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Results for “nucleoprotein complex”

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PubMed2026

Molecular determinants of AR-enhancer interaction and cistrome reprogramming in prostate cancer progression.

The androgen receptor (AR) is a key transcription factor in prostate cancer (PCa), whose enhanced and altered functions are known drivers of cancer progression. A key aspect of this is reprogramming of the AR cistrome, which consists of genome-wide enhancer-binding sites through which AR regulates gene expression. The magnitude and biological impact of the AR cistrome are impacted by the AR itself, including the responses to ligand, as well as the organization of the associated DNA response elements, and availability of pioneer factors, cofactors, and noncoding RNAs, all of which contribute to a functional transcription complex. In this review, we will examine, in the context of PCa progression, the factors that affect the binding of AR and its interacting partners at enhancers, with a focus on AR cistrome reprogramming. We also discuss the clinical utility of targeting the AR-enhancer nucleoprotein complex and the potential of using the AR cistrome as a prognostic tool.

Humans↗

PubMed1975

Binding of D-alpha-tocopherol to rat liver nuclear components.

When D-alpha-tocopherol is administered i.v. to vitamin E deficient rats, significant amounts of this vitamin are bound to a nucleoprotein complex in hepatic nuclei, and this complex can be solubilized by high concentrations of sodium chloride (0.6 M). The bound vitamin in this complex, extractable by ethanol, was found to be identical with authentic alpha-tocopherol by thin layer chromatography.

Animals↗

PubMed1988

Electron spectroscopic imaging of DNA.

Electron spectroscopic imaging (ESI) was carried out using a fixed beam electron microscope equipped with a parallel electron energy filter to form micrographs of purified plasmid DNA without the use of heavy metal stains and shadows. Inelastically scattered electrons that have ionized the phosphorus LII,III shell electrons were used to form phosphorus distribution maps of DNA and deoxyribonucleoprotein complexes. Signal-to-noise values of the net phosphorus content over single DNA molecules compared to two and four interwound DNA strands directly reflect the known stoichiometric levels of phosphorus content, illustrating that ESI can be used to determine the relative levels of nucleic acid in nucleoprotein complexes. An initial attempt to characterize nucleosomal and transcriptionally active chromatin from Saccharomyces cerevisiae with this technique reveals three distinct ultrastructural classes of the basic chromatin fiber.

DNA↗

PubMed1991

Gene expression and chromatin structure in the cellular slime mold, Dictyostelium discoideum.

Micrococcal nuclease digestion of chromatin from growing cells reveals a structural organization which differs for genes transcribed at diverse rates. The late cAMP dependent prespore genes which are not transcribed in growing cells are found in growing cells in a regular nucleosomal repeat with an average spacing of 168 nucleotides. By contrast genes expressed at a low level in growing cells show an irregular pattern of bands with an average distance between bands of 80 nucleotides. The sizes of the bands generated from the transcribed genes are consistent with the concept that transcription results in the loss of the linker region histone H1 with concomitant sliding of nucleosomes to generate close packed ("slipped") di, tri, and tetra nucleosomes lacking the linker region. Further analysis of dinucleosomes released by micrococcal nuclease digestion reveals that transcriptionally active genes are found associated with dinucleosomes species which may be lacking histone H1. The length of DNA protected by these dinucleosomes is heterogeneous, ranging from 250 to 300 nucleotides. Methodology is described which has been adapted to allow two dimensional hybridization mapping of nucleoprotein complexes on single copy Dictyostelium genes.

Chromatin↗

PubMed1993

Integration of murine leukemia virus DNA depends on mitosis.

In synchronized rat or mouse cells infected with Moloney murine leukemia virus (MLV), integration of viral DNA and production of viral proteins occur only after the cells traverse mitosis. Integration is blocked when cells are prevented from progressing through mitosis. Viral nucleoprotein complexes isolated from arrested cells contain full-length viral DNA and can integrate this viral DNA in vitro, showing that the block to integration in arrested cells is not due to a lack of mature integration machinery. When infected cells traverse mitosis, there is a sharp increase in nuclear accumulation of viral DNA. The dependence of integration on mitosis may therefore be due to a requirement for mitosis and nuclear envelope breakdown for entry of the viral integration complex into the nucleus.

Animals↗

PubMed1999

DNA structure and transcription.

Regulation of transcription occurs through complex interactions of RNA polymerase and accessory proteins with specific DNA sequences and with each other. The DNA template topology influences the interaction of RNA polymerase with the promoter and its response to repressors, activators and the intracellular milieu through the formation of altered DNA structures or of nucleoprotein complexes. Recent developments on the role of DNA structures in transcription regulation are discussed.

DNA↗

PubMed2006

Intracytoplasmic maturation of the human immunodeficiency virus type 1 reverse transcription complexes determines their capacity to integrate into chromatin.

BACKGROUND: The early events of the HIV-1 life cycle include entry of the viral core into target cell, assembly of the reverse transcription complex (RTCs) performing reverse transcription, its transformation into integration-competent complexes called pre-integration complexes (PICs), trafficking of complexes into the nucleus, and finally integration of the viral DNA into chromatin. Molecular details and temporal organization of these processes remain among the least investigated and most controversial problems in the biology of HIV. RESULTS: To quantitatively evaluate maturation and nuclear translocation of the HIV-1 RTCs, nucleoprotein complexes isolated from the nucleus (nRTC) and cytoplasm (cRTC) of HeLa cells infected with MLV Env-pseudotyped HIV-1 were analyzed by real-time PCR. While most complexes completed reverse transcription in the cytoplasm, some got into the nucleus before completing DNA synthesis. The HIV-specific RNA complexes could get into the nucleus when reverse transcription was blocked by reverse transcriptase inhibitor, although nuclear import of RNA complexes was less efficient than of DNA-containing RTCs. Analysis of the RTC nuclear import in synchronized cells infected in the G2/M phase of the cell cycle showed enrichment in the nuclei of RTCs containing incomplete HIV-1 DNA compared to non-synchronized cells, where RTCs with complete reverse transcripts prevailed. Immunoprecipitation assays identified viral proteins IN, Vpr, MA, and cellular Ini1 and PML associated with both cRTCs and nRTCs, whereas CA was detected only in cRTCs and RT was diminished in nRTCs. Cytoplasmic maturation of the complexes was associated with increased immunoreactivity with anti-Vpr and anti-IN antibodies, and decreased reactivity with antibodies to RT. Both cRTCs and nRTCs carried out endogenous reverse transcription reaction in vitro. In contrast to cRTCs, in vitro completion of reverse transcription in nRTCs did not increase their integration into chromatin. CONCLUSION: These results suggest that RTC maturation occurs predominantly in the cytoplasm. Immature RTCs containing RT and incomplete DNA can translocate into the nucleus during mitosis and complete reverse transcription, but are defective for integration.

Cell Division↗

PubMed1995

Binding of the Escherichia coli UvrAB proteins to the DNA mono- and diadducts of cis-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatinum(II ) and cisplatin. Analysis of the factors controlling recognition and proof of monoadduct-mediated UvrB-DNA cross-linking.

The interactions of the Escherichia coli endonuclease UvrAB proteins with the DNA mono- and diadducts of both the cis-racemic exo-[N-2-amino-N-2-methylamino-2,2,1-bicycloheptane]dichloroplatin um(II) (complex 1) and cisplatin (cis-diamminedichloroplatinum(II) (cis-DDP)), have been studied. Complex 1 reacts faster with DNA than cis-DDP and gives monoadducts with a longer lifetime (8 h 20 min chelation t 1/2 compared with 2 h 40 min for cis-DDP). Using pSP65 plasmid [3H]DNA, the filter binding assay was associated with the analysis of the nucleoprotein complexes to characterize the UvrAB recognition of the platinum adducts and to demonstrate the occurrence of platinum-mediated DNA-protein cross-linking. First, it is shown that the UvrAB proteins recognize the complex 1 mono- and diadducts with a higher affinity than those of cis-DDP. Fifteen times more cis-DDP adducts per plasmid are required than complex 1 adducts, to lead to similar UvrAB binding. However, the UvrAB proteins recognize monoadducts and diadducts of each complex with a similar affinity. Second, it is shown that UvrB is the protein involved in the nucleo-protein complexes formed from mono- and diadducts of complex 1 and cis-DDP. This protein is also partly cross-linked to DNA with a similar efficiency by monoadducts derived from complex 1 and cis-DDP. However, as UvrB has a greater affinity for the DNA adducts of complex 1 than for those of cis-DDP, more UvrB-platinum-DNA cross-links are formed with complex 1 than with cis-DDP. This study, using a bacterial repair system as a model, points to a possible strategy for making new cytotoxic platinum complexes for mammalian cells.

Adenosine Triphosphatases↗

PubMed1997

A three-protein-DNA complex on a B cell-specific domain of the immunoglobulin mu heavy chain gene enhancer.

The lymphoid-specific immunoglobulin mu heavy chain gene intron enhancer (muE) contains multiple binding sites for trans-acting nuclear factors. We have used a combination of in vitro and in vivo assays to reconstruct protein-DNA interactions on a minimal B cell-specific mu enhancer that contains three motifs, muA, muB, and muE3. Using ETS-domain proteins that transactivate the minimal enhancer in non-lymphoid cells, we show that (i) PU.1 binds coordinately to both muA and muB sites in vitro and (ii) in the presence of Ets-1, this factor binds to the muA site and PU.1 to the muB site. Two factors, TFE3 and USF, bind to the muE3 element. When the ETS proteins are present together with muE3 binding proteins, a three-protein-DNA complex is generated. Furthermore, we provide evidence for protein-protein interactions between Ets-1 and PU.1 proteins that bind to muA and muB sites, and between Ets-1 and TFE3 bound to the muA and mu3 sites. We propose that this domain of the mu enhancer is assembled into a nucleoprotein complex that contains two tissue-restricted ETS domain proteins that recognize DNA from the same side of the helix and one ubiquitously expressed bHLH-leucine zipper protein that binds between them, recognizing its site from a different side of the helix.

3T3 Cells↗

PubMed1993

Photocrosslinking of 5-iodouracil-substituted RNA and DNA to proteins.

5-Iodouracil-substituted RNA and DNA were crosslinked regiospecifically to associated proteins in yields of 70 to 94% of bound nucleic acid. Irradiation of the iodouracil chromophore with monochromatic, long-wavelength ultraviolet radiation (325 nanometers) eliminates excitation of other nucleic acid and protein chromophores. The combination of high crosslinking yields, excellent specificity, and elimination of photodamage to other chromophores represents an important advance toward the precise identification of contacts in nucleoprotein complexes.

Animals↗

PubMed1981

[Chromatin structure with no nucleosomes: dissociated SV40 virus].

The mild dissociation of SV40 virus particles at pH 7.5 yields a nucleoprotein complex containing all the capsid proteins and histones of the intact virion. Electron microscopic observations show a bead-on-a-string structure comprising about 70 particles 7 nm in diameter along the viral DNA. No particle of nucleosome size was visible. The compaction of DNA in this complex is lower than 2 and is likely to be close to unity.

Animals↗

PubMed1999

Global structure similarities of intact and nicked DNA complexed with IHF measured in solution by fluorescence resonance energy transfer.

We have analyzed the structure of two related protein-DNA complexes consisting of integration host factor (IHF) bound to two different versions of the H' site of bacteriophage lambda. Both DNA substrates were 55 bp in length. While one was native duplex the other possessed a nick in one strand at a crucial position within the IHF consensus at the same position as in the reported crystal structure of the DNA-IHF complex. By labeling the 5'-ends of these DNA molecules with donor and acceptor fluorescent dyes, we were able to measure the distance between the dyes by fluorescence resonance energy transfer (FRET) and model DNA distortion. The FRET efficiency decreased from 0.49 +/- 0.01 (nicked DNA) to 0.37 +/- 0.01 (intact DNA) when the gap in the DNA strand was closed. The measured dye-to-dye distance of IHF in complex with nicked DNA was in agreement with the expected value from the crystal structure. Although we found that the two structures were distinguishable, the global shape induced by IHF was retained between the two DNA molecules. Furthermore, our FRET and modeling techniques have sufficiently high resolution to distinguish subtle changes in nucleoprotein complexes with biological relevance.

Bacterial Proteins↗

PubMed1994

Sex-reversing mutations affect the architecture of SRY-DNA complexes.

The testis determining factor, SRY, is a DNA binding protein that causes a large distortion of its DNA target sites. We have analysed the biochemical properties of the DNA binding domains (HMG-boxes) of mutant SRY proteins from five patients with complete gonadal dysgenesis. The mutant proteins fall into three categories: two bind and bend DNA almost normally, two bind inefficiently but bend DNA normally and one binds DNA with almost normal affinity but produces a different angle. The mutations with moderate effect on complex formation can be transmitted to male progeny, the ones with severe effects on either binding or bending are de novo. The angle induced by SRY depends on the exact DNA sequence and thus adds another level of discrimination in target site recognition. These data suggest that the exact spatial arrangement of the nucleoprotein complex organized by SRY is essential for sex determination.

Base Sequence↗

PubMed1989

Signals at the bacteriophage phi 29 DNA replication origins required for protein p6 binding and activity.

Protein p6 of Bacillus subtilis phage phi 29 binds specifically to the ends of the viral DNA that contain the replication origins, giving rise to a nucleoprotein structure. DNA regions recognized by protein p6 have been mapped by deletion analysis and DNase I footprinting. Main protein p6-recognition signals have been located between nucleotides 62 and 125 at the right phi 29 DNA end and between nucleotides 46 and 68 at the left end. In addition, recognition signals are also present at other sites within 200-300 bp at each phi 29 DNA end. Protein p6 does not seem to recognize a specific sequence in the DNA, but rather a structural feature, which could be bendability. The formation of the protein p6-DNA nucleoprotein complex is likely to be the structural basis for the protein p6 activity in the initiation of replication.

Bacteriophages↗

PubMed1999

Xer site-specific recombination. DNA strand rejoining by recombinase XerC.

Xer site-specific recombination functions in the stable maintenance of circular replicons in Escherichia coli. Each of two related recombinase proteins, XerC and XerD, cleaves a specific pair of DNA strands, exchanges them, and rejoins them to the partner DNA molecule during a complete recombination reaction. The rejoining activity of recombinase XerC has been analyzed using isolated covalent XerC-DNA complexes resulting from DNA cleavage reactions upon Holliday junction substrates. These covalent protein-DNA complexes are competent in the rejoining reaction, demonstrating that covalently bound XerC can catalyze strand rejoining in the absence of other proteins. This contrasts with a recombinase-mediated cleavage reaction, which requires the presence of both recombinases, the recombinase mediating catalysis at any given time requiring activation by the partner recombinase. In a recombining nucleoprotein complex, both cleavage and rejoining can occur prior to dissociation of the complex.

Base Sequence↗

PubMed1992

Protein complexes bearing myc-like antigenicity recognize two distinct DNA sequences.

The products of the myc proto-oncogene family have been suggested to carry functions important for cell proliferation, differentiation and neoplasia, but the molecular mechanisms of such biological effects have not yet been clarified. We have previously reported that the c-myc protein or protein(s) complexed with the c-myc protein bind to a specific sequence in the region upstream of the c-myc gene, where there exist an origin of cellular DNA replication (ori) and also a transcriptional enhancer. It was recently reported that the c-myc protein forms complexes with a novel helix-loop-helix leucine zipper protein (Max), and that the Myc-Max complex specifically recognizes a DNA sequence different from the sequence we have defined in the c-myc gene. In this report we examine the nuclear extract prepared from human Raji cells for binding to the two different sequences which have been reported as specific binding sequences for c-myc protein or c-myc protein complexes. The binding to one sequence was inhibited in the presence of excess amount of the other sequence, suggesting that the same protein(s) may be necessary for binding to either of the two sequences. Since binding to both sequences was cancelled by pretreatment of the Raji extract with an anti-c-myc protein antibody, it is suggested that proteins carrying myc-like antigenicity are necessary for efficient binding of the nucleoprotein complexes to the two distinct DNA sequences.

Base Sequence↗

PubMed1995

Functional structure of the promoter region of the human thymidylate synthase gene and nuclear factors that regulate the expression of the gene.

To identify the essential motifs of the promoter of the human thymidylate synthase (hTS) gene, we constructed a set of deletion mutants that covers the region from -441 to +28 of the hTS gene. (The nucleotide positions are numbered from the first position of the initiation codon of the hTS gene.) From the results of chloramphenicol acetyltransferase (CAT) assay of these mutants, two positive elements for the promoter activity were identified: one contains CACCC box (CCACACCC) that is found in the SV40 enhancer motif and the other contains the sequence that is homologous to the Sp1 binding site in the mouse TS gene. Furthermore, two negative regulatory sequences were identified between the two positive elements and upstream from the CACCC box. Cassette mutations were introduced into these motifs and the function of the motifs was confirmed. From the results of gel mobility shift analysis, we found that three nucleoprotein complexes were formed in the promoter region of the hTS gene. The formation of one of the complexes was competed by the DNA fragment bearing the GC box. The gel mobility shift analyses using the DNA fragments with cassette mutations revealed that the complex was formed on the Sp1 binding site of the hTS gene and the formation of the complex correlated with the promoter activity of the fragments with cassette mutations measured by the CAT assay.

Animals↗

PubMed1995

Electron microscopic visualization of RecT protein and its complexes with DNA.

Electron microscopy has been used to examine Escherichia coli RecT protein alone and in the complexes it forms with DNA substrates, with which it catalyzes strand exchange in vitro. Negative staining has revealed that the 33 kDa RecT protein monomers form open C-shaped and closed O-shaped particles. RecT protein monomers assemble into donut-shaped oligomers containing seven or eight protein monomers and rod-like structures. When bound to single-stranded DNA, RecT forms highly twisted nucleoprotein filaments that are 18 nm in diameter and have a helical pitch of 10 nm. When added to linear duplex DNA in the presence of active RecE protein (exonuclease VIII), filamentous nucleoprotein complexes are formed on the DNA ends and the DNA molecules are frequently cyclized through protein-protein interactions.

Bacterial Proteins↗