Seven groups of features are extracted, which can be computed directly from the primary sequence, and each group is evaluated by the fold cross-validation test and Jackknife test. Comparing the results of numerical experiments, the group of amino acid composition and fractal dimension dimension vector gets the best result, the average accuracy is This resulting predictor is also compared with existing method DNA-Prot and shows better performances.
Published by Elsevier Ltd All rights reserved. DNA-binding proteins are fundamentally important in cellular processes. Several computational-based methods have been developed to improve the prediction of DNA-binding proteins in previous years. However, insufficient work has been done on the prediction of DNA-binding proteins from protein sequence information.
The hybrid feature contains two types of novel sequence features, which reflect information about the conservation of physicochemical properties of the amino acids, and the binding propensity of DNA-binding residues and non-binding propensities of non-binding residues. The comparisons with each feature demonstrated that these two novel features contributed most to the improvement in predictive ability.
Furthermore, to improve the prediction performance of the DNABP model, feature selection using the minimum redundancy maximum relevance mRMR method combined with incremental feature selection IFS was carried out during the model construction. High prediction accuracy and performance comparisons with previous research suggested that DNABP could be a useful approach to identify DNA-binding proteins from sequence information.
DNA-binding proteins play vital roles in cellular processes, such as DNA packaging, replication, transcription, regulation, and other DNA-associated activities. The current main prediction method is based on machine learning, and its accuracy mainly depends on the features extraction method. Therefore, using an efficient feature representation method is important to enhance the classification accuracy.
However, existing feature representation methods cannot efficiently distinguish DNA-binding proteins from non- DNA-binding proteins. In this paper, a multi-feature representation method, which combines three feature representation methods, namely, K-Skip-N-Grams, Information theory, and Sequential and structural features SSF , is used to represent the protein sequences and improve feature representation ability.
In addition, the classifier is a support vector machine. The mixed-feature representation method is evaluated using fold cross-validation and a test set. Feature vectors, which are obtained from a combination of three feature extractions, show the best performance in fold cross-validation both under non-dimensional reduction and dimensional reduction by max-relevance-max-distance. Moreover, the reduced mixed feature method performs better than the non-reduced mixed feature technique. Among these methods, mixed features exhibit superiority over the single features.
Structure and DNA-binding of meiosis-specific protein Hop2. Here we report structure elucidation of the DNA binding domain of homologous pairing protein 2 Hop2 , which is important to gene diversity when sperms and eggs are produced. Together with another protein Mnd1, Hop2 enhances the strand invasion activity of recombinase Dmc1 by over 30 times, facilitating proper synapsis of homologous chromosomes. However, the structural and biochemical bases for the function of Hop2 and Mnd1 have not been well understood.
As a first step toward such understanding, we recently solved the structure for the N-terminus of Hop2 using solution NMR. This fragment shows a typical winged-head conformation with recognized DNA binding activity. DNA interacting sites were then investigated by chemical shift perturbations in a titration experiment.
Information of these sites was used to guide protein -DNA docking with MD simulation, revealing that helix 3 is stably lodged in the DNA major groove and that wing 1 connecting strands 2 and 3 transiently comes in contact with the minor groove in nanosecond time scale. Mutagenesis analysis further confirmed the DNA binding sites in this fragment of the protein.
Therefore, the identification and characterization of these proteins are of great importance. We present here a random forests classifier for identifying DBPs among proteins with known three-dimensional structures. First, clusters of evolutionarily conserved regions patches on the protein 's surface are detected using the PatchFinder algorithm; previous studies showed that these regions are typically the proteins ' functionally important regions.
Next, we train a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein including its dipole moment. Using fold cross validation on a dataset of DNA-binding proteins and proteins which do not bind DNA, the classifier achieved a sensitivity and a specificity of 0. Furthermore, when we tested 5 different methods on 11 new DBPs which did not appear in the original dataset, only our method annotated all correctly.
The resulting classifier was applied to a collection of proteins of known structure and unknown function. Of these proteins , were predicted to bind DNA, and we anticipate that some of them interact with DNA using new structural motifs. The use of complementary computational tools supports the notion that at least some of them do bind DNA.
Expression, purification and biochemical characterization of a single-stranded DNA binding protein from Herbaspirillum seropedicae. An open reading frame encoding a protein similar in size and sequence to the Escherichia coli single-stranded DNA binding protein SSB protein was identified in the Herbaspirillum seropedicae genome.
This open reading frame was cloned into the expression plasmid pET14b. The SSB protein from H. Mass spectrometry data confirmed the identity of this protein. The production of this recombinant protein in good yield opens up the possibility of obtaining its 3D-structure and will help further investigations into DNA metabolism. The structure solution of DNA-binding protein structures and complexes based on the combination of location of DNA-binding protein motif fragments with density modification in a multi-solution frame is described.
Protein—DNA interactions play a major role in all aspects of genetic activity within an organism, such as transcription, packaging, rearrangement, replication and repair. The molecular detail of protein—DNA interactions can be best visualized through crystallography, and structures emphasizing insight into the principles of binding and base-sequence recognition are essential to understanding the subtleties of the underlying mechanisms.
Crystallographic structure solution of protein—DNA complexes therefore remains a challenging area that is in need of optimized experimental and computational methods. The method is based on the combination of locating small, very accurate fragments using the program Phaser and density modification with the program SHELXE.
Negatively supercoiled plasmid pUC19 did not compete, whereas an otherwise identical plasmid pUC19 CG , which contained a dG-dC 7 segment in the Z-form was an excellent competitor. A Southwestern blot using [32P]poly dG-m5dC as a probe in the presence of MgCl2 identified a protein having a molecular weight of 51 kDa. The 51 kDa zuotin was partially sequenced at the N-terminal and the gene, ZUO1, was cloned, sequenced and expressed in Escherichia coli; the expressed zuotin showed similar Z- DNA binding activity, but with lower affinity than zuotin that had been partially purified from yeast.
Zuotin was deduced to have a number of potential phosphorylation sites including two CDC28 homologous to the human and Schizosaccharomyces pombe cdc2 phosphorylation sites. A 60 amino acid segment of zuotin has similarity to several histone H1 sequences.
Disruption of ZUO1 in yeast resulted in a slow growth phenotype. The gel mobility shift assay method revealed a specifically ultraviolet UV damage recognizing, DNA-binding protein in nuclear extracts of normal human cells. This superfamily plays important roles in plant disease resistance, abiotic stress, senescence as well as in some developmental processes. These results provided us with structural information to understand the mechanism of transcriptional control and signal transduction events of the WRKY proteins.
Identification of DNA-binding proteins using multi-features fusion and binary firefly optimization algorithm. Therefore, the developing of effective computational tools for identifying DBPs is becoming highly desirable.
In this study, we proposed an accurate method for the prediction of DBPs. Firstly, we focused on the challenge of improving DBP prediction accuracy with information solely from the sequence. Secondly, we used multiple informative features to encode the protein. These features included evolutionary conservation profile, secondary structure motifs, and physicochemical properties. Thirdly, we introduced a novel improved Binary Firefly Algorithm BFA to remove redundant or noisy features as well as select optimal parameters for the classifier.
The experimental results of our predictor on two benchmark datasets outperformed many state-of-the-art predictors, which revealed the effectiveness of our method. The promising prediction performance on a new-compiled independent testing dataset from PDB and a large-scale dataset from UniProt proved the good generalization ability of our method. In addition, the BFA forged in this research would be of great potential in practical applications in optimization fields, especially in feature selection problems.
A highly accurate method was proposed for the identification of DBPs. A user-friendly web-server named iDbP identification of DNA-binding Proteins was constructed and provided for academic use. NtrC has a three domain structure typical of EBP family. In Herbaspirillum seropedicae, an endophytic diazotroph, NtrC regulates several operons involved in nitrogen assimilation, including glnAntrBC.
Histone H1 in Leishmania presents relevant differences compared to higher eukaryote counterparts, such as the lack of a DNA-binding central globular domain. Despite that, it is apparently fully functional since its differential expression levels have been related to changes in chromatin condensation and infectivity, among other features.
The localization and the aggregation state of L. Analysis of H1 sequences from the Leishmania Genome Database revealed that our protein is included in a very divergent group of histones H1 that is present only in L. An antibody raised against recombinant L. Mass spectrometry analysis and in vitro DNA-binding experiments have also proven that L. Finally, despite the lack of a globular domain, L. A simple microplate method was designed for rapid testing DNA-binding activity of proteins.
The principle of the assay involves binding of tested DNA by his-tagged protein immobilized on a nickel-coated ELISA plate, following colorimetric detection of biotinylated DNA with avidin conjugated to horseradish peroxidase. The method was used to compare DNA mismatch binding activities of MutS proteins from three bacterial species. The assay required relatively low amounts of tested protein approximately 0. Sensitive colorimetric detection enables naked eye observations and quantitation with an ELISA reader.
The performance of the assay, which we believe is a distinguishing trait of the method, is based on two strong and specific molecular interactions: binding of a his-tagged protein to a nickel-coated microplate and binding of biotinylated DNA to avidin. In the reported experiments, the solution was used to optimize the conditions for DNA mismatch binding by MutS protein ; however, the approach could be implemented to test nucleic acids interactions with any protein of interest.
Coupled binding-bending-folding: The complex conformational dynamics of protein-DNA binding studied by atomistic molecular dynamics simulations. While thermodynamic aspects of this behavior are understood, and its biological function is often known, the mechanism by which the conformational changes occur is generally unclear. By providing detailed structural and energetic data, molecular dynamics simulations have been helpful in elucidating and rationalizing protein-DNA binding.
This review will summarize recent atomistic molecular dynamics simulations of the conformational dynamics of DNA and protein-DNA binding. A brief overview of recent developments in DNA force fields is given as well. Simulations have been crucial in rationalizing the intrinsic flexibility of DNA, and have been instrumental in identifying the sequence of binding events, the triggers for the conformational motion, and the mechanism of binding for a number of important DNA-binding proteins.
Molecular dynamics simulations are an important tool for understanding the complex binding behavior of DNA-binding proteins. With recent advances in force fields and rapid increases in simulation time scales, simulations will become even more important for future studies. This article is part of a Special Issue entitled Recent developments of molecular dynamics. Published by Elsevier B. Isolation from genomic DNA of sequences binding specific regulatory proteins by the acceleration of protein electrophoretic mobility upon DNA binding.
We report an efficient and flexible in vitro method for the isolation of genomic DNA sequences that are the binding targets of a given DNA binding protein. This method takes advantage of the fact that binding of a protein to a DNA molecule generally increases the rate of migration of the protein in nondenaturing gel electrophoresis.
We have applied this method to isolate a binding site for FadR, a global regulator of fatty acid metabolism in E. Toward rules relating zinc finger protein sequences and DNA binding site preferences. Zinc finger proteins of the Cys2-His2 type consist of tandem arrays of domains, where each domain appears to contact three adjacent base pairs of DNA through three key residues.
We have designed and prepared a series of variants of the central zinc finger within the DNA binding domain of Sp1 by using information from an analysis of a large data base of zinc finger protein sequences. These results provide the basis for rules that may develop into a code that will allow the design of zinc finger proteins with preselected DNA site specificity.
Using all-atom molecular dynamics simulations, we characterized the molecular mechanism of ssDNA association with SSB. Placed in solution, ssDNA—SSB assemblies were observed to change their structure spontaneously; such structural changes were suppressed in the crystallographic environment. Such reptation-like motion was confined by DNA binding to high-affinity spots, suggesting a two-step mechanism for SSB diffusion.
The known three-dimensional structure of a related protein was used in devising a scheme of site-directed mutagenesis that led to the creation of a temperature-sensitive mutation in the TF1 gene. At the nonpermissive temperature, this mutation disrupted the temporal regulation of viral protein synthesis and processing, altered the kinetics of accumulation of at least one viral transcript, and prohibited the production of infective progeny phage.
We suggest that TF1 function is required to shut off the expression of several early-middle and middle viral genes and that TF1 plays a role in phage head morphogenesis. Spontaneous second-site mutations of the temperature-sensitive mutant TF1 allele that suppressed its associated phenotypes were analyzed.
These suppressor mutations conferred greater amino acid sequence homology with the type II DNA-binding protein from the thermophile Bacillus stearothermophilus. Genome-wide survey of DNA-binding proteins in Arabidopsis thaliana: analysis of distribution and functions.
The interaction of proteins with their respective DNA targets is known to control many high-fidelity cellular processes. Performing a comprehensive survey of the sequenced genomes for DNA-binding proteins DBPs will help in understanding their distribution and the associated functions in a particular genome. Availability of fully sequenced genome of Arabidopsis thaliana enables the review of distribution of DBPs in this model plant genome.
This resulted in proteins , identified as DNA-binding in Arabidopsis genome, which are distributed across different PFam families. Our search protocol helped to assign DNA-binding property to several proteins that were previously marked as unknown, putative or hypothetical in function. The distribution of Arabidopsis genes having a role in plant DNA repair were particularly studied and noted for their functional mapping. The functions observed to be overrepresented in the plant genome harbour DNAmethyladenine glycosylase activity, alkylbase DNA N-glycosylase activity and DNA- apurinic or apyrimidinic site lyase activity, suggesting their role in specialized functions such as gene regulation and DNA repair.
Purification and general properties of the DNA-binding protein P16 from rat liver mitochondria. The mitochondrial DNA-binding protein P16 was isolated from rat liver mitochondrial lysates by affinity chromatography on single strand DNA agarose and separated from DNA in the preparation by alkaline CsCl isopycnic gradients. Digestion of single strand DNA-bound P16 with proteinase K produced a protease-insensitive, DNA-binding fragment Mr approximately equal to 6, that has been purified by essentially the same procedures used for intact P The partial amino acid compositions for P16 and the DNA-binding fragment were obtained by conventional methods.
Analysis of subcellular fractions revealed that nearly all of the cellular P16 was located in the mitochondria and that only trace amounts of protein of comparable electrophoretic mobility could be isolated from the nuclear or cytoplasmic fractions. These results indicate that P16 is synthesized on cytoplasmic ribosomes and imported into the mitochondria. The addition of purified P16 to deproteinized mitochondrial DNA resulted in the complete protection of the labeled nascent strands of displacement loops against branch migrational loss during cleavage of parental DNA with SstI, thus providing strong evidence that P16 is the single entity required for this in vitro function.
HMG-D is a major high mobility group chromosomal protein present during early embryogenesis in Drosophila melanogaster. Six peptides obtained after digestion of the isolated protein with Achromobacter protease I were partially or completely sequenced. DBP showed a tendency to multimerization in the course of purification and was found to bind preferentially to single-stranded DNA.
The sizes of the protected fragments indicated that a binding site size for DBP is about 30 nt per protein monomer. This helix-destabilizing ability is consistent with the prediction that DBP functions as a single-stranded DNA binding protein in virus replication.
Mikhailov, Victor S. Purified DBP formed oligomers that were crosslinked by redox reagents resulting in predominantly protein dimers and tetramers. In gel retardation assays, DBP showed a high affinity for single-stranded oligonucleotides and was able to compete with another baculovirus SSB protein , LEF-3, for binding sites. The unwinding and renaturation activities of DBP, as well as the DNA binding activity, were sensitive to sulfhydryl reagents and were inhibited by oxidation of thiol groups with diamide or by alkylation with N-ethylmaleimide.
These activities and a tight association with subnuclear structures suggests that DBP is a component of the virogenic stroma that is involved in the processing of replicative intermediates. Identification of DNA-binding proteins using structural, electrostatic and evolutionary features. DNA-binding proteins DBPs participate in various crucial processes in the life-cycle of the cells, and the identification and characterization of these proteins is of great importance.
We present here a random forests classifier for identifying DBPs among proteins with known 3D structures. First, clusters of evolutionarily conserved regions patches on the surface of proteins were detected using the PatchFinder algorithm; earlier studies showed that these regions are typically the functionally important regions of proteins. Next, we trained a classifier using features like the electrostatic potential, cluster-based amino acid conservation patterns and the secondary structure content of the patches, as well as features of the whole protein , including its dipole moment.
Using fold cross-validation on a dataset of DBPs and proteins that do not bind DNA, the classifier achieved a sensitivity and a specificity of 0. Furthermore, when we tested five different methods on 11 new DBPs that did not appear in the original dataset, only our method annotated all correctly. Method for nucleic acid hybridization using single-stranded DNA binding protein. Method of nucleic acid hybridization for detecting the presence of a specific nucleic acid sequence in a population of different nucleic acid sequences using a nucleic acid probe.
The nucleic acid probe hybridizes with the specific nucleic acid sequence but not with other nucleic acid sequences in the population. The method includes contacting a sample potentially including the nucleic acid sequence with the nucleic acid probe under hybridizing conditions in the presence of a single-stranded DNA binding protein provided in an amount which stimulates renaturation of a dilute solution i.
An immunoassay was used to examine the interaction between a herpes simplex virus protein , ICP8, and various types of DNA. The advantage of this assay is that the protein is not subjected to harsh purification procedures. No evidence for sequence-specific ds DNA binding was obtained for either the entire herpes simplex virus genome or cloned viral sequences. Neighboring genes for DNA-binding proteins rescue male sterility in Drosophila hybrids.
Crosses between closely related animal species often result in male hybrids that are sterile, and the molecular and functional basis of genetic factors for hybrid male sterility is of great interest. Here, we report a molecular and functional analysis of HMS1, a region of 9. The HMS1 region contains two strong candidate genes for the genetic incompatibility, agt and Taf1 Both encode unrelated DNA-binding proteins , agt for an alkyl-cysteine-S-alkyltransferase and Taf1 for a subunit of transcription factor TFIID that serves as a multifunctional transcriptional regulator.
The contribution of each gene to hybrid male sterility was assessed by means of germ-line transformation, with constructs containing complete agt and Taf1 genomic sequences as well as various chimeric constructs. Both agt and Taf1 contribute about equally to HMS1 hybrid male sterility. Transgenes containing either locus rescue sterility in about one-half of the males, and among fertile males the number of offspring is in the normal range.
This finding suggests compensatory proliferation of the rescued, nondysfunctional germ cells. Results with chimeric transgenes imply that the hybrid incompatibilities result from interactions among nucleotide differences residing along both agt and Taf1 Our results challenge a number of preliminary generalizations about the molecular and functional basis of hybrid male sterility, and strongly reinforce the role of DNA-binding proteins as a class of genes contributing to the maintenance of postzygotic reproductive isolation.
The HMS1 region contains two strong candidate genes for the genetic incompatibility, agt and Taf1. Both encode unrelated DNA-binding proteins , agt for an alkyl-cysteine-S-alkyltransferase and Taf1 for a subunit of transcription factor TFIID that serves as a multifunctional transcriptional regulator. Results with chimeric transgenes imply that the hybrid incompatibilities result from interactions among nucleotide differences residing along both agt and Taf1.
Our results challenge a number of preliminary generalizations about the molecular and functional basis of hybrid male sterility, and strongly reinforce the role of DNA-binding proteins as a class of genes contributing to the maintenance of postzygotic reproductive isolation.
Immobilization of proteins onto microbeads using a DNA binding tag for enzymatic assays. The resulting protein -coated microbeads can be utilized for functional analysis of the enzymatic activity using flow cytometry. The quantity of bead-bound proteins can be enhanced by increasing the number of ORCs. In addition, proteins with the scCro-tag that were synthesized using a cell-free protein synthesis system were also immobilized onto the beads, thus indicating that this bead-based system would be applicable to high-throughput analysis of various enzymatic activities.
Naturally, TALEs are injected by Xanthomonas bacteria into plant cells to manipulate the host transcriptome. We show that Bat proteins can be adapted for use as transcription factors and nucleases and that sequence preferences can be reprogrammed. This feature allowed us to explore alternative strategies for the design of custom Bat repeat arrays, providing novel insights into the functional relevance of non-RVD residues. The Bat proteins offer fertile grounds for research into the creation of improved programmable DNA-binding proteins and comparative insights into TALE-like evolution.
First, the algorithm predicts the functional region of the protein based on its evolutionary profile; the assumption is that large clusters of conserved residues are good markers of functional regions. Next, various characteristics of the predicted functional region as well as global features of the protein are calculated, such as the average surface electrostatic potential, the dipole moment and cluster-based amino acid conservation patterns. Finally, a random forests classifier is used to predict whether the query protein is likely to bind DNA and to estimate the prediction confidence.
We have trained and tested the classifier on various datasets and shown that it outperformed related methods. The application of the server to an updated version of the N-Func database, which contains proteins of unknown function with solved 3D-structure, suggested new putative DBPs for experimental studies.
Vanarsdall, Adam L. Whereas LEF-3 is a multi-functional protein essential for viral DNA replication, transporting helicase into the nucleus, and forms a stable complex with the baculovirus alkaline nuclease, the role for DBP in baculovirus replication remains unclear. The lack of DBP does not cause a general shutdown of the expression of viral genes, as was revealed by accumulation of early LEF-3 , late VP39 , and very late P10 proteins in cells transfected with the dbp knockout construct.
In addition, analysis of the viral DNA replicated by the dbp knockout by using field inversion gel electrophoresis failed to detect the presence of genome-length DNA. Assessment of the cellular localization of DBP relative to replicated viral DNA by immunoelectron microscopy indicated that, at 24 h post-infection, DBP co-localized with nascent DNA at distinct electron-dense regions within the nucleus. Finally, immunoelectron microscopic analysis of cells transfected with the dbp.
We have assessed their DNA-binding properties and modelled repeat structures. Sequence alignments show that there are only three residues conserved between repeats of all TALE-like proteins including the two new additions. This conserved motif could prove useful as an identifier for future TALE-likes. POZ-domain transcription factors are characterized by the presence of a protein-protein interaction domain called the POZ or BTB domain at their N terminus and zinc fingers at their C terminus.
Despite the large number of POZ-domain transcription factors that have been identified to date and the significant insights that have been gained into their cellular functions, relatively little is known about their DNA binding properties. Remarkably, it binds efficiently to probes carrying these repeats in various orientations and spacings with no particular rotational alignment, indicating that its interaction with DNA is highly flexible.
The high mobility group protein 1 enhances binding of the estrogen receptor DNA binding domain to the estrogen response element. Because HMG1 dramatically enhanced estrogen receptor DBD binding to the ERE, and the DBD is the most highly conserved region among the nuclear receptor superfamily members, HMG1 may function to enhance binding of other nuclear receptors to their respective response elements and act in concert with coactivator proteins to regulate expression of hormone-responsive genes.
Pax-3, a novel murine DNA binding protein expressed during early neurogenesis. We describe the isolation and characterization of Pax-3, a novel murine paired box gene expressed exclusively during embryogenesis. Pax-3 encodes a amino acid protein with an Mr of 56 kd containing both a paired domain and a paired-type homeodomain. The Pax-3 protein is a DNA binding protein that specifically recognizes the e5 sequence present upstream of the Drosophila even-skipped gene.
Pax-3 transcripts are first detected in 8. During early neurogenesis, Pax-3 expression is limited to mitotic cells in the ventricular zone of the developing spinal cord and to distinct regions in the hindbrain, midbrain and diencephalon. In day embryos, expression of Pax-3 is also seen in neural crest cells of the developing spinal ganglia, the craniofacial mesectoderm and in limb mesenchyme of 10 and 11 day embryos.
Genome-wide profiling of DNA-binding proteins using barcode-based multiplex Solexa sequencing. Because ChIP-chip introduces several biases, most notably due to the use of a fixed number of probes, ChIP-Seq has quickly become the method of choice as, depending on the sequencing depth, it is more sensitive, quantitative, and provides a greater binding site location resolution. With the ever increasing number of reads that can be generated per sequencing run, it has now become possible to analyze several samples simultaneously while maintaining sufficient sequence coverage, thus significantly reducing the cost per ChIP-Seq experiment.
In this chapter, we provide a step-by-step guide on how to perform multiplexed ChIP-Seq analyses. As a proof-of-concept, we focus on the genome-wide profiling of RNA Polymerase II as measuring its DNA occupancy at different stages of any biological process can provide insights into the gene regulatory mechanisms involved. However, the protocol can also be used to perform multiplexed ChIP-Seq analyses of other DNA-binding proteins such as chromatin modifiers and transcription factors. Correct repair of damaged DNA is critical for genomic integrity.
Deficiencies in DNA repair are linked with human cancer. Here we report a novel mechanism by which a virus manipulates DNA damage responses. This results in activated stress responses and apoptosis. This leads to failure to recruit repair proteins such as Rad51 or Rad9, explaining why LT prevents repair of double strand DNA breaks by homologous recombination. A targeted intervention directed at RPA based on this viral mechanism could be useful in circumventing the resistance of cancer cells to therapy.
During animal development, distinct tissues, organs, and appendages are specified through differential gene transcription by Hox transcription factors. However, the conserved Hox homeodomains bind DNA with high affinity yet low specificity. We have therefore explored the structure of the Drosophila melanogaster Hox protein Ultrabithorax and the impact of its nonhomeodomain regions on DNA binding properties.
Computational and experimental approaches identified several conserved, intrinsically disordered regions outside the homeodomain of Ultrabithorax that impact DNA binding by the homeodomain. Full-length Ultrabithorax bound to target DNA 2. Binding is restored almost to homeodomain affinity by the mostly disordered N-terminal amino acids R region in a length-dependent manner.
Both the I2 and R regions contain portions of the activation domain, functionally linking DNA binding and transcription regulation. The amino acid sequences of I2 and much of the I1 and R regions vary significantly among Ultrabithorax orthologues, potentially diversifying Hox-DNA interactions.
We report a dual illumination, single-molecule imaging strategy to dissect directly and in real-time the correlation between nanometer-scale domain motion of a DNA repair protein and its interaction with individual DNA substrates. Conformational dynamics was assessed via FeS-mediated quenching of a fluorophore site-specifically incorporated into XPD.
Simultaneously, binding of DNA molecules labeled with a spectrally distinct fluorophore was detected by colocalization of the DNA- and protein -derived signals. We show that XPD undergoes thermally driven conformational transitions that manifest in spatial separation of its two auxiliary domains. DNA binding does not strictly enforce a specific conformation.
Our imaging strategy will be a valuable tool to study other FeS-containing nucleic acid processing enzymes. Activator Protein redox switch controlling structure and DNA-binding. The transcription factor, activator protein -1 AP-1 , binds to cognate DNA under redox control; yet, the underlying mechanism has remained enigmatic.
However, while JunD is competent to bind DNA, the FosB bZIP domain must undergo a large conformational rearrangement that is controlled by a 'redox switch' centered on an inter-molecular disulfide bond.
Protein -DNA interactions are involved in many fundamental biological processes essential for cellular function. Most of the existing computational approaches employed only the sequence context of the target residue for its prediction. In the present study, for each target residue, we applied both the spatial context and the sequence context to construct the feature space.
Finally, a predictor PDNAsite was developed through the integration of the support vector machines SVM classifier and ensemble learning. Results on the PDNA and the PDNA datasets demonstrate that features extracted from spatial context provide more information than those from sequence context and the combination of them gives more performance gain.
An analysis of the number of binding sites in the spatial context of the target site indicates that the interactions between binding sites next to each other are important for protein -DNA recognition and their binding ability. Quantitative characterization of conformational-specific protein-DNA binding using a dual-spectral interferometric imaging biosensor. DNA-binding proteins play crucial roles in the maintenance and functions of the genome and yet, their specific binding mechanisms are not fully understood.
Recently, it was discovered that DNA-binding proteins recognize specific binding sites to carry out their functions through an indirect readout mechanism by recognizing and capturing DNA conformational flexibility and deformation. High-throughput DNA microarray-based methods that provide large-scale protein-DNA binding information have shown effective and comprehensive analysis of protein-DNA binding affinities, but do not provide information of DNA conformational changes in specific protein -DNA complexes.
Building on the high-throughput capability of DNA microarrays, we demonstrate a quantitative approach that simultaneously measures the amount of protein binding to DNA and nanometer-scale DNA conformational change induced by protein binding in a microarray format. Both measurements rely on spectral interferometry on a layered substrate using a single optical instrument in two distinct modalities. In the first modality, we quantitate the amount of binding of protein to surface-immobilized DNA in each DNA spot using a label-free spectral reflectivity technique that accurately measures the surface densities of protein and DNA accumulated on the substrate.
In the second modality, for each DNA spot, we simultaneously measure DNA conformational change using a fluorescence vertical sectioning technique that determines average axial height of fluorophores tagged to specific nucleotides of the surface-immobilized DNA. The approach presented in this paper, when combined with current high-throughput DNA microarray-based technologies, has the potential to serve as a rapid and simple method for quantitative and large-scale characterization of conformational specific protein -DNA interactions.
Therefore, elucidating the regulatory mechanism of these signaling steps of unphosphorylated STAT3 is a potential step in the discovery of a novel cancer drug. However, the mechanism of unphosphorylated STAT3 binding to the promoter of target genes remains unclear. Theoretical studies of protein-protein and protein-DNA binding rates. Proteins are folded chains of amino acids. Some of the amino acids e.
Lys, Arg, His, Asp, and Glu carry charges under physiological conditions. Proteins almost always function through binding to other proteins or ligands, for example barnase is a ribonuclease protein , found in the bacterium Bacillus amyloliquefaceus. Barnase degrades RNA by hydrolysis. For the bacterium to inhibit the potentially lethal action of Barnase within its own cell it co-produces another protein called barstar which binds quickly, and tightly, to barnase. The biological function of this binding is to block the active site of barnase.
The speeds rates at which proteins associate are vital to many biological processes. They span a wide range from less than to M-1s A different paradigm appears in the case of protein binding to DNA. The rate in this case is enhanced through attractive surface potential that effectively reduces the dimensionality of the available search space for the diffusing protein.
For protein-protein association we present a general strategy for computing protein-protein rates of association. The main achievements of this strategy is the ability to obtain a stringent reaction criteria based on the landscape of short-range interactions between the associating proteins , and the ability to compute the effect of the electrostatic interactions on the rates of association accurately using the best known solvers for Poisson-Boltzmann equation presently available.
For protein -DNA association we present a mathematical model for proteins targeting specific sites on a circular DNA topology. The main achievements are the realization that a linear DNA with reflecting ends. Mutations in the DDB2 gene inactivate UV-DDB in individuals from complementation group E of xeroderma pigmentosum XP-E , an autosomal recessive disease characterized by sun sensitivity, severe risk for skin cancer and defective nucleotide excision repair.
UV-DDB is also deficient in many rodent tissues, exposing a shortcoming in rodent models for cancer. The C-terminal domain of the DNA-binding domain, Pot1pC, exhibits non-specific ssDNA recognition, achieved through thermodynamically equivalent alternative binding conformations. Given this flexibility, it is unclear how specificity for ssDNA over RNA, an activity required for biological function, is achieved.
Examination of the ribose-position specificity of Pot1pC shows that ssDNA specificity is additive but not uniformly distributed across the ligand. These alternative conformations, accessed through both ligand and protein flexibility, recover much, but not all, of the binding energy, leading to the observed reduction in affinities.
These findings suggest that intermolecular interfaces are remarkably sophisticated in their tuning of specificity toward flexible ligands. The mechanisms by which plants restrict the growth of pathogens include transient activation of numerous defense-related genes.
Box P is a putative cis-acting element of a distinct group of such genes, including those encoding the enzyme phenylalanine ammonialyase PAL. BPF-1 binds to this element with specificity similar to that of the binding activity in nuclear extracts. BPF-1 mRNA accumulates rapidly in elicitor-treated parsley cells and around fungal infection sites on parsley leaves. This accumulation is, at least partly, due to a rapid and transient increase in the transcription rate of BPF These results are consistent with the hypothesis that BPF-1 is involved in disease resistance by modulating plant defense gene expression.
A simple-prepare, single-use and cost-effective, in vitro biosensor for the detection of TAR DNA-binding protein 43 TDP , a biomarker of neuro-degenerative disorders, was designed, manufactured and tested. This study reports the first biosensor application for the detection of TDP using a novel biosensor fabrication methodology.
Bioconjugation mechanism was applied by conjugating anti-TDP 43 with N-succinimidyl S-acetylthioacetate SATA producing a thiol-linked anti-TDP 43, which was used to directly link with gold electrode surface, minimizing the preparation steps for biosensor fabrication and simplifying the biosensor surface. The effectiveness of this bioconjugation mechanism was evaluated and confirmed by FqRRM12 protein , using nuclear magnetic resonance NMR. Human TDP peptide of 0.
Surface shapes and surrounding environment analysis of single- and double-stranded DNA-binding proteins in protein -DNA interface. Protein-DNA bindings are critical to many biological processes. However, the structural mechanisms underlying these interactions are not fully understood. In the results, we found that the interface shapes, hydrogen bonds, and the surrounding environment present significant differences between the two kinds of proteins.
Proteins ; We reported earlier that the tobacco early ethylene-responsive gene NtER1 encodes a calmodulin-binding protein Yang, T. Here we demonstrate that there is one NtER1 homolog as well as five related genes in Arabidopsis. These six genes are rapidly and differentially induced by environmental signals such as temperature extremes, UVB, salt, and wounding; hormones such as ethylene and abscisic acid; and signal molecules such as methyl jasmonate, H 2 O 2 , and salicylic acid.
Hence, they were designated as AtSR Arabidopsis thaliana signal-responsive genes. The multiple CGCG cis-elements are found in promoters of genes such as those involved in ethylene signaling, abscisic acid signaling, and light signal perception. Segregation analysis with partially diploid phage heterozygous for TF1 showed that phage bearing only these null alleles was inviable. Deletion of the nine C-proximal amino acids of TF1 prohibited phage multiplication in vivo and abolished its site-specific DNA-binding activity in vitro.
To determine the effect of human DNA binding protein dbpA on the biology of gastric cancer cells. DbpA expression was analyzed by Western blot analysis and immunofluorescence staining in gastric cancer tissues and cell lines. A dbpA-specific small interference si RNA was designed and synthesized. Transwell migration and colony formation assays were used to assess the inhibitory effects of dbpA siRNA on cell invasion and tumorigenesis in vitro.
Drug-sensitivity was evaluated using a conventional 3- 4,5-dimethylthiazolyl -2,5-diphenyl tetrazolium bromide MTT assay. The expression of dbpA was upregulated in gastric cancer tissues and cell lines as compared to adjacent normal tissues or gastric epithelial cells. Silencing of dbpA increased expression of E-cadherin, decreased expression of adenomatous polyposis coli APC , beta-catenin and cyclin D1, but had no effect on expression of NF-kappaB.
Silencing of dbpA also suppressed cell invasion and colony formation of SGC cells, and enhanced their chemosensitivity to 5-fluorouracil. DbpA plays an important role in the pathogenesis and development of gastric cancer, and the process involves E-cadherin, APC, beta-catenin and cyclin D1.
Silencing of dbpA might be a novel therapeutic strategy for increasing chemosensitivity to 5-fluorouracil in gastric cancer. Acid or erythromycin stress significantly improves transformation efficiency through regulating expression of DNA binding proteins in Lactococcus lactis F Lactococcus lactis is a gram-positive bacterium used extensively in the dairy industry and food fermentation, and its biological characteristics are usually improved through genetic manipulation.
However, poor transformation efficiency was the main restriction factor for the construction of engineered strains. In this study, the transformation efficiency of L. Notably, the transformation efficiency of F44e L. Especially for radA, Overexpression of some DNA binding proteins could improve the transformation efficiency. The results suggested that acid or erythromycin stress could improve the transformation efficiency of L.
We have proposed a simple but promising strategy for improving the transformation efficiency of L. Published by Elsevier Inc. Numerous in vivo and in vitro studies have demonstrated formation of these structures in telomeric and non-telomeric regions of the genome. Telomeric GQs protect the chromosome ends whereas non-telomeric GQs either act as road blocks or recognition sites for DNA metabolic machinery. These observations suggest the significance of these structures in regulation of different metabolic processes, such as replication and repair.
GQs are typically thermodynamically more stable than the corresponding Watson-Crick base pairing formed by G-rich and C-rich strands, making protein activity a crucial factor for their destabilization. Inside the cell, GQs interact with different proteins and their enzymatic activity is the determining factor for their stability.
We studied interactions of several proteins with GQs to understand the underlying principles of protein -GQ interactions using single-molecule FRET and other biophysical techniques. One of these sequences is the human telomeric repeat and the other two, located in the promoter region of tyrosine hydroxylase gene, are highly heterogeneous sequences that better represent PQS in the genome.
The thermal stability of these structures do not necessarily correlate with their stability against protein -mediated unfolding. We conclude that thermal stability is not necessarily an adequate criterion for predicting the physiological viability of GQ structures.
To determine the critical structural factors that influence protein -GQ interactions we studied two groups of GQ structures that have systematically varying loop lengths and number of G-tetrad layers. It is recruited to replication forks via an interaction with replication protein A RPA , the major ss DNA-binding protein in eukaryotic cells.
A calmodulin binding protein from Arabidopsis is induced by ethylene and contains a DNA-binding motif. Calmodulin CaM , a key calcium sensor in all eukaryotes, regulates diverse cellular processes by interacting with other proteins. To isolate CaM binding proteins involved in ethylene signal transduction, we screened an expression library prepared from ethylene-treated Arabidopsis seedlings with 35S-labeled CaM.
These include a DNA-binding domain at the N terminus, an acidic region at the C terminus, and nuclear localization signals. The structural features of EICBP are conserved in all EICBP-related proteins in Arabidopsis, suggesting that they may constitute a new family of DNA binding proteins and are likely to be involved in modulating gene expression in the presence of ethylene.
Structural analysis of DNA binding by C. CspI, a member of a novel class of R-M controller proteins regulating gene expression. The structure of the new class of controller proteins exemplified by C. CspI in complex with its 21 bp DNA-recognition sequence is presented, and the molecular basis of sequence recognition in this class of proteins is discussed. An unusual extended spacer between the dimer binding sites suggests a novel interaction between the two C- protein dimers. Using X-ray crystallography, the structure of the protein in complex with its 21 bp DNA-recognition sequence was solved to 1.
An unusual aspect of the promoter sequence is the extended spacer between the dimer binding sites, suggesting a novel interaction between the two C- protein dimers when bound to both recognition sites correctly spaced on the DNA. A U-bend model is proposed for this tetrameric complex, based on the results of gel-mobility assays, hydrodynamic analysis and the observation of key contacts at the interface between dimers in the crystal.
In order to study the regulatory mechanism of DBP on viral proliferation, genome replication, and gene transcription, a BmNPV dbp gene knockout virus dbp-ko-Bacmid was generated by the means of Red recombination system. In addition, dbp-repaired virus dbp-re-Bacmid was constructed by the means of the Bac to Bac system.
Then, the Bacmids were transfected into BmN cells. The results of the latter experiment revealed that the transcript of dbp gene was first detected at 3 h post-infection. Furthermore, the replication level of virus genome and the transcriptional level of virus early, late, and very late genes in BmN cells, which had been transfected with 3 kinds of Bacmids, were analyzed by Real-Time PCR.
Rad52 is the prototype of recombination mediators, and the human Rad52 protein has two distinct DNA-binding sites: the first site binds to single-stranded DNA, and the second site binds to either double- or single-stranded DNA. We previously showed that yeast Rad52 extensively stimulates Radcatalyzed D-loop formation even in the absence of replication protein A, by forming a stoichiometric complex with Rad However, the precise roles of Rad52 and Rad51 within the complex are unknown.
In the present study, we constructed yeast Rad52 mutants in which the amino acid residues corresponding to the second DNA-binding site of the human Rad52 protein were replaced with either alanine or aspartic acid. We found that the second DNA-binding site is important for the yeast Rad52 function in vivo. RadRad52 complexes consisting of these Rad52 mutants were defective in promoting the formation of D-loops, and the ability of the complex to associate with double-stranded DNA was specifically impaired.
Our studies suggest that Rad52 within the complex associates with double-stranded DNA to assist Radmediated homologous pairing. Breaking the DNA-binding code of Ralstonia solanacearum TAL effectors provides new possibilities to generate plant resistance genes against bacterial wilt disease. Tags: endow, dota2, hero, abaddon, gaming, game, valve, steam, international, Tags: abaddon, demon, supernaural, captainshroom. Tags: supernatural, abaddon, alaina huffman.
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The start of MLS season is being delayed two weeks and Commissioner Don Garber said teams will play in empty stadiums or before limited crowds. Op-Ed: Mark Cuban was right the first time. We should stop singing the national anthem at ballgames. USC Sports. USC point guard Ethan Anderson missed six weeks because of back pain. Finally recovered, he has helped the Trojans into a first-place tie in the Pac About Us.
Brand Publishing. Times Events. Times News Platforms. Times Store. Second Opinion. Facebook Twitter Show more sharing options Share Close extra sharing options. This Saturday, Santa Anita will run eight races in front of only a handful of owners and state officials. So, does this spell disaster for an already struggling sport? Not necessarily. Enter email address. John Cherwa. Follow Us twitter instagram email facebook. More From the Los Angeles Times. Soccer Start of MLS season is being delayed two weeks.
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