SEARCH · PubMed Health

Results for “nucleoprotein complex”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 721 records · Page 40Linked to original sources

PubMed2004

DNA looping and catalysis; the IHF-folded arm of Tn10 promotes conformational changes and hairpin resolution.

DNA loops and bends are common features of DNA processing machines. The bacterial transposon Tn10 has recruited integration host factor (IHF), a site-specific DNA-bending protein, as an architectural component for assembly of the higher-order nucleoprotein complex within which the transposition reaction takes place. Here, we demonstrate additional roles for the IHF loop during the catalytic steps of the reaction. We show that metal ion-dependent unfolding of the IHF-bent transposon arm is communicated to the catalytic center, inducing a substantial conformational change in the DNA. Partial disruption of the IHF loop shows that this step promotes resolution of the hairpin intermediate on one transposon end and initiation of catalysis at the other. Further evidence suggests that the molecular mechanism responsible may be mechanical stress in the IHF loop, related to a change in the relative position of the transposase contacts that anchor the loop on either side.

Base Sequence↗

PubMed2002

Fluorescence resonance energy transfer studies of U-shaped DNA molecules.

Fluorescence resonance energy transfer studies allow to determine global shape properties of nucleic acids and nucleoprotein complexes. In many DNA-protein complexes, the DNA is more or less bent and the degree of bending can be obtained by FRET. For example, the DNA in complex with the integration host factor (IHF) is kinked by approximately 160 degrees building a U-shaped structure. The two DNA helix ends come close to one another in space in a distance range easily measurable by FRET. The global DNA structure of this complex can be mimicked by introducing two regions with unpaired bases ('bulges') into the DNA each producing a sharp kink of approximately 80 degrees. These U-shaped DNA constructs were used to measure the electrostatic interaction of the two nearly parallel negatively charged DNA helix arms. The electrostatic repulsion between the helix arms, and as a consequence their distance, decreases with growing salt concentration of mono- or divalent cations. This experimental approach also allows the sensitive study of the local structure of DNA sequences positioned between the two bulges.

Acrylic Resins↗

PubMed2005

Why repetitive DNA is essential to genome function.

There are clear theoretical reasons and many well-documented examples which show that repetitive, DNA is essential for genome function. Generic repeated signals in the DNA are necessary to format expression of unique coding sequence files and to organise additional functions essential for genome replication and accurate transmission to progeny cells. Repetitive DNA sequence elements are also fundamental to the cooperative molecular interactions forming nucleoprotein complexes. Here, we review the surprising abundance of repetitive DNA in many genomes, describe its structural diversity, and discuss dozens of cases where the functional importance of repetitive elements has been studied in molecular detail. In particular, the fact that repeat elements serve either as initiators or boundaries for heterochromatin domains and provide a significant fraction of scaffolding/matrix attachment regions (S/MARs) suggests that the repetitive component of the genome plays a major architectonic role in higher order physical structuring. Employing an information science model, the 'functionalist' perspective on repetitive DNA leads to new ways of thinking about the systemic organisation of cellular genomes and provides several novel possibilities involving repeat elements in evolutionarily significant genome reorganisation. These ideas may facilitate the interpretation of comparisons between sequenced genomes, where the repetitive DNA component is often greater than the coding sequence component.

Animals↗

PubMed2001

Peptidic determinants and structural model of human NDP kinase B (Nm23-H2) bound to single-stranded DNA.

Isoform B of human NDP kinase (NDPK-B) was previously identified as a transcription factor stimulating in vitro and ex vivo the transcription of the c-myc oncogene, which involves this enzyme in carcinogenesis. We have studied the enzymatic properties of NDPK-B in the presence of several single-stranded oligonucleotides. We show that the oligonucleotides are competitive inhibitors of the catalytic activity, indicating that the active site acts as a binding template for the anchorage of the oligonucleotide. Furthermore, the presence of a guanine at the 3'-end of several different aptamers increases its affinity 10-fold. To define the surface of the protein contacting the DNA within the nucleoprotein complex, we used single nanosecond laser pulses as the cross-linking reagent and MALDI-TOF mass spectrometry to identify cross-linked peptides purified from proteolytic digests of the cross-linked complex. Using 11-mer and 30-mer single-stranded oligonucleotides, the same three different nucleopeptides were identified after irradiation of the complexes, indicating a common binding mode for these two aptamers. Taken together, these results allowed us to propose a structural model of NDPK-B bound to single-stranded DNA.

Amino Acid Sequence↗

PubMed1988

Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization.

Factor D, a DNA binding protein that enhances the activities of diverse DNA polymerases with a common restricted set of templates, was initially characterized in mouse liver but has resisted extensive purification. In this paper, we report that a similar stimulatory activity can be obtained in highly purified form from nuclei of rabbit hepatocytes. The rabbit liver protein increases the rates at which several DNA polymerases copy sparsely primed natural DNA templates and primed synthetic poly(dT), but it has no effect on the rates of copying of activated DNA or of poly(dG), poly(dA), and poly(dC). Direct binding of the purified stimulatory protein to an oligomer that contains a (dT)16 base stretch is visualized by retardation of the nucleoprotein complex on nondenaturing electrophoretograms. In the presence of the enhancing factor, Michaelis constants, Km, of responsive polymerase for singly primed bacteriophage M13 DNA and for poly(dT), but not for poly(dA), are decreased. Product analysis of M13 DNA primer extension indicates that the rabbit factor augments the apparent processivity of DNA polymerase by decreasing the extent of enzyme pausing at a tract of four consecutive thymidine residues in the template. Gel filtration of the native stimulatory protein yields an apparent relative molecular size of 58 +/- 2 kilodaltons. Stimulatory activity is readily inactivated by heat or by trypsin digestion, but it is resistant to micrococcal nuclease, N-ethyl-maleimide, or calcium ions.

Animals↗

PubMed1981

Ribonucleic acid and other polyanions facilitate chromatin assembly in vitro.

Crude extracts of Drosophila embryos are a rich source of both DNA topoisomerase I and chromatin assembly activity [Nelson, T., Hsieh, T., & Brutlag, D.L. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 5510-5514; Hseih, T., & Brutlag, D. L. (1980) Cell (Cambridge, Mass.) 21, 115-125]. Purified topoisomerase I from Drosophila embryos, however, is not sufficient for chromatin assembly. Rather, the ability of Drosophila embryo extracts to mediate chromatin assembly in vitro requires an anionic fraction which we demonstrate to be RNA. Exogenous natural and homopolymer RNAs, if of sufficient length, can also mediate chromatin assembly in vitro. The RNA acts stoichiometrically in assembly, being required in amounts at least equal in weight to the amount of histones present. Natural and homopolymer DNAs, whether single or double stranded, are inactive under the same conditions. The arginine-rich histones H3 and H4 or histone H4 alone is sufficient to produce nucleoprotein complexes with physiological numbers of supertwists in the DNA. Complexes containing these subsets of the core histones also resemble assembled complexes containing all four core histones with respect to some patterns of nuclease sensitivity, although complexes containing all four core histones more closely resemble native chromatin in nuclease digestions.

Animals↗

PubMed2003

Nucleosome positioning in the human c-Fos promoter analyzed by in vivo footprinting with psoralen and ionizing radiation.

We performed detailed footprinting analysis of nucleosome positioning in the c-FOS promoter of living human fibroblasts. The translational position was determined by terminal transferase-dependent PCR with 4,5',8-trimethylpsoralen. The rotational position was determined by ligation-mediated PCR with ionizing radiation. In the middle of the c-FOS promoter, a nucleosome was positioned not only translationally but also rotationally. The comparison of the results of our in vivo footprinting with those of a previous report on the in vitro footprinting of reconstituted nucleosomes revealed that the major in vivo translational position was approximately 70 bp upstream of the in vitro position, whereas the rotational position was unchanged. The in vivo translational position appears to be strongly influenced by the presence of transcription factors, which may function as boundaries, while the rotational position appears to be determined predominantly by the DNA sequence. We also investigated the influence of the transcriptional activation of the c-FOS gene on the positioning of this nucleosome. Although it is well-known that there are rapid changes in general nuclease sensitivity and chemical modifications of histone in the c-FOS gene upon activation, we could not detect any change in the translational or rotational position of this nucleosome. The nucleoprotein complex in the c-FOS promoter containing the positioned nucleosome and several transcription factors seems to be structurally unaltered upon activation, despite the rapid chemical modifications of the nucleosome and some of the transcription factors.

Base Sequence↗

PubMed1996

Effect of human immunodeficiency virus type 1 (HIV-1) nucleocapsid protein on HIV-1 reverse transcriptase activity in vitro.

Conversion of human immunodeficiency virus type 1 (HIV-1) genomic RNA to viral DNA is a requisite step in the virus life cycle. This conversion is catalyzed by reverse transcriptase (RT) associated with a large nucleoprotein complex composed of several viral proteins including nucleocapsid (NC). To better characterize the biochemical mechanisms of viral DNA synthesis, we overexpressed and purified recombinant HIV-1 NC and studied its effect on the activity and processivity of HIV-1 RT during polymerization of HIV-1 template sequences in vitro. The effect of NC on steady-state RT activity was dependent on the order of addition of reaction components. Addition of NC prior to formation of RT-primer.template-dNTP ternary complexes inhibited primer extension and reduced total product yields by slowing steady-state RT turnover. In contrast, addition of NC to preformed ternary complexes resulted in efficient primer extension and increased RT processivity at specific DNA template sites. NC stimulated polymerization (2-4 times) through eight of 13 sites examined in the cRRE region of HIV-1 env and increased the rate of polymerization through the D3/CTS region of HIV-1 pol 10 times. The data suggest that NC affects RT processivity by facilitating polymerization through regions of template secondary structure. Thus, NC functions as a single-strand binding (SSB)-like accessory replication factor for RT in vitro and may be part of a multicomponent retroviral replication complex.

Base Sequence↗

PubMed2004

Characterization of nonspecific protein-DNA interactions by 1H paramagnetic relaxation enhancement.

Nonspecific protein-DNA interactions play an important role in a variety of contexts related to DNA packaging, nucleoprotein complex formation, and gene regulation. Biophysical characterization of nonspecific protein-DNA interactions at the atomic level poses significant challenges owing to the dynamic nature of such complexes. Although NMR spectroscopy represents a powerful tool for the analysis of dynamic systems, conventional NMR techniques have provided little information on nonspecific protein-DNA interactions. We show that intermolecular (1)H paramagnetic relaxation enhancement (PRE) arising from Mn(2+) chelated to an EDTA-group covalently attached to a thymine base (dT-EDTA-Mn(2+)) in DNA provides a unique approach for probing the global dynamics and equilibrium distribution of nonspecific protein-DNA interactions. For nonspecific DNA binding, similar intermolecular (1)H-PRE profiles are observed on the (1)H resonances of the bound protein when dT-EDTA-Mn(2+) is located at either end of a DNA oligonucleotide duplex. We demonstrate the applicability of this approach to HMG-box proteins and contrast the results obtained for nonspecific DNA binding of the A-box of HMGB-1 (HMGB-1A) with sequence-specific DNA binding of the related SRY protein. Intermolecular (1)H-PRE data demonstrate unambiguously that HMGB-1A binds to multiple sites in multiple orientations even on a DNA fragment as short as 14 base pairs. Combining the (1)H-PRE data with the crystal structure of the HMGB-1 A-box/cisplatin-modified DNA complex allows one to obtain a semiquantitative estimate of the equilibrium populations at the various sites.

Amino Acid Sequence↗

PubMed1999

The 68 kDa DNA compacting nucleoid protein from soybean chloroplasts inhibits DNA synthesis in vitro.

Nucleoids were purified from chloroplasts of dividing soybean cells and their polypeptide composition analyzed by SDS-polyacrylamide gel electrophoresis. Of the 15-20 nucleoid-associated polypeptides, several demonstrated DNA binding activity. Upon disruption of the nucleoids with high concentrations of NaCl, a subset of these proteins and the majority of chloroplast DNA were recovered in the supernatant after centrifugation. Removal of the salt by dialysis resulted in formation of nucleoprotein complexes resembling genuine nucleoids. Purification of these structures revealed three major proteins of 68, 35 and 18 kDa. After purification of the 68 kDa protein to homogeneity, this protein was able to compact purified chloroplast DNA into a nucleoid-like structure in a protein concentration-dependent fashion. Addition of the 68 kDa protein to an in vitro chloroplast DNA replication system resulted in complete inhibition of nucleotide incorporation at concentrations above 300 ng of 68 kDa protein per microg of template DNA. These results led to in situ immunofluorescence studies of chloroplasts replicating DNA which suggested that newly synthesized DNA is not co-localized with nucleoids. Presumably, either the plastid replication machinery has means of removing nucleoid proteins prior to replication or the concentration of nucleoid proteins is tightly regulated and the proteins turned over in order to allow replication to proceed.

Cell Fractionation↗

PubMed2002

Telomeres, telomerase, and stability of the plant genome.

Telomeres, the complex nucleoprotein structures at the ends of linear eukaryotic chromosomes, along with telomerase, the enzyme that synthesizes telomeric DNA, are required to maintain a stable genome. Together, the enzyme and substrate perform this essential service by protecting chromosomes from exonucleolytic degradation and end-to-end fusions and by compensating for the inability of conventional DNA replication machinery to completely duplicate the ends of linear chromosomes. Telomeres are also important for chromosome organization within the nucleus, especially during mitosis and meiosis. The contributions of telomeres and telomerases to plant genome stability have been confirmed by analysis of Arabidopsis mutants that lack telomerase activity. These mutants have unstable genomes, but manage to survive up to ten generations with increasingly shortened telomeres and cytogenetic abnormalities. Comparisons between telomerase-deficient Arabidopsis and telomerase-deficient mice reveal distinct differences in the consequences of massive genome damage, probably reflecting the greater developmental and genomic plasticity of plants.

Genome, Plant↗

PubMed2004

Centromere DNA, proteins and kinetochore assembly in vertebrate cells.

The centromere is a specialized region of the chromosome that is essential for faithful chromosome segregation during mitosis and meiosis in eukaryotic cells. It is the site at which the kinetochore, the functional nucleoprotein complex responsible for microtubule binding and chromosome movement, is assembled through complex molecular mechanisms. Herein, I review recent advances in our understanding of centromeric DNAs as sites for kinetochore assembly and the mechanisms underlying kinetochore assembly in vertebrate cells.

Animals↗

PubMed1998

DNA bending determines Fos-Jun heterodimer orientation.

Heterodimeric transcription factors can bind to palindromic recognition elements in two opposite orientations with potentially distinct effects on transcriptional activity. We have determined the orientation of Fos-Jun binding at different AP-1 sites using a novel gel-based fluorescence resonance energy transfer assay. The orientation preference of heterodimer binding varied over a greater than 10-fold range. Single base pair substitutions that alter bending of flanking sequences reversed the orientation of heterodimer binding. Single amino acid substitutions that reduce the difference in DNA bending between Fos and Jun also reduced the orientation preference. Consequently, indirect read-out mediated by differences in DNA structure can contribute to the structural organization of nucleoprotein complexes.

Binding Sites↗

PubMed1988

Transcriptional autoregulation of the proto-oncogene fos.

Serum-induced transcription of the proto-oncogene fos is under negative feedback regulation mediated by the fos protein. The fos promoter region responsive to repression is also required for serum inducibility and binds a nucleoprotein complex in which the nuclear factor AP-1 is associated with fos protein.

Animals↗

PubMed2000

Telomere dysfunction promotes non-reciprocal translocations and epithelial cancers in mice.

Aged humans sustain a high rate of epithelial cancers such as carcinomas of the breast and colon, whereas mice carrying common tumour suppressor gene mutations typically develop soft tissue sarcomas and lymphomas. Among the many factors that may contribute to this species variance are differences in telomere length and regulation. Telomeres comprise the nucleoprotein complexes that cap the ends of eukaryotic chromosomes and are maintained by the reverse transcriptase, telomerase. In human cells, insufficient levels of telomerase lead to telomere attrition with cell division in culture and possibly with ageing and tumorigenesis in vivo. In contrast, critical reduction in telomere length is not observed in the mouse owing to promiscuous telomerase expression and long telomeres. Here we provide evidence that telomere attrition in ageing telomerase-deficient p53 mutant mice promotes the development of epithelial cancers by a process of fusion-bridge breakage that leads to the formation of complex non-reciprocal translocations--a classical cytogenetic feature of human carcinomas. Our data suggest a model in which telomere dysfunction brought about by continual epithelial renewal during life generates the massive ploidy changes associated with the development of epithelial cancers.

Adenocarcinoma↗

PubMed2000

Structure of the 30S ribosomal subunit.

Genetic information encoded in messenger RNA is translated into protein by the ribosome, which is a large nucleoprotein complex comprising two subunits, denoted 30S and 50S in bacteria. Here we report the crystal structure of the 30S subunit from Thermus thermophilus, refined to 3 A resolution. The final atomic model rationalizes over four decades of biochemical data on the ribosome, and provides a wealth of information about RNA and protein structure, protein-RNA interactions and ribosome assembly. It is also a structural basis for analysis of the functions of the 30S subunit, such as decoding, and for understanding the action of antibiotics. The structure will facilitate the interpretation in molecular terms of lower resolution structural data on several functional states of the ribosome from electron microscopy and crystallography.

Bacterial Proteins↗

PubMed1992

Identification of a Fab interaction footprint site on an icosahedral virus by cryoelectron microscopy and X-ray crystallography.

Biological processes frequently require the formation of multi-protein or nucleoprotein complexes. Some of these complexes have been produced in homogeneous form, crystallized, and analysed at high resolution by X-ray crystallography (for example, see refs 1-3). Most, however, are too large or too unstable to crystallize. Individual components of such complexes can often be purified and analysed by crystallography. Here we report how the coordinated application of cryoelectron microscopy, three-dimensional image reconstruction, and X-ray crystallography provides a powerful approach to study large, unstable macromolecular complexes. Three-dimensional reconstructions of native cowpea mosaic virus (CMPV) and a complex of CPMV saturated with a Fab fragment of a monoclonal antibody against the virus have been determined at 23 A resolution from low-irradiation images of unstained, frozen-hydrated samples. Despite the nominal resolution of the complex, the physical footprint of the Fab on the capsid surface and the orientation and position of the Fab have been determined to within a few ångstroms by fitting atomic models of CPMV4 and Fab (Kol)5 to reconstructed density maps.

Antibodies, Monoclonal↗

PubMed1995

Structural basis for DNA bending by the architectural transcription factor LEF-1.

Lymphoid enhancer-binding factor (LEF-1) and the closely related T-cell factor 1 (TCF-1) are sequence-specific and cell-type-specific DNA-binding proteins that play important regulatory roles in organogenesis and thymocyte differentiation. LEF-1 participates in regulation of the enhancer associated with the T cell receptor (TCR)-alpha gene by inducing a sharp bend in the DNA and facilitating interactions between Ets-1, PEBP2-alpha, and ATF/CREB, transcription factors bound at sites flanking the LEF-1 site. It seems that LEF-1 plays an architectural role in the assembly and function of this regulatory nucleoprotein complex. LEF-1 recognizes a specific nucleotide sequence through a high-mobility-group (HMG) domain. Proteins containing HMG domains bind DNA in the minor groove, bend the double helix, and recognize four-way junctions and other irregular DNA structures. Here we report the solution structure of a complex of the LEF-1 HMG domain and adjacent basic region with its cognate DNA. The structure reveals the HMG domain bound in the widened minor groove of a markedly distorted and bent double helix. The basic region binds across the narrowed major groove and contributes to DNA recognition.

Amino Acid Sequence↗