The human DNA ends proteome uncovers an unexpected entanglement of functional pathways.

DNA ends get exposed in cells upon either normal or dysfunctional cellular processes or molecular events. Telomeres need to be protected by the shelterin complex to avoid junctions occurring between chromosomes while failing topoisomerases or clustered DNA damage processing may produce double-strand breaks, thus requiring swift repair to avoid cell death. The rigorous study of the great many proteins involved in the maintenance of DNA integrity is a challenging task because of the innumerous unspecific electrostatic and/or hydrophobic DNA-protein interactions that arise due to the chemical nature of DNA.

Monitoring DNA triplex formation using multicolor fluorescence and application to insulin-like growth factor I promoter downregulation.

Inhibition of insulin-like growth factor I (IGF-I) signaling is a promising antitumor strategy and nucleic acid-based approaches have been investigated to target genes in the pathway. Here, we sought to modulate IGF-I transcriptional activity using triple helix formation. The IGF-I P1 promoter contains a purine/pyrimidine (R/Y) sequence that is pivotal for transcription as determined by deletion analysis and can be targeted with a triplex-forming oligonucleotide (TFO).

Differential RNA-binding activity of the hnRNP G protein correlated with the sex genotype in the amphibian oocyte.

A proteomic approach has enabled the identification of an orthologue of the splicing factor hnRNP G in the amphibians Xenopus tropicalis, Ambystoma mexicanum, Notophthalmus viridescens and Pleurodeles walt, which shows a specific RNA-binding affinity similar to that of the human hnRN G protein. Three isoforms of this protein with a differential binding affinity for a specific RNA probe were identified in the P. walt oocyte.

Proteomic study of the effects of microcystin-LR on organelle and membrane proteins in medaka fish liver.

The microcystin-leucine-arginine toxin (MC-LR) is produced by cyanobacteria that sometimes bloom in water reservoirs. It targets the liver, thus posing potential health risks to human and animals. Microcystin inhibits the protein phosphatases PP1 and PP2A, leading to diverse cellular deregulation processes.

Global quantitative analysis of protein phosphorylation status in fish exposed to microcystin.

The hepatotoxins, microcystins (MCs) are potent inhibitors of protein phosphatases PP1 and PP2A. These nonribosomal peptides are getting more and more attention because of their acute toxicity and potent tumor-promoting activity. These toxins are produced by freshwater cyanobacteria.

Proteomic and phosphoproteomic analysis of cellular responses in medaka fish (Oryzias latipes) following oral gavage with microcystin-LR.

Chronic and subchronic toxicity resulting from exposure to microcystins (MCs) receives increasing attention due to the risk of bioaccumulation of these toxins by aquatic animals, including fish. The mechanisms of action of MCs that target the liver, involve modifications of protein phosphorylation resulting from phosphatases 1 and 2A inhibition. Therefore, studying phosphoprotein modifications by using a specific phosphoprotein stain Pro-Q Diamond in fish liver contaminated with MC-leucine-arginine (MC-LR), the most toxic MC, should help dissecting disturbed signaling and metabolic networks.

Modern tools for identification of nucleic acid-binding proteins.

Numerous biological mechanisms depend on nucleic acid--protein interactions. The first step to the understanding of these mechanisms is to identify interacting molecules. Knowing one partner, the identification of other associated molecular species can be carried out using affinity-based purification procedures.

Global quantitative analysis of protein expression and phosphorylation status in the liver of the medaka fish (Oryzias latipes) exposed to microcystin-LR I. Balneation study.

Microcystins (MCs) are hepatotoxins with potent inhibitor activity of protein phosphatases PP1 and PP2A. These non-ribosomal peptides are getting more and more attention due to their acute toxicity and potent tumor-promoting activity. These toxins are produced by freshwater cyanobacteria.

Analytical biochemistry of DNA--protein assemblies from crude cell extracts.

Purification of specific DNA-protein complexes is a challenging task, as the involved interactions can be both electrostatic/H-bond and hydrophobic. The chromatographic stringency needed to obtain reasonable purifications uses salts and detergents. However, these components elicit the removal of proteins unspecifically bound to the chromatographic support itself, thus contaminating the purification products.

An in vitro enzymatic assay coupled to proteomics analysis reveals a new DNA processing activity for Ewing sarcoma and TAF(II)68 proteins.

Based on structural and functional similarities, translocated in liposarcoma/fusion (TLS/FUS) protein, Ewing sarcoma (EWS) protein and human TATA binding protein-associated factor (hTAF(II)68) have been grouped in the TLS-EWS-TAF(II)68 (TET) protein family. Translocations involving their genes lead to sarcomas. Polypyrimidine tract-binding protein-associated splicing factor (PSF), although not grouped in this family, presents structural and functional similarities with TET proteins and is involved in translocation leading to carcinoma.

Psoralen interstrand cross-link repair is specifically altered by an adjacent triple-stranded structure.

Targeting DNA-damaging agents to specific DNA sites by using sequence-specific DNA ligands has been successful in directing genomic modifications. The understanding of repair processing of such targeted damage and the influence of the adjacent complex is largely unknown. In this way, directed interstrand cross-links (ICLs) have already been generated by psoralen targeting.

Contributions of mass spectrometry in the study of nucleic acid-binding proteins and of nucleic acid-protein interactions.

Nucleic-acid-protein (NA-P) interactions play essential roles in a variety of biological processes-gene expression regulation, DNA repair, chromatin structure regulation, transcription regulation, RNA processing, and translation-to cite only a few. Such biological processes involve a broad spectrum of NA-P interactions as well as protein-protein (P-P) interactions. These interactions are dynamic, in terms of the chemical composition of the complexes involved and in terms of their mere existence, which may be restricted to a given cell-cycle phase.

A reliable and simple method for two-dimensional electrophoresis and identification of HeLa nuclear alkaline nucleic acid-binding proteins using immobilized pH gradient.

Alkaline proteins were separated by two-dimensional electrophoresis, using isoelectric focusing in commercial pH 6-11 immobilized pH gradients (IPG), in order to identify nucleic acid-binding proteins by South- or Northwestern blotting. The corresponding spots were chosen according to their DNA or RNA binding properties, excised, and submitted to a simplified tryptic digestion and peptide extraction protocols. Matrix assisted laser desorption/lonization-time of flight (MALDI-TOF)-mass spectrometry was used to identify 36 out of 39 excised protein spots.

Selection and identification of proteins bound to DNA triple-helical structures by combination of 2D-electrophoresis and MALDI-TOF mass spectrometry.

Identification of proteins binding specifically to peculiar nucleic acid structures can lead to comprehension of their role in vivo and contribute to the discovery of structure-related gene regulation. This work was devoted to establishing a reliable procedure to select proteins on the basis of their interaction with a nucleic acid probe chosen to fold into a given structure. 2D-electrophoresis and mass spectrometry were combined for protein identification.

Triple helix formation and the antigene strategy for sequence-specific control of gene expression.

Specific gene expression involves the binding of natural ligands to the DNA base pairs. Among the compounds rationally designed for artificial regulation of gene expression, oligonucleotides can bind with a high specificity of recognition to the major groove of double helical DNA by forming Hoogsteen type bonds with purine bases of the Watson-Crick base pairs, resulting in triple helix formation. Although the potential target sequences were originally restricted to polypurine-polypyrimidine sequences, considerable efforts were devoted to the extension of the repertoire by rational conception of appropriate derivatives.

Psoralen adducts induced by triplex-forming oligonucleotides are refractory to repair in HeLa cells.

The use of triple helix-forming oligonucleotides constitutes an attractive strategy to regulate gene expression by inhibition of transcription. Psoralen-oligonucleotide conjugates form, upon irradiation, covalent triplexes and thereby modify the specific target sequence. The processing of such photoproducts on the promoter of the gene coding for the interleukin-2 receptor alpha chain was investigated in HeLa cells and HeLa nuclear extracts.

Identification of a triplex DNA-binding protein from human cells.

Intramolecular or intermolecular triple helices could be recognized by specific proteins that stabilize triplex structures and might play a role in gene regulation. In order to identify such proteins, we designed a 55 nucleotide-long DNA oligomer that could fold on itself to form an intramolecular triple helix of the Py Pu x Py motif. The stability of this triplex under physiological conditions was demonstrated by gel retardation and thermal denaturation experiments.

Sequence-specific control of gene expression by antigene and clamp oligonucleotides.

Control of gene expression at the transcriptional level can be achieved with triplex-forming oligonucleotides provided that the target sequence is accessible within the chromatin structure of cell nuclei. Using oligonucleotide-psoralen conjugates as probes we have shown that the promoter region of the gene encoding the alpha subunit of the interleukin 2 receptor and the polypurine tract of integrated HIV provirus can form sequence-specific, triple-helical complexes in cell cultures. Oligonucleotide-intercalator conjugates can inhibit transcription initiation by competing with transcription factor binding.

Peptide nucleic acids directed to the promoter of the alpha-chain of the interleukin-2 receptor.

Two 10-mer oligopyrimidine peptide nucleic acids (PNAs) were designed to interfere with IL-2R alpha promoter expression by binding to the regulatory sequences overlapping SRF and NF-kappa B transcription factor sites. Specific complexes were formed on each target sequence, and clearly involved (1) Hoogsteen hydrogen bonds as shown by experiments in which the purine strand of a single or double-stranded target was substituted with 7-deazadeoxyguanosine, (2) P-loop formation on double-helical DNA as evidenced by susceptibility to a single-strand-specific nuclease. When formed on a single-stranded DNA target, these highly stable complexes were responsible for efficient physical blockage of T7 DNA polymerase elongation on the template DNA containing the target oligopurine sequence.

Detection of covalent triplex within human cells.

Triple helix-forming oligonucleotides covalently linked to psoralen can be specifically cross-linked to both strands of DNA at the triplex-duplex junction following UV irradiation. We have previously shown that a 15mer psoralen-oligonucleotide conjugate forming a triple helix on the promoter of the alpha subunit gene of the interleukin-2 receptor inhibits transcription of reporter plasmids transfected into living cells after irradiation. In the present work, we directly demonstrate covalent triple helix formation at the target site inside cells.

Inhibition of replication initiation by triple helix-forming oligonucleotides.

Oligonucleotide-directed triple helix formation constitutes a new approach to block gene expression via transcription inhibition. In addition triple helices might inhibit replication. We have examined the capacity of triple helix-forming oligonucleotides to inhibit the initiation of replication on a single-stranded DNA template using T7 DNA polymerase (Sequenase).

Triplex formation with alpha anomers of purine-rich and pyrimidine-rich oligodeoxynucleotides.

Nuclease-resistant alpha anomers of pyrimidine-rich CT- and purine-rich GA- and GT-containing oligonucleotides were investigated for their triplex-forming potential and compared with their corresponding nuclease-sensitive beta anomers. Both 23mer CT-alpha and 23mer CT-beta had quite similar triplex binding affinities. Synthetic 23mer GT-alpha oligonucleotides were capable of triplex formation with binding affinities slightly lower than corresponding 23mer GT-beta oligonucleotides.

Oligonucleotide-directed switching of DNA polymerases to a dead-end track.

During DNA replication, the presence of oligonucleotides with partial homology to the template strand was shown to induce a switch of the polymerase from the normal template to the oligonucleotide. The latter acted as a dead-end template and led to abortive replication. The only prerequisite was that the oligonucleotide could form 7-9 base pairs with the newly synthesized DNA strand in order to switch templates.

Inhibition of gene expression by triple helix-directed DNA cross-linking at specific sites.

Synthetic oligodeoxynucleotides represent promising tools for gene inhibition in live systems. Triple helix-forming oligonucleotides, which bind to double-stranded DNA, are of special interest since they are targeted to the gene itself rather than to its mRNA product, as in the antisense strategy. Triple helix-forming oligonucleotides can be coupled to DNA-modifying agents and used to introduce modifications in the DNA target in a highly sequence-specific manner.

Unexpected effect of an anti-human immunodeficiency virus intermolecular triplex-forming oligonucleotide in an in vitro transcription system due to RNase H-induced cleavage of the RNA transcript.

A 16-mer oligodeoxynucleotide (ODN) which specifically recognizes the polypurine tract (PPT) located upstream of the 3' long terminal repeat (LTR) of human immunodeficiency virus (HIV) proviral DNA via triplex formation is shown to have a dramatic effect on in vitro transcription from the HIV-LTR promoter. In the presence of HeLa cell extracts, a shorter RNA transcript is obtained in the presence of the 16-mer ODN. This truncated RNA lacks about 200 nucleotides from its 3' region.