's syndrome protein in cigarette smoke–induced cellular senescence.Methods: Cellular senescence and amounts of Werner's syndrome protein were measured in fibroblasts isolated from patients with emphysema and compared with age-matched nonsmokers. The in vitro effects of cigarette smoke on amounts of Werner's syndrome protein, function, and senescence were also evaluated in primary human lung fibroblasts and epithelial cells.Measurements and Main Results: Cultured lung fibroblasts isolated from patients with emphysema exhibited a senescent phenotype accompanied by a decrease in Werner's syndrome protein. Cigarette smoke extract decreased Werner's syndrome protein in cultured fibroblasts and epithelial cells. Werner's syndrome protein–deficient fibroblasts were more susceptible to cigarette smoke–induced cellular senescence and cell migration impairment. In contrast, exogenous overexpression of Werner's syndrome protein attenuated the cigarette smoke effects.Conclusions: Cigarette smoke induces cellular senescence and cell migration impairment via Werner's syndrome protein down-regulation. Rescue of Werner's syndrome protein down-regulation may represent a potential therapeutic target for smoking-related diseases.
2004-01-01
Full Text Available.Minor histocompatibility antigens (mHAs) recognized by donor T cells play a central role as immunologic targets of graft-versus-host disease (GVHD) and graft versus leukemia after allogeneic hematopoietic stem cell transplantation (HSCT). Men who have undergone sex-mismatched allogeneic HSCT are at high risk for GVHD because of immune responses directed against mHAs encoded by genes on the Y chromosome (termed H-Y antigens). We hypothesized that the immunogenicity of mHAs results in a coordinated response involving B cells as well as T cells. To test this, we measured antibody responses to a well-characterized H-Y antigen, dead box RNA helicase Y (DBY), and its homolog, DBX, in 150 HSCT patients. Using Western blot and enzyme-linked immunosorbent assay (ELISA), we found that 50% of male patients who received stem cell grafts from female donors developed antibody responses to recombinant DBY protein. Antibodies to DBY were also detected in 17% of healthy women, but not in healthy men. Antibody responses were directed primarily against areas of amino acid disparity between DBY and DBX. These studies demonstrate that the immune response to mHA includes the generation of specific antibodies and suggests that the serologic response to these antigens may also be useful in the identification of new mHAs.
Natural epitope variants of the hepatitis C virus impair cytotoxic T lymphocyte activity
2010-04-28
Full Text Available.AIM: To understand how interactions between hepatitis C virus (HCV) and the host’s immune system might lead to viral persistence or effective elimination of HCV.METHODS: Nucleotides 3519-3935 of the non-structural 3 (NS3) region were amplified by using reverse transcription polymerase chain reaction (PCR). PCR products of the HCV NS3 regions were integrated into a PCR
In vitro studies have attempted to identify dengue virus (DEN) target cells in peripheral blood; however, extensive phenotyping of peripheral blood mononuclear cells (PBMCs) from dengue patients has not been reported. PBMCs collected from hospitalized children suspected of acute dengue were analyzed for DEN prM, CD32, CD86, CD14, CD11c, CD16, CD209, CCR7, CD4, and CD8 by flow cytometry to detect DEN antigen in PBMCs and to phenotype DEN-positive cells. DEN prM was detected primarily in activated monocytes (CD14(+), CD32(+), CD86(+), CD11c(+)). A subset of samples analyzed for DEN nonstructural protein 3 (NS3) confirmed that approximately half of DEN antigen-positive cells contained replicating virus. A higher percentage of PBMCs from DHF patients expressed prM, CD86, CD32, and CD11c than did those from DF patients. Increased activation of monocytes and greater numbers of DEN-infected cells were associated with more severe dengue, implicating a role for monocyte activation in dengue immunopathogenesis.
Full Text Available.BackgroundCancer associated with smoking and drinking remains a serious health problem worldwide. The survival of patients is very poor due to the lack of effective early biomarkers. FOXM1 overexpression is linked to the majority of human cancers but its mechanism remains unclear in head and neck squamous cell carcinoma (HNSCC).Methodology/Principal FindingsFOXM1 mRNA and protein expressions were investigated in four independent cohorts (total 75 patients) consisting of normal, premalignant and HNSCC tissues and cells using quantitative PCR (qPCR), expression microarray, immunohistochemistry and immunocytochemistry. Effect of putative oral carcinogens on FOXM1 transcriptional activity was dose-dependently assayed and confirmed using a FOXM1-specific luciferase reporter system, qPCR, immunoblotting and short-hairpin RNA interference. Genome-wide single nucleotide polymorphism (SNP) array was used to ‘trace’ the genomic instability signature pattern in 8 clonal lines of FOXM1-induced malignant human oral keratinocytes. Furthermore, acute FOXM1 upregulation in primary oral keratinocytes directly induced genomic instability. We have shown for the first time that overexpression of FOXM1 precedes HNSCC malignancy. Screening putative carcinogens in human oral keratinocytes surprisingly showed that nicotine, which is not perceived to be a human carcinogen, directly induced FOXM1 mRNA, protein stabilisation and transcriptional activity at concentrations relevant to tobacco chewers. Importantly, nicotine also augmented FOXM1-induced transformation of human oral keratinocytes. A centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, both located within a consensus loci (10q23), were found to be novel targets of FOXM1 and their expression correlated tightly with HNSCC progression.Conclusions/SignificanceThis study cautions the potential co-carcinogenic effect of nicotine in tobacco replacement therapies. We hypothesise that aberrant upregulation of FOXM1 may be inducing genomic instability through a program of malignant transformation involving the activation of CEP55 and HELLS which may facilitate aberrant mitosis and epigenetic modifications. Our finding that FOXM1 is upregulated early during oral cancer progression renders FOXM1 an attractive diagnostic biomarker for early cancer detection and its candidate mechanistic targets, CEP55 and HELLS, as indicators of malignant conversion and progression.
BackgroundCancer associated with smoking and drinking remains a serious health problem worldwide. The survival of patients is very poor due to the lack of effective early biomarkers. FOXM1 overexpression is linked to the majority of human cancers but its mechanism remains unclear in head and neck squamous cell carcinoma (HNSCC).Methodology/Principal FindingsFOXM1 mRNA and protein expressions were investigated in four independent cohorts (total 75 patients) consisting of normal, premalignant and HNSCC tissues and cells using quantitative PCR (qPCR), expression microarray, immunohistochemistry and immunocytochemistry. Effect of putative oral carcinogens on FOXM1 transcriptional activity was dose-dependently assayed and confirmed using a FOXM1-specific luciferase reporter system, qPCR, immunoblotting and short-hairpin RNA interference. Genome-wide single nucleotide polymorphism (SNP) array was used to ‘trace’ the genomic instability signature pattern in 8 clonal lines of FOXM1-induced malignant human oral keratinocytes. Furthermore, acute FOXM1 upregulation in primary oral keratinocytes directly induced genomic instability. We have shown for the first time that overexpression of FOXM1 precedes HNSCC malignancy. Screening putative carcinogens in human oral keratinocytes surprisingly showed that nicotine, which is not perceived to be a human carcinogen, directly induced FOXM1 mRNA, protein stabilisation and transcriptional activity at concentrations relevant to tobacco chewers. Importantly, nicotine also augmented FOXM1-induced transformation of human oral keratinocytes. A centrosomal protein CEP55 and a DNA helicase/putative stem cell marker HELLS, both located within a consensus loci (10q23), were found to be novel targets of FOXM1 and their expression correlated tightly with HNSCC progression.Conclusions/SignificanceThis study cautions the potential co-carcinogenic effect of nicotine in tobacco replacement therapies. We hypothesise that aberrant upregulation of FOXM1 may be inducing genomic instability through a program of malignant transformation involving the activation of CEP55 and HELLS which may facilitate aberrant mitosis and epigenetic modifications. Our finding that FOXM1 is upregulated early during oral cancer progression renders FOXM1 an attractive diagnostic biomarker for early cancer detection and its candidate mechanistic targets, CEP55 and HELLS, as indicators of malignant conversion and progression.
2008-02-01
Full Text Available. Escherichia coli contains five members of the DEAD-box RNA helicase family, a ubiquitous class of proteins characterized by their ability to unwind RNA duplexes. Although four of these proteins have been implicated in RNA turnover or ribosome biogenesis, no cellular function for the RhlE DEAD-box protein has been described as yet. During an analysis of the cold-sensitive growth defect of a strain lacking the DeaD/CsdA RNA helicase, rhlE plasmids were identified from a chromosomal library as multicopy suppressors of the growth defect. Remarkably, when tested for allele specificity, RhlE overproduction was found to exacerbate the cold-sensitive growth defect of a strain that lacks the SrmB RNA helicase. Moreover, the absence of RhlE exacerbated or alleviated the cold-sensitive defect of deaD or srmB strains, respectively. Primer extension and ribosome analysis indicated that RhlE regulates the accumulation of immature ribosomal RNA or ribosome precursors when deaD or srmB strains are grown at low temperatures. By using an epitope-tagged version of RhlE, the majority of RhlE in cell extracts was found to cosediment with ribosome-containing fractions. Since both DeaD and SrmB have been recently shown to function in ribosome assembly, these findings suggests that rhlE genetically interacts with srmB and deaD to modulate their function during ribosome maturation. On the basis of the available evidence, I propose that RhlE is a novel ribosome assembly factor, which plays a role in the interconversion of ribosomal RNA-folding intermediates that are further processed by DeaD or SrmB during ribosome maturation.
The Bacillus subtilis SPPI phage encoded protein G39P is a loader and inhibitor of the phage G40P replicative helicase involved in the initiation of phage DNA replication. The 2.4A crystal structure of a C-terminal truncated variant of G39P was solved using multiple wavelength anomalous dispersion exploiting the anomalous signal of seleno- methionine substituted protein. Inspection of the electron density maps revealed the asymmetric unit contained three independent G39P monomers, composed of 3 alpha-helices and their connecting loops. However, the model only accounted for the first 67 residues of the protein, as there was no interpretable electron density for residues 68 to 112. A preliminary NMR investigation revealed the C-terminal region of the protein had rapid internal motion and formed no well-defined stable fold that involved immobilized side chains. This is consistent with the X-ray analysis that displayed no electron density for these residues. A detailed comparison of NMR spectra from the C-terminal truncated variant and from intact wild-type G39P revealed no major differences between the two forms. Thus these investigations have revealed that G39P has a bipartite structure comprising a folded N-terminal domain and an unfolded C-terminal domain that has been shown, through genetic and biochemical analysis, to be essential for helicase interaction. In addition, the packing of the G39P monomers around a non-crystallographic 6 sub 1 -screw axis presents an intriguing insight into the possible organization of the helicase loader when bound to its helicase partner.
RNA helicases play important roles in RNA metabolism. Human immunodeficiency virus type 1 (HIV-1) does not carry its own RNA helicase, the virus thus needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport. In this study, we report that knockdown of a DEAD-box protein named DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to the viral RNA packaging stage during virus assembly.
2008-01-01
RNA helicases play important roles in RNA metabolism. Human immunodeficiency virus type 1 (HIV-1) does not carry its own RNA helicase, the virus thus needs to exploit cellular RNA helicases to promote the replication of its RNA at various steps such as transcription, folding and transport. In this study, we report that knockdown of a DEAD-box protein named DDX24 inhibits the packaging of HIV-1 RNA and thus diminishes viral infectivity. The decreased viral RNA packaging as a result of DDX24-knockdown is observed only in the context of the Rev/RRE (Rev response element)-dependent but not the CTE (constitutive transport element)-mediated nuclear export of viral RNA, which is explained by the specific interaction of DDX24 with the Rev protein. We propose that DDX24 acts at the early phase of HIV-1 RNA metabolism prior to nuclear export and the consequence of this action extends to ...
In budding yeast, the Pif1 DNA helicase is involved in the maintenance of both nuclear and mitochondrial genomes, but its role in these processes is still poorly understood. Here, we provide evidence for a new Pif1 function by demonstrating that its absence promotes genetic instability of alleles of the G-rich human minisatellite CEB1 inserted in the Saccharomyces cerevisiae genome, but not of other tandem repeats. Inactivation of other DNA helicases, including Sgs1, had no effect on CEB1 stability. In vitro, we show that CEB1 repeats formed stable G-quadruplex (G4) secondary structures and the Pif1 protein unwinds these structures more efficiently than regular B-DNA. Finally, synthetic CEB1 arrays in which we mutated the potential G4-forming sequences were no longer destabilized in pif1Δ cells. Hence, we conclude that CEB1 instability in pif1Δ cells depends on the potential to form G-quadruplex structures, suggesting that Pif1 could play a role in the metabolism of G4-forming sequences.
PcrA Helicase Dismantles RecA Filaments by Reeling in DNA in Uniform Steps
2010-01-01
Summary Translocation of helicase-like proteins on nucleic acids underlies key cellular functions. However, it is still unclear how translocation can drive removal of DNA-bound proteins, and basic properties like the elementary step size remain controversial. Using single-molecule fluorescence analysis on a prototypical superfamily 1 helicase, Bacillus stearothermophilus PcrA, we discovered that PcrA preferentially translocates on the DNA lagging strand instead of unwinding the template duplex. PcrA anchors itself to the template duplex using the 2B subdomain and reels in the lagging strand, extruding a single-stranded loop. Static disorder limited previous ensemble studies of a PcrA stepping mechanism. Here, highly repetitive looping revealed that PcrA translocates in uniform steps of 1 n...
Mammalian BLM helicase is critical for integrating multiple pathways of meiotic recombination
Bloom’s syndrome (BS) is an autosomal recessive disorder characterized by growth retardation, cancer predisposition, and sterility. BS mutated (Blm), the gene mutated in BS patients, is one of five mammalian RecQ helicases. Although BLM has been shown to promote genome stability by assisting in the repair of DNA structures that arise during homologous recombination in somatic cells, less is known about its role in meiotic recombination primarily because of the embryonic lethality associated with Blm deletion. However, the localization of BLM protein on meiotic chromosomes together with evidence from yeast and other organisms implicates a role for BLM helicase in meiotic recombination events, prompting us to explore the meiotic phenotype of mice bearing a conditional mutant allele of Blm. In this study, we show that BLM deficiency does not affect entry into prophase I but causes severe defects in meiotic progression. This is exemplified by improper pairing and synapsis of homologous chromosomes and altered processing of recombination intermediates, resulting in increased chiasmata. Our data provide the first analysis of BLM function in mammalian meiosis and strongly argue that BLM is involved in proper pairing, synapsis, and segregation of homologous chromosomes; however, it is dispensable for the accumulation of recombination intermediates.
MRE11 complex links RECQ5 helicase to sites of DNA damage
2009-05-01
Full Text Available.RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11–RAD50–NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3′→5′ exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation of RECQ5 at DSBs was neither dependent on MDC1 that mediates binding of MRN to DSB-flanking chromatin nor on CtIP that acts in conjunction with MRN to promote resection of DSBs for repair by HR. Collectively, these data suggest that the MRN complex recruits RECQ5 to sites of DNA damage to regulate DNA repair.
MRE11 complex links RECQ5 helicase to sites of DNA damage
2009-01-01
RECQ5 DNA helicase suppresses homologous recombination (HR) possibly through disruption of RAD51 filaments. Here, we show that RECQ5 is constitutively associated with the MRE11–RAD50–NBS1 (MRN) complex, a primary sensor of DNA double-strand breaks (DSBs) that promotes DSB repair and regulates DNA damage signaling via activation of the ATM kinase. Experiments with purified proteins indicated that RECQ5 interacts with the MRN complex through both MRE11 and NBS1. Functional assays revealed that RECQ5 specifically inhibited the 3′→5′ exonuclease activity of MRE11, while MRN had no effect on the helicase activity of RECQ5. At the cellular level, we observed that the MRN complex was required for the recruitment of RECQ5 to sites of DNA damage. Accumulation...
2009-01-01
Helicobacter pylori, an important bacterial pathogen, causes gastric ulcer and gastric adenocarcinoma in humans. The fundamentals of basic biology such as DNA replication are poorly understood in this pathogen. In the present study, we report the cloning and functional characterization of the single-stranded DNA (ssDNA) binding protein from H. pylori. The N-terminal DNA binding domain shows significant homology with E. coli single-stranded DNA binding protein (SSB), whereas the C-terminal domain shows less homology. The overall DNA-binding activity and tetramerization properties, however, remain unaffected. In in vitro experiments with purified proteins, H. pylori (Hp) SSB bound specifically to ssDNA and modulated the enzymatic ATPase and helicase activity of HpDnaB helicase. HpSSB and HpD...
Conserved helicase domain of human RecQ4 is required for strand annealing-independent DNA unwinding
2010-01-01
Humans have five members of the well conserved RecQ helicase family: RecQ1, Bloom syndrome protein (BLM), Werner syndrome protein (WRN), RecQ4, and RecQ5, which are all known for their roles in maintaining genome stability. BLM, WRN, and RecQ4 are associated with premature aging and cancer predisposition. Of the three, RecQ4's biological and cellular roles have been least thoroughly characterized. Here we tested the helicase activity of purified human RecQ4 on various substrates. Consistent with recent results, we detected ATP-dependent RecQ4 unwinding of forked duplexes. However, our results provide the first evidence that human RecQ4's unwinding is independent of strand annealing, and that it does not require the presence of excess ssDNA. Moreover, we demonstrate that a point mutation of...
Yeast as a model system to study RecQ helicase function
2010-01-01
Mutations in the highly conserved RecQ helicase, BLM, cause the rare cancer predisposition disorder, Bloom's syndrome. The orthologues of BLM in Saccharomyces cerevisiae and Schizosaccharomyces pombe are SGS1 and rqh1^+, respectively. Studies in these yeast species have revealed a plethora of roles for the Sgs1 and Rqh1 proteins in repair of double strand breaks, restart of stalled replication forks, processing of aberrant intermediates that arise during meiotic recombination, and maintenance of telomeres. In this review, we focus on the known roles of Sgs1 and Rqh1 and how studies in yeast species have improved our knowledge of how BLM suppresses neoplastic transformation.
Widespread expression of the Supv3L1 mitochondrial RNA helicase in the mouse
2010-01-01
Supv3L1 is an evolutionarily conserved helicase that plays a critical role in the mitochondrial RNA surveillance and degradation machinery. Conditional ablation of Supv3L1 in adult mice leads to premature aging phenotypes including loss of muscle mass and adipose tissue and severe skin abnormalities. To get insights into the spatial and temporal expression of Supv3L1 in the mouse, we generated knock-in and transgenic strains in which an EGFP reporter was placed under control of the Supv3L1 native promoter. During development, expression of Supv3L1 begins at the blastocyst stage, becomes widespread and strong in all fetal tissues and cell types, and continues during postnatal growth. In mature animals reporter expression is only slightly diminished in most tissues and continues to be highly...
Werner syndrome gene variants in human sarcomas
2010-01-01
Werner syndrome is an autosomal inherited disease that is characterized by premature aging. The gene mutated in Werner syndrome (WS), WRN, encodes both a 3prime 5prime DNA helicase and a 3prime 5prime DNA exonuclease. Among the WS phenotypes is an exceptionally high incidence of sarcomas. We asked whether spontaneous sarcomas, not known to be associated with WS, also harbor mutations or unreported single nucleotide polymorphisms (SNPs) in WRN. We analyzed RNA or DNA sequences within the helicase and exonuclease domains from 51 and 69 matched sarcoma and adjacent normal tissues, respectively. Among a total of 13 nucleotide variants detected, we identified three novel nonsynonymous substitutions: c.611C
The Rothmund-Thomson gene product RECQL4 localizes to the nucleolus in response to oxidative stress
2006-01-01
Mutations in the RECQL4 helicase gene have been linked to Rothmund-Thomson syndrome (RTS), which is characterized by poikiloderma, growth deficiency, and a predisposition to cancer. Examination of RECQL4 subcellular localization in live cells demonstrated a nucleoplasmic pattern and, to a lesser degree, staining in nucleoli. Analysis of RECQL4-GFP deletion mutants revealed two nuclear localization regions in the N-terminal region of RECQL4 and a nucleolar localization signal at amino acids 376-386. RECQL4 localization did not change after treatment with the DNA-damaging agents bleomycin, etoposide, UV irradiation and gamma irradiation, in contrast to the Bloom and Werner syndrome helicases that relocate to distinct nuclear foci after damage. However, in a significant number of cells exposed to hydrogen peroxide or streptonigrin, RECQL4 ...
2010-01-01
Summary The RecQ family of DNA helicases including WRN (Werner syndrome protein) and BLM (Bloom syndrome protein) protects the genome against deleterious changes. Here we report the cocrystal structure of the RecQ C-terminal (RQC) domain of human WRN bound to a DNA duplex. In the complex, the RQC domain specifically interacted with a blunt end of the duplex and, surprisingly, unpaired a Watson-Crick base pair in the absence of an ATPase domain. The b wing, an extended hairpin motif that is characteristic of winged-helix motifs, was used as a "separating knife" to wedge between the first and second base pairs, whereas the recognition helix, a principal component of helix-turn-helix motifs that are usually embedded within DNA grooves, was unprecedentedly excluded from the interaction. Our re...
2010-01-01
Gonadotropin-regulated testicular RNA helicase (GRTH/DDX25), a multifunctional protein and a component of ribonucleoprotein complexes, is essential for the completion of spermatogenesis. We investigated the nuclear/cytoplasmic shuttling of GRTH in germ cells and its impact on the chromatoid body (CB)-a perinuclear organelle viewed as a storage/processing site of mRNAs. GRTH resides in the nucleus, cytoplasm and CB of round spermatids. Treatment of these cells with inhibitors of nuclear export or RNA synthesis caused nuclear retention of GRTH and its absence in the cytoplasm and CB. The nuclear levels of GRTH bound RNA messages were significantly enhanced and major reduction was observed in the cytoplasm. This indicated GRTH main transport function of mRNAs to the cytoplasm and CB. MVH, a g...
Progress on chromatin remodeling factor CHD
2006-01-01
Chromatin remodeling plays an important role in DNA repair, gene transcriptional regulation. Remodeling resultes in a series of important changes in chromatin structure, e.g. loosing the condensed chromatin and opening the structure of nucleoscome, which leads to increased accessibility of the transcription factors to nucleosomal DNA. CHD (chromodomain, helicase, DNA-binding) genes compose a subfamily of the known chromatin remodeling complexes. Up to now, six human CHD members have been cloned. They contain three main domains: Chromodomain which locates in the N terminus of the proteins, SW12/SNF2-related ATPase/helicase domain in the middle region, DNA-binding domain in the C terminus. The mutation or abnor-mal expression of CHD gene is thought to be related to some human diseases. (authors)
2007-01-01
Abstract: Human progeroid Werner syndrome provides the current best model for analysis of human aging, recapitulating many aspects of normal aging as a result of mutation of the WRN gene. This gene encodes a RecQ-type helicase with additional exonuclease activity. While biochemical studies in vitro have proven invaluable in determining substrate specificities of the WRN exonuclease and helicase, it has been difficult to dissociate the two key enzyme activities in vivo. We are developing Drosophila as a model system for analysis of WRN function; the suitability of Drosophila for extensive and sophisticated genetic manipulation permits us to investigate regulatory pathways and the impact of WRN loss at organismal, cellular, and molecular levels. BLASTP screening of the Drosophila genome with...
Ku complex interacts with and stimulates the Werner protein
2000-04-15
Full Text Available.Werner syndrome (WS) is the hallmark premature aging disorder in which affected humans appear older than their chronological age. The protein WRNp, defective in WS, has helicase function, DNA-dependent ATPase, and exonuclease activity. Although WRNp functions in nucleic acid metabolism, there is little or no information about the pathways or protein interactions in which it participates. Here we identify Ku70 and Ku86 as proteins that interact with WRNp. Although Ku proteins had no effect on ATPase or helicase activity, they strongly stimulated specific exonuclease activity. These results suggest that WRNp and the Ku complex participate in a common DNA metabolic pathway.
2008-01-01
Summary The premature human aging Werner syndrome (WS) is caused by mutation of the RecQ-family WRN helicase, which is unique in possessing also 3prime-5prime exonuclease activity. WS patients show significant genomic instability with elevated cancer incidence. WRN is implicated in restraining illegitimate recombination, especially during DNA replication. Here we identify a Drosophila ortholog of the WRN exonuclease encoded by the CG7670 locus. The predicted DmWRNexo protein shows conservation of structural motifs and key catalytic residues with human WRN exonuclease, but entirely lacks a helicase domain. Insertion of a piggyBac element into the 5prime UTR of CG7670 severely reduces gene expression. DmWRNexo mutant flies homozygous for this insertional allele of CG7670 are thus severely hy...
2010-01-01
Werner syndrome (WS) is a premature aging disorder caused by mutations in a RecQ-like DNA helicase. Mice lacking the helicase domain of the WRN homologue exhibit many phenotypic features of WS. Importantly, mutant WrnDhel/Dhel mice show abnormal increases in visceral fat deposition and fasting blood triglyceride levels followed by insulin resistance and high blood glucose levels. These mice also exhibit increased heart and liver tissue reactive oxygen species concomitantly with oxidative DNA damage, indicating a pro-oxidant status. We treated mice with either ascorbate or catechin hydrate for 9 months. Vitamin C supplementation reduced oxidative stress in liver and heart tissues and reversed hypertriglyceridemia, hyperglycemia, and insulin resistance and reduced fat weight in mutant WrnDhe...
Structure of West Nile virus NS3 protease: Ligand stabilization of the catalytic conformation
2009-01-01
Over the last decade, West Nile virus has spread rapidly via mosquito transmission from infected migratory birds to humans. One potential therapeutic approach to treating infection is to inhibit the virally encoded serine protease that is essential for viral replication. Here we report the crystal structure of the viral NS3 protease tethered to its essential NS2B cofactor and bound to a potent substrate-based tripeptide inhibitor, 2-naphthoyl-Lys-Lys-Arg-H (Ki = 41 nM), capped at the N-terminus by 2-naphthoyl and capped at the C-terminus by aldehyde. An important and unexpected feature of this structure is the presence of two conformations of the catalytic histidine suggesting a role for ligand stabilization of the catalytically competent His conformation. Analysis of other West Nile virus NS3 protease structures and related serine proteases supports this hypothesis, suggesting that the common catalytic mechanism involves an induced-fit mechanism. Crown copyright © 2008 Published by Elsevier Ltd. Publisher: Elsevier Contributor: Peter Wright Coverage: 2009-02-06T00:00:00Z
The MPH1 Gene of Saccharomyces cerevisiae Functions in Okazaki Fragment Processing*S⃞
2009-04-17
Saccharomyces cerevisiae MPH1 was first identified as a gene encoding a 3′ to 5′ DNA helicase, which when deleted leads to a mutator phenotype. In this study, we isolated...Full Text Available
2010-10-01
The production of aberrant RNA (aRNA) is the initial step in several RNAi pathways. How aRNA is produced and specifically recognized by RNA-dependent RNA polymerases (RdRPs) to generate double-stranded...Full Text Available
2010-01-01
WRN-1 is the Caenorhabditis elegans homolog of the human Werner syndrome protein, a RecQ helicase, mutations of which are associated with premature aging and increased genome instability....Full Text Available
Structural basis of the 3′-end recognition of a leading strand in stalled replication forks by PriA
2007-05-16
In eubacteria, PriA helicase detects the stalled DNA replication forks. This critical role of PriA is ascribed to its ability to bind to the 3′ end of a nascent leading DNA strand in the stalled...Full Text Available
2010-09-15
The chromatin remodelling factor chromodomain helicase DNA-binding protein 4 (CHD4) is a catalytic subunit of the NuRD transcriptional repressor complex. Here, we reveal novel functions for CHD4 in...Full Text Available
The minichromosome maintenance (MCM) helicase, composed of subunits Mcm2–7, is essential for the initiation and elongation phases of DNA replication. Even when DNA synthesis is blocked, MCM continues DNA unwinding to some extent for activation of the replication checkpoint and then stops. However, the mechanism of regulation of MCM-helicase activity remains unknown. Here, we show that truncation of the Mcm4 C-terminal domain (CTD) in fission yeast results in hypersensitivity to replication block caused by dNTP depletion. The truncation mcm4-c84 does not affect the activation of the replication checkpoint pathway but delays its attenuation during recovery from replication block. Two dimensional gel electrophoresis showed that mcm4-c84 delays the disappearance of replication intermediates, indicating that the Mcm4 CTD is required for efficient recovery of stalled replication forks. Remarkably, chromatin immunoprecipitation revealed that mcm4-c84 brings about an increase rather than a decrease in the association of the single-stranded DNA-binding protein RPA to stalled forks, and MCM and the accessory complex GINS are unaffected. These results suggest that the Mcm4 CTD is required to suspend MCM-helicase activity after the formation of single-stranded DNA sufficient for checkpoint activation.
Human RECQL5β stimulates flap endonuclease 1
2010-05-01
Human RECQL5 is a member of the RecQ helicase family which is implicated in genome maintenance. Five human members of the family have been identified; three of them, BLM, WRN and RECQL4 are associated...Full Text Available
The protein DDX3X is a DEAD-box RNA helicase that is essential for the hepatitis C virus (HCV) life cycle. The HCV core protein has been shown to bind to DDX3X both in vitro and in...Full Text Available
2009-12-01
Antigenic variation plays a vital role in the pathogenesis of many infectious bacteria and protozoa including Borrelia burgdorferi, the causative agent of Lyme disease. VlsE, a 35 kDa...Full Text Available
2009-09-01
A minor core protein, VP6, of bluetongue virus (BTV) possesses nucleoside triphosphatase, RNA binding, and helicase activities. Although the enzymatic functions of VP6 have been documented in vitro...Full Text Available
A novel RNA helicase gene tightly linked to the Triplo-lethal locus of Drosophila.
1990-09-25
The Triplo-lethal (Tpl) locus of Drosophila is the only known locus which is lethal when present in three copies rather than the normal two. After recovering a hybrid-dysgenesis-induced mutation of...Full Text Available
2010-01-01
The viral genome-linked protein, VPg, of potyviruses is a multifunctional protein involved in viral genome translation and replication. Previous studies have shown that both eukaryotic translation initiation...Full Text Available
Novel macrocyclic HCV NS3 protease inhibitors derived from a-amino cyclic boronates
2010-01-01
A novel series of P2-P4 macrocyclic HCV NS3/4A protease inhibitors with a-amino cyclic boronates as warheads at the P1 site was designed and synthesized. When compared to their linear analogs, these macrocyclic inhibitors exhibited a remarkable improvement in cell-based replicon activities, with compounds 9a and 9e reaching sub-micromolar potency in replicon assay. The SAR around a-amino cyclic boronates clearly established the influence of ring size, chirality and of the substitution pattern. Furthermore, X-ray structure of the co-crystal of inhibitor 9a and NS3 protease revealed that Ser-139 in the enzyme active site traps boron in the warhead region of 9a, thus establishing its mode of action.
Identification of palmatine as an inhibitor of West Nile virus
2010-01-01
In this study, we investigated the specific inhibition of West Nile virus (WNV) NS2B-NS3 protease and viral propagation by palmatine, a chemical compound from Coptis chinensis Franch. It was demonstrated that palmatine could inhibit WNV NS2B-NS3 protease activity in an uncompetitive manner, with a 50% inhibitory concentration (IC50) of 96?M. Palmatine suppressed WNV without detectable cytotoxicity (a 50% effective concentration [EC50] of 3.6?M and a 50% cytotoxicity concentration [CC50] of 1,031?M). Furthermore, palmatine could also suppress dengue virus and yellow fever virus in a dose-dependent manner. This compound could potentially be developed for the treatment of flavivirus infections.
2009-12-01
The flavivirus genome comprises a single strand of positive-sense RNA, which is translated into a polyprotein and cleaved by a combination of viral and host proteases to yield functional proteins. One...Full Text Available
2010 JPC Abstract: Ares I First Stage Propulsion System Status
Ares I First Stage Propulsion System Status" href="?R=952341&id=1&as=false&or=false&qs=Ntt%3Dsystems%2Btechnical%2Bprogress%26Ntk%3Dall%26Ntx%3Dmode%2Bmatchall%26Ns%3DHarvestDate%257c1%26N%3D0"
2010 JPC Abstract: Ares I First Stage Propulsion System Status
Ares I First Stage Propulsion System Status" href="?R=370547&id=1&as=false&or=false&qs=Ntt%3Dgrain%2Bpropellant%2Bgeometry%26Ntk%3Dall%26Ntx%3Dmode%2Bmatchall%26Ns%3DHarvestDate%257c1%26N%3D0"
2000-01-01
DEAD-box RNA helicases, by unwinding duplex RNA in bacteria and eukaryotes, are involved in essential cellular processes, including translation initiation and ribosome biogenesis, and have recently been implicated in enabling bacteria to survive cold-shock and grow at low temperature. Despite these critical physiological roles, they have not been characterized in archaea. Due to their presumed importance in removing cold-stabilised secondary structures in mRNA, we have characterised a putative DEAD-box RNA helicase gene (deaD) from the Antarctic methanogen, Methanococcoides burtonii. The encoded protein, DeaD is predicted to contain a core element involved in ATP hydrolysis and RNA-binding, and an unusual C-terminal domain that contains seven perfect, trideca-peptide, direct repeats that may be involved in RNA binding. Alignment and phylogenetic analyses were performed on the core regions of the M. burtonii and other DEAD-box RNA helicases. These revealed a loose but consistent clustering of archaeal and bacterial sequences and enabled the generation of a prokaryotic-specific consensus sequence. The consensus highlights the importance of residues other than the eight motifs that are often associated with DEAD-box RNA helicases, as well as de-emphasising the importance of the "A" residue within the "DEAD" motif. Cells growing at 4oC contained abundant levels of deaD mRNA, however no mRNA was detected in cells growing at 23oC (the optimal temperature for growth). The transcription initiation site was mapped downstream from an archaeal box-A element (TATA box), which preceded a long (113 nucleotides) 5'-untranslated region (5'-UTR). Within the 5'-UTR was an 11 bp sequence that closely matches (nine out of 11) cold-box elements that are present in the 5'-UTRs of cold-shock induced genes from bacteria. To determine if the archaeal 5'-UTR performs an analagous function to the bacterial 5'-UTRs, the archaeal deaD 5'-UTR was transcribed in E. coli under the control of the cspA promoter and transcriptional terminator. It has previously been reported that overexpression of the cspA 5'-UTR leads to an extended cold-shock response due to the 5'-UTR titrating cellular levels of a cold-shock repressor protein(s). In our hands, the cold-shock protein profiles resulting from overexpression of Escherichia coli cspA and M. burtonii deaD 5'-UTRs were similar, however they did not differ from those for the overexpression of a control plasmid lacking a 5'-UTR. In association with other recent data from E. coli, our results indicate that the role of the 5'-UTR in gene regulation is presently unclear. Irrespective of the mechanisms, it is striking that highly similar 5'-UTRs with cold-box elements are present in cold induced genes from E. coli, Anabaena and M. burtonii. This is the first study examining low temperature regulation in archaea and provides initial evidence that gene expression from a cold adapted archaeon involves a bacterial-like transcriptional regulatory mechanism. In addition, it provides the foundation for further studies into the function and regulation of DEAD-box RNA helicases in archaea, and in particular, their roles in low temperature adaptation. Relation: Vol:297; pp.553-567; Journal:Journal of Molecular Biology Other identifier: ISSN:0022-2836; unsworks:4208 Language: EN Rights: http://unsworks.unsw.edu.au/copyright
The two eIF4A helicases in Trypanosoma brucei are functionally distinct
2006-01-01
Protozoan parasites belonging to the family Trypanosomatidae are characterized by an unusual pathway for the production of mRNAs via polycistronic transcription and trans-splicing of a 5prime capped mini-exon which is linked to the 3prime cleavage and polyadenylation of the upstream transcript. However, little is known of the mechanism of protein synthesis in these organisms, despite their importance as agents of a number of human diseases. Here we have investigated the role of two Trypanosoma brucei homologues of the translation initiation factor eIF4A (in the light of subsequent experiments these were named as TbEIF4AI and TbEIF4AIII). eIF4A, a DEAD-box RNA helicase, is a subunit of the translation initiation complex eIF4F which binds to the cap structure of eukaryotic mRNA and recruits ...
Srs2: The “Odd-Job Man” in DNA repair
AbstractHomologous recombination plays a key role in the maintenance of genome integrity, especially during DNA replication and the repair of double-stranded DNA breaks (DSBs). Just a single un-repaired break can lead to aneuploidy, genetic aberrations or cell death. DSBs are caused by a vast number of both endogenous and exogenous agents including genotoxic chemicals or ionizing radiation, as well as through replication of a damaged template DNA or the replication fork collapse. It is essential for cell survival to recognise and process DSBs as well as other toxic intermediates and launch most appropriate repair mechanism. Many helicases have been implicated to play role in these processes, however their detail roles, specificities and co-operativity in the complex protein-protein interaction networks remain unclear. In this review we summarize the current knowledge about Saccharomyces cerevisiae helicase Srs2 and its effect on multiple DNA metabolic processes that generally affect genome stability. It would appear that Srs2 functions as an “Odd-Job Man” in these processes to make sure that the jobs proceed when and where they are needed.
2010-06-01
Full Text Available.The AddAB helicase and nuclease complex is used for repairing double-strand DNA breaks in the many bacteria that do not possess RecBCD. Here, we show that AddAB, from the Gram-negative opportunistic pathogen Bacteroides fragilis, can rescue the ultraviolet sensitivity of an Escherichia coli recBCD mutant and that addAB is required for survival of B. fragilis following DNA damage. Using single-molecule observations we demonstrate that AddAB can translocate along DNA at up to 250 bp per second and can unwind an average of 14 000 bp, with some complexes capable of unwinding 40 000 bp. These results demonstrate the importance of processivity for facilitating encounters with recognition sequences that modify enzyme function during homologous recombination.
Rm62, a DEAD-box RNA helicase, complexes with DSP1 in Drosophila embryos
2010-01-01
Two main classes of proteins, Polycomb group (PcG) and Trithorax group (TrxG), play a key role in the regulation of homeotic genes. These proteins act in multimeric complexes to remodel chromatin. A third class of proteins named Enhancers of Trithorax and Polycomb (ETP) modulates the activity of TrxG and PcG, but their role remains largely unknown. We previously identified an HMGB-like protein, DSP1 (Dorsal Switch Protein 1), which was classified as an ETP. Preliminary studies have revealed that DSP1 is involved in multimeric complexes. Here we identify a DEAD-box RNA helicase, Rm62, as partner of DSP1 in a 250-kDa complex. Coimmunoprecipitation assays performed on embryo extracts indicate that DSP1 and Rm62 are associated in 3- to 12-h embryos. Furthermore, DSP1 and Rm62 colocalize on pol...
Release of SF3 from the intron branchpoint activates the first step of pre-mRNA splicing
2010-03-01
Full Text Available.Eukaryotic pre-mRNA splicing is a complex process requiring the precise timing and action of >100 trans-acting factors. It has been known for some time that the two steps of splicing chemistry require three DEAH-box RNA helicase-like proteins; however, their mechanism of action at these steps has remained elusive. Spliceosomes arrested in vivo at the three helicase checkpoints were purified, and first step-arrested spliceosomes were functionally characterized. We show that the first step of splicing requires a novel ATP-independent conformational change. Prp2p then catalyzes an ATP-dependent rearrangement displacing the SF3a and SF3b complexes from the branchpoint within the spliceosome. We propose a model in which SF3 prevents premature nucleophilic attack of the chemically reactive hydroxyl of the branchpoint adenosine prior to the first transesterification. When the spliceosome attains the proper conformation and upon the function of Prp2p, SF3 is displaced from the branchpoint allowing first step chemistry to occur.
2008-01-01
SummarySaccharomyces cerevisiae RecQ helicase, Sgs1, and XPF family endonuclease, Mus81-Mms4, are implicated in processing joint molecule (JM) recombination intermediates. We show that cells lacking either enzyme frequently experience chromosome segregation problems during meiosis and that when both enzymes are absent attempted segregation fails catastrophically. In all cases, segregation appears to be impeded by unresolved JMs. Analysis of the DNA events of recombination indicates that Sgs1 limits aberrant JM structures that result from secondary strand-invasion events and often require Mus81-Mms4 for their normal resolution. Aberrant JMs contain high levels of single Holliday junctions and include intersister JMs, multichromatid JMs comprising three and four chromatids, and newly identif...
2008-08-08
Full Text Available.SUMMARYBudding yeast lacking the Sgs1 helicase and the Mus81/Mms4 endonuclease are inviable, and indirect studies implicate homologous recombination gone awry as the cause of death. We show that mutants lacking both enzymes have profound defects in meiotic recombination intermediate metabolism and crossover (CO) formation. Recombination intermediates (joint molecules; JMs) accumulate in these cells, many with structures that are infrequent in wild type cells. These JMs persist, preventing nuclear division. Using an inducible expression system, we restored Mus81 or Sgs1 to sgs1 mus81 cells at a time when JMs are forming. Mus81 expression did not prevent JM formation, but restored JM resolution, CO formation, and nuclear division. In contrast, Sgs1 expression reduced the extent of JM accumulation. These results indicate that Sgs1 and Mus81/Mms4 collaborate to direct meiotic recombination towards interhomolog interactions that promote proper chromosome segregation, and also indicate that Mus81/Mms4 promotes JM resolution in vivo.
SUMMARYBudding yeast lacking the Sgs1 helicase and the Mus81/Mms4 endonuclease are inviable, and indirect studies implicate homologous recombination gone awry as the cause of death. We show that mutants lacking both enzymes have profound defects in meiotic recombination intermediate metabolism and crossover (CO) formation. Recombination intermediates (joint molecules; JMs) accumulate in these cells, many with structures that are infrequent in wild type cells. These JMs persist, preventing nuclear division. Using an inducible expression system, we restored Mus81 or Sgs1 to sgs1 mus81 cells at a time when JMs are forming. Mus81 expression did not prevent JM formation, but restored JM resolution, CO formation, and nuclear division. In contrast, Sgs1 expression reduced the extent of JM accumulation. These results indicate that Sgs1 and Mus81/Mms4 collaborate to direct meiotic recombination towards interhomolog interactions that promote proper chromosome segregation, and also indicate that Mus81/Mms4 promotes JM resolution in vivo.
Mechanism of DNA Translocation in a Replicative Hexameric Helicase
2006-01-01
The E1 protein of papillomavirus is a hexameric ring helicase belonging to the AAA + family. The mechanism that couples the ATP cycle to DNA translocation has been unclear. Here we present the crystal structure of the E1 hexamer with single-stranded DNA discretely bound within the hexamer channel and nucleotides at the subunit interfaces. This structure demonstrates that only one strand of DNA passes through the hexamer channel and that the DNA-binding hairpins of each subunit form a spiral 'staircase' that sequentially tracks the oligonucleotide backbone. Consecutively grouped ATP, ADP and apo configurations correlate with the height of the hairpin, suggesting a straightforward DNA translocation mechanism. Each subunit sequentially progresses through ATP, ADP and apo states while the associated DNA-binding hairpin travels from the top staircase position to the bottom, escorting one nucleotide of single-stranded DNA through the channel. These events permute sequentially around the ring from one subunit to the next.
2008-01-01
The first step of homology-dependent DNA double-strand break (DSB) repair is the 5′ strand-specific processing of DNA ends to generate 3′ single-strand tails. Despite extensive effort, the nuclease(s) that is directly responsible for the resection of 5′ strands in eukaryotic cells remains elusive. Using nucleoplasmic extracts (NPE) derived from the eggs of Xenopus laevis as the model system, we have found that DNA processing consists of at least two steps: an ATP-dependent unwinding of ends and an ATP-independent 5′→3′ degradation of single-strand tails. The unwinding step is catalyzed by DNA helicases, the major one of which is the Xenopus Werner syndrome protein (xWRN), a member of the RecQ helicase family. In this study, we report the puri...
2008-01-01
Rothmund-Thomson syndrome (RTS), a rare recessive autosomal disorder, presents genome instability and clinical heterogeneity with growth deficiency, skin and bone defects, premature aging symptoms and cancer susceptibility. A subset of RTS patients presents mutations of the RECQL4 gene, member of the RecQ family of DNA helicases, including the RECQL2 (BLM) and RECQL3 (WRN) genes, defective in the cancer prone Bloom and Werner syndromes, respectively. Analysis of the RECQL4 gene in six clinically diagnosed RTS patients shows five patients, including two siblings, with eight mutations mainly located in the helicase domain, three patients presenting two mutations. The alterations include four missense mutations, one nonsense mutation and the same frameshift deletion, g.2881delG in exon 9 found in three patients. Seven RECQL4 polymorphisms, two being new, have also ...
2009-01-01
The E1 helicase from BPV and HPV16 interacts with Ubc9 to facilitate viral genome replication. We report that HPV11 E1 also interacts with Ubc9 in vitro and in the yeast two-hybrid system. Residues in E1 involved in oligomerization (353-435) were sufficient for binding to Ubc9 in vitro, but the origin-binding and ATPase domains were additionally required in yeast. Nuclear accumulation of BPV E1 was shown previously to depend on its interaction with Ubc9 and sumoylation on lysine 514. In contrast, HPV11 and HPV16 E1 mutants defective for Ubc9 binding remained nuclear even when the SUMO pathway was inhibited. Furthermore, we found that K514 in BPV E1 and the analogous K559 in HPV11 E1 are not essential for nuclear accumulation of E1. These results suggest that the interaction of E1 with Ubc9 is not essential for its nuclear accumulation but, rather, depends on its oligomerization and ...
Cell Cycle Regulation of DNA Replication
Eukaryotic DNA replication is regulated to ensure all chromosomes replicate once and only once per cell cycle. Replication begins at many origins scattered along each chromosome. Except for budding yeast, origins are not defined DNA sequences and probably are inherited by epigenetic mechanisms. Initiation at origins occurs throughout the S phase according to a temporal program that is important in regulating gene expression during development. Most replication proteins are conserved in evolution in eukaryotes and archaea, but not in bacteria. However, the mechanism of initiation is conserved and consists of origin recognition, assembly of pre-replication (pre-RC) initiative complexes, helicase activation, and replisome loading. Cell cycle regulation by protein phosphorylation ensures that pre-RC assembly can only occur in G1 phase, whereas helicase activation and loading can only occur in S phase. Checkpoint regulation maintains high fidelity by stabilizing replication forks and preventing cell cycle progression during replication stress or damage.
2009-01-01
RecQ helicases play a central role in maintaining genome stability and may interact with some important cancer-related proteins such as BRCA1. Mutations of the human RecQ helicase genes WRN and BLM lead to rare autosomal recessive disorders, Werner and Bloom syndromes, which are associated with premature aging and cancer predisposition, including breast cancer. In this casecontrol study of 1,004 breast cancer cases and 1,008 controls, we tested the hypothesis that non-conservative amino acid exchanges in WRN (leu1074Phe), BLM (Met298Thr) and BRCA1 (Pro871Leu) are independently or jointly associated with the risk of breast cancer in Chinese women. We found that the variant genotype of WRN Leu1074Phe was associated with a 1.36-fold significantly increased risk of breast cancer (OR=1.3...
Pathways of mammalian replication fork restart
2010-01-01
Single-molecule analyses of DNA replication have greatly advanced our understanding of mammalian replication restart. Several proteins that are not part of the core replication machinery promote the efficient restart of replication forks that have been stalled by replication inhibitors, suggesting that bona fide fork restart pathways exist in mammalian cells. Different models of replication fork restart can be envisaged, based on the involvement of DNA helicases, nucleases, homologous recombination factors and the importance of DNA double-strand break formation.
Interfacial instability and DNA fork reversal by repair proteins
2010-01-01
A repair protein like RecG moves the stalled replication fork in the direction from the zipped to the unzipped state of DNA. It is proposed here that a softening of the zipped-unzipped interface at the fork results in the front propagating towards the unzipped side. In this scenario, an ordinary helicase destabilizes the zipped state locally near the interface and the fork propagates towards the zipped side. The softening of the interface can be produced by the aromatic interaction, predicted from the crystal structure, between RecG and the nascent broken base pairs at the Y-fork. A numerical analysis of the model also reveals the possibility of a stop and go type motion
The role of AtMUS81 in DNA repair and its genetic interaction with the helicase AtRecQ4A.
The endonuclease MUS81 has been shown in a variety of organisms to be involved in DNA repair in mitotic and meiotic cells. Homologues of the MUS81 gene exist in the genomes of all eukaryotes, pointing to a conserved role of the protein. However, the biological role of MUS81 varies between different eukaryotes. For example, while loss of the gene results in strongly impaired fertility in Saccharomyces cerevisiae and nearly complete sterility in Schizosaccharomyces pombe, it is not essential for meiosis in mammals. We identified a functional homologue (AtMUS81/At4g30870) in the genome of Arabidopsis thaliana and isolated a full-length cDNA of this gene. Analysing two independent T-DNA insertion lines of AtMUS81, we found that they are sensitive to the mutagens MMS and MMC. Both mutants have a deficiency in homologous recombination in somatic cells but only after induction by genotoxic stress. In contrast to yeast, no meiotic defect of AtMUS81 mutants was detectable and the mutants are viable. Crosses with a hyperrecombinogenic mutant of the AtRecQ4A helicase resulted in synthetic lethality in the double mutant. Thus, the nuclease AtMUS81 and the helicase AtRecQ4A seem to be involved in two alternative pathways of resolution of replicative DNA structures in somatic cells.
The involvement of host proteins in the replication and transcription of viral RNA is a poorly understood area for many RNA viruses. For coronaviruses, it was long speculated that replication of the giant RNA genome and transcription of multiple subgenomic mRNA species by a unique discontinuous transcription mechanism may require host cofactors. To search for such cellular proteins, yeast two-hybrid screening was carried out by using the nonstructural protein 14 (nsp14) from the coronavirus infectious bronchitis virus (IBV) as a bait protein, leading to the identification of DDX1, a cellular RNA helicase in the DExD/H helicase family, as a potential interacting partner. This interaction was subsequently confirmed by coimmunoprecipitation assays with cells coexpressing the two proteins and with IBV-infected cells. Furthermore, the endogenous DDX1 protein was found to be relocated from the nucleus to the cytoplasm in IBV-infected cells. In addition to its interaction with IBV nsp14, DDX1 could also interact with the nsp14 protein from severe acute respiratory syndrome coronavirus (SARS-CoV), suggesting that interaction with DDX1 may be a general feature of coronavirus nsp14. The interacting domains were mapped to the C-terminal region of DDX1 containing motifs V and VI and to the N-terminal portion of nsp14. Manipulation of DDX1 expression, either by small interfering RNA-induced knockdown or by overexpression of a mutant DDX1 protein, confirmed that this interaction may enhance IBV replication. This study reveals that DDX1 contributes to efficient coronavirus replication in cell culture.
1995-03-20
The BAT1 gene has previously been identified about 30 kb upstream from the tumor necrosis factor (TNF) locus and close to a NF{sub kb}-related gene of the nuclear factor family in the major histocompatibility complex (MHC) of human, mouse, and pig. We now show that the BAT1 translation product is the homolog of the rat p47 nuclear protein, the WM6 Drosophila gene product, and probably also Ce08102 of Caenorhabditis elegans, all members of the DEAD protein family of ATP-dependent RNA helicases. This family has more than 40 members, including the eukaryotic translation initiation factor-4A (eIF-4A), the human nuclear protein p68, and the Drosophila oocyte polar granule component vasa. BAT1 spans about 10 kb, is split into 10 exons of varying length, and encodes a protein of 428 amino acids ({approximately}48 kDa). Human and pig BAT1 cDNAs display 95.6% identity in the coding region and 80% identity in the 5{prime} and 3{prime} noncoding regions. Several repeat sequences of different types were identified in introns of the porcine BAT1 gene. Three different mRNAs, 4.1,1.7, and 0.9 kb, respectively, were detected in all tissues analyzed upon hybridization with porcine BAT1 cDNA. Transfection and expression of human BAT1 cDNA after tagging with a heterologous antibody recognition epitope revealed a nuclear localization of the hybrid protein. An MspI RFLP was detected in an SLA class I typed family, confirming the localization of the BAT1 gene in the porcine MHC. BAT1 thus encodes a putative nuclear ATP-dependent RNA helicase and is likely to have an indispensable function. 35 refs., 6 figs., 1 tab.
Nuclear VCP/p97-like protein 2 (NVL2) is a member of the chaperone-like AAA-ATPase family with two conserved ATP-binding modules. Our previous studies have shown that NVL2 is localized to the nucleolus by interacting with ribosomal protein L5 and may participate in ribosome synthesis, a process involving various non-ribosomal factors including chaperones and RNA helicases. Here, we show that NVL2 is associated with pre-ribosomal particles in the nucleus. Moreover, we used yeast two-hybrid and co-immunoprecipitation assays to identify an NVL2-interacting protein that could yield insights into NVL2 function in ribosome biogenesis. We found that NVL2 interacts with DOB1, a DExD/H-box RNA helicase, whose yeast homologue functions in a late stage of the 60S subunit synthesis. DOB1 can interact with a second ATP-binding module mutant of NVL2, which shows a dominant negative effect on ribosome synthesis. In contrast, it cannot interact with a first ATP-binding module mutant, which does not show the dominant negative effect. When the dominant negative mutant of NVL2 was overexpressed in cells, DOB1 appeared to remain associated with nuclear pre-ribosomal particles. Such accumulation was not observed upon overexpression of wild-type NVL2 or a nondominant-negative mutant. Taken together, our results suggest that NVL2 might regulate the association/dissociation reaction of DOB1 with pre-ribosomal particles by acting as a molecular chaperone.
2007-07-27
Full Text Available.SummaryBloom’s helicase (BLM) is thought to prevent crossing-over during DNA double-strand-break repair (DSBR) by disassembling double Holliday Junctions (dHJs) or by preventing their formation. We show that Saccharomyces cerevisiae BLM ortholog, Sgs1, prevents aberrant crossing-over during meiosis by suppressing formation of joint molecules (JMs) comprising three and four interconnected duplexes. Sgs1 and pro-crossover factors, Msh5 and Mlh3, are antagonistic, since Sgs1 prevents dHJ formation in msh5 cells and sgs1 mutation alleviates crossover defects of both msh5 and mlh3 mutants. We propose that differential activity of Sgs1 and pro-crossover factors at the two DSB-ends effects productive formation of dHJs and crossovers, and prevents multi-chromatid JMs and counterproductive crossing-over. Strand-invasion of different templates by both DSB-ends may be a common feature of DSBR that increases repair efficiency but also the likelihood of associated crossing-over. Thus, by disrupting aberrant JMs, BLM-related helicases maximize repair efficiency while minimizing the risk of deleterious crossing-over.
2010-04-01
Full Text Available.Plasmid encoded replication initiation (Rep) proteins recruit host helicases to plasmid replication origins. Previously, we showed that RepD recruits directionally the PcrA helicase to the pC221 oriD, remains associated with it, and increases its processivity during plasmid unwinding. Here we show that RepD forms a complex extending upstream and downstream of the core oriD. Binding of RepD causes remodelling of a region upstream from the core oriD forming a ‘landing pad’ for the PcrA. PcrA is recruited by this extended RepD–DNA complex via an interaction with RepD at this upstream site. PcrA appears to have weak affinity for this region even in the absence of RepD. Upon binding of ADPNP (non-hydrolysable analogue of ATP), by PcrA, a conformational rearrangement of the RepD–PcrA–ATP initiation complex confines it strictly within the boundaries of the core oriD. We conclude that RepD-mediated recruitment of PcrA at oriD is a three step process. First, an extended RepD–oriD complex includes a region upstream from the core oriD; second, the PcrA is recruited to this upstream region and thirdly upon ATP-binding PcrA relocates within the core oriD.
Functional significance of the Rad51-Srs2 complex in Rad51 presynaptic filament disruption
2009-11-01
Full Text Available.The SRS2 (Suppressor of RAD Six screen mutant 2) gene encodes an ATP-dependent DNA helicase that regulates homologous recombination in Saccharomyces cerevisiae. Mutations in SRS2 result in a hyper-recombination phenotype, sensitivity to DNA damaging agents and synthetic lethality with mutations that affect DNA metabolism. Several of these phenotypes can be suppressed by inactivating genes of the RAD52 epistasis group that promote homologous recombination, implicating inappropriate recombination as the underlying cause of the mutant phenotype. Consistent with the genetic data, purified Srs2 strongly inhibits Rad51-mediated recombination reactions by disrupting the Rad51-ssDNA presynaptic filament. Srs2 interacts with Rad51 in the yeast two-hybrid assay and also in vitro. To investigate the functional relevance of the Srs2-Rad51 complex, we have generated srs2 truncation mutants that retain full ATPase and helicase activities, but differ in their ability to interact with Rad51. Importantly, the srs2 mutant proteins attenuated for Rad51 interaction are much less capable of Rad51 presynaptic filament disruption. An internal deletion in Srs2 likewise diminishes Rad51 interaction and anti-recombinase activity. We also present evidence that deleting the Srs2 C-terminus engenders a hyper-recombination phenotype. These results highlight the importance of Rad51 interaction in the anti-recombinase function of Srs2, and provide evidence that this Srs2 function can be uncoupled from its helicase activity.
A RecB-like helicase in Helicobacter pylori is important for DNA repair and host colonization.
The human gastric pathogen Helicobacter pylori encounters frequent oxidative and acid stress in its specific niche, and this causes bacterial DNA damage. H. pylori exhibits a very high degree of DNA recombination, which is required for repairing both DNA double-stranded (ds) breaks and blocked replication forks. Nevertheless, few genes encoding components of DNA recombinational repair processes have been identified in H. pylori. An H. pylori mutant defective in a putative helicase gene (HP1553) was constructed and characterized herein. The HP1553 mutant strain was much more sensitive to mitomycin C than the WT strain, indicating that HP1553 is required for repair of DNA ds breaks. Disruption of HP1553 resulted in a significant decrease in the DNA recombination frequency, suggesting that HP1553 is involved in DNA recombination processes, probably functioning as a RecB-like helicase. HP1553 was shown to be important for H. pylori protection against oxidative stress-induced DNA damage, as the exposure of the HP1553 mutant cells to air for 6 h caused significant fragmentation of genomic DNA and led to cell death. In a mouse infection model, the HP1553 mutant strain displayed a greatly reduced ability to colonize the host stomachs, indicating that HP1553 plays a significant role in H. pylori survival/colonization in the host.
2009-01-01
The seventh year of the Zyvox® Annual Appraisal of Potency and Spectrum Program (2008) continues to monitor the in vitro activities of linezolid and comparator agents tested against Gram-positive pathogens in Latin America, Europe, Canada, and the Asia-Pacific region. Linezolid is an oxazolidinone approved for the treatment of vancomycin-resistant Enterococcus faecium infections, complicated skin and soft tissue infections, and nosocomial pneumonia caused by various Gram-positive species including methicillin-resistant Staphylococcus aureus (MRSA). Surveillance isolates were submitted from 64 medical centers (24 countries) for a total of 6121 strains. Each country was requested to send 200 consecutive isolates in 6 targeted pathogen categories to a central processing laboratory, except the United Kingdom, Japan, and China where more strains were processed (400, 400, and 800, respectively). Reference broth microdilution susceptibility testing methods were used to test the following organism groups: S. aureus (3240), coagulase-negative staphylococci (CoNS) (748), enterococci (864), Streptococcus pneumoniae (655), viridans group (297), and β-hemolytic streptococci (317). Eight linezolid-resistant (LZD-R) isolates were detected in 7 countries (Italy [2], France, China, Brazil, Sweden, Germany, and United Kingdom) among the enterococci (Enterococcus faecalis [3] and E. faecium [2]) and CoNS (3 Staphylococcus epidermidis). Five LZD-R isolates contained 23S rRNA mutations (G2576T or G2447T), and 2 strains had undeterminable resistance mechanisms. One CoNS (Italy) contained the mobile cfr gene. Vancomycin-resistant enterococci rates ranged from 0.0% in several countries to 59.4% in Taiwan. All streptococci were linezolid susceptible (MIC90, 1 μg/mL). In conclusion, the activity of linezolid remained uniform and stable across the sampled geographic regions studied when compared to the 2006 to 2007 results. Documented LZD-R remains rare (only 0.13% overall but highest for CoNS [0.41%] and enterococci [0.69%]) among the 24 countries sampled for the 6 different pathogen groups. Rates of clindamycin resistance and the frequency of MRSA varied by geographic region and between nations; therefore, like oxazolidinones, it requires continued surveillance for changing resistance patterns.Ronald N. Jones, James E. Ross, Jan M. Bell, Uchino Utsuki, Ikeda Fumiaki, Intetsu Kobayashi and John D. Turnidgehttp://www.elsevier.com/wps/find/journaldescription.cws_home/505759/description#description Publisher: Elsevier Contributor: School of Paediatrics and Reproductive Health : Paediatrics Other identifier: Diagnostic Microbiology and Infectious Disease, 2009; 65(4):404-413; 0732-8893; 0020093941; 10.1016/j.diagmicrobio.2009.10.001; 000272112200008 Language: en
2007-01-01
Pathogenic members of the flavivirus family, including West Nile Virus (WNV) and Dengue Virus (DV), are growing global threats for which there are no specific treatments. The two-component flaviviral enzyme NS2B-NS3 cleaves the viral polyprotein precursor within the host cell, a process that is required for viral replication. Here, we report the crystal structure of WNV NS2B-NS3pro both in a substrate-free form and in complex with the trypsin inhibitor aprotinin/BPTI. We show that aprotinin binds in a substrate-mimetic fashion in which the productive conformation of the protease is fully formed, providing evidence for an 'induced fit' mechanism of catalysis and allowing us to rationalize the distinct substrate specificities of WNV and DV proteases. We also show that the NS2B cofactor of WNV can adopt two very distinct conformations and that this is likely to be a general feature of flaviviral proteases, providing further opportunities for regulation. Finally, by comparing the flaviviral proteases with the more distantly related Hepatitis C virus, we provide insights into the evolution of the Flaviviridae fold. Our work should expedite the design of protease inhibitors to treat a range of flaviviral infections.
Nuclear Structure and Spectroscopic Analysis of Some Even-Even W, Os, pt and Hg Isotopes
2008-01-01
In this work, the spectral anomalies of some even-even W, Os, Pt and Hg isotopes were studied. The application of several nuclear models such as variable moment of inertia (VMI) model, nuclear softness (NS3) model, dynamic version model and simple model of backbending, revealed the following notations: the predictions of the VMI-model and NS3-model gave good results for 1190Hg, 192Hg and 194Hg isotopes ...
2010 JPC Abstract: Ares I First Stage Propulsion System Status
Ares I First Stage Propulsion System Status" href="?R=11478&id=2&as=false&or=false&qs=No%3D30%26Ntt%3Dsystem%2Bsystems%26Ntk%3Dall%26Ntx%3Dmode%2Bmatchall%26Ns%3DHarvestDate%257c1%26N%3D0"
Single-Stranded DNA Transposition Is Coupled to Host Replication
2010-01-01
Summary DNA transposition has contributed significantly to evolution of eukaryotes and prokaryotes. Insertion sequences (ISs) are the simplest prokaryotic transposons and are divided into families on the basis of their organization and transposition mechanism. Here, we describe a link between transposition of IS608 and ISDra2, both members of the IS200/IS605 family, which uses obligatory single-stranded DNA intermediates, and the host replication fork. Replication direction through the IS plays a crucial role in excision: activity is maximal when the "top" IS strand is located on the lagging-strand template. Excision is stimulated upon transient inactivation of replicative helicase function or inhibition of Okazaki fragment synthesis. IS608 insertions also exhibit an orientation preference...
2010-01-01
Salmonella enterica is an important pathogen that causes a variety of infectious diseases in animals and humans. Live attenuated vaccines generally confer better protection than killed or subunit vaccines; however, the former are limited by their inherent toxicity. We evaluated the potential of a novel candidate Salmonella vaccine strain that lacks the ruvB gene. The ruvB gene encodes a Holliday junction helicase that is required to resolve junctions that arise during the repair of non-arresting lesions after DNA replication. The deletion of this gene in Salmonella significantly impaired cell survival and proliferation within epithelial cells and macrophages. The defective virulence in ruvB mutant may be partially due to decreased expression of ssaG, a Salmonella pathogenicity island-2 gen...
RIG-I-like receptors: Sensing and responding to RNA virus infection
2009-01-01
Viral and microbial pathogens contain specific motifs or pathogen-associated molecular patterns (PAMPs) that are recognized by cell surface- and endosome-associated Toll-like receptors (TLRs). RNA virus infection is also detected through TLR-independent mechanisms. Early viral replicative intermediates are detected by two recently characterized cystolic viral RNA receptors-RIG-I and MDA-5. Both are DExDH/box RNA helicases, and RIG-I specifically recognizes 5prime-triphosphate containing viral RNA and transmits signals that induce type I interferon-mediated host immunity against virus infection. In this review, we will focus on RIG-I-like receptor (RLR) signal transduction and the regulatory mechanisms - ubiquitination, deubiquitination, ISGylation - underlying this important host response.
Epigenetic silencers are enriched in dormant desert frog muscle
2008-01-01
Green-striped burrowing frogs, Cyclorana alboguttata, survive droughts by entering a metabolic depression called aestivation, characterised by a reduction in resting oxygen consumption by 80%. Aestivation in C. alboguttata is manifest by transcriptional silencing of skeletal muscle bioenergetic genes, such as NADH ubiquinone oxidoreductase 1, ATP synthase and superoxide dismutase 2. In this study, we hypothesised that aestivation is associated with epigenetic change in frog muscle. We assessed mRNA transcript abundance of seven genes that code for proteins with established roles in epigenetically-mediated gene silencing [transcriptional co-repressor SIN3A, DNA (cytosine-5-) methyltransferase 1, methyl CpG binding protein 2, chromodomain helicase DNA binding protein 4, histone binding prote...
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