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. We devised a technique that discriminates the proteins recruited specifically at DNA ends from those that bind to DNA because of a generic affinity for the double helix. Our study shows that the DNA ends proteome comprises proteins of an unexpectedly wide functional spectrum, ranging from DNA repair to ribosome biogenesis and cytoskeleton, including novel proteins of undocumented function. A global mapping of the identified proteome on published DNA repair protein networks demonstrated the excellent specificity and functional coverage of our purification technique. Finally, the native nucleoproteic complexes that assembled specifically onto DNA ends were shown to be endowed with a highly efficient DNA repair activity.

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 ). We designed modified purine‐ and pyrimidine‐rich TFO s to bind to the R/Y sequence. To monitor TFO binding, we developed a fluorescence‐based gel‐retardation assay that allowed independent detection of each strand in three‐stranded complexes using end‐labeling with Alexa 488, cyanine (Cy)3 and Cy5 fluorochromes. We characterized TFO s for their ability to inhibit restriction enzyme activity, compete with DNA ‐binding proteins and inhibit IGF ‐I transcription in reporter assays. TFO s containing modified nucleobases, 5‐methyl‐2′‐deoxycytidine and 5‐propynyl‐2′‐deoxyuridine, specifically inhibited restriction enzyme cleavage and formed triplexes on the P1 promoter fragment. In cells, deletion of the R/Y‐rich sequence led to 48% transcriptional inhibition of a reporter gene. Transfection with TFO s inhibited reporter gene activity to a similar extent, whereas transcription from a mutant construct with an interrupted R/Y region was unaffected, strongly suggesting the involvement of triplex formation in the inhibitory mechanisms. Our results indicate that nuclease‐resistant TFO s will likely inhibit endogenous IGF ‐I gene function in cells.

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. In situ hybridization to lampbrush chromosomes of P. waltl revealed the presence of a family of hnRNP G genes, which were mapped on the Z and W chromosomes and one autosome. This indicates that the isoforms identified in this study are possibly encoded by a gene family linked to the evolution of sex chromosomes similarly to the hnRNP G/RBMX gene family in mammals.

Proteomics aims to understand gene function and molecular processes of the living cell through a large-scale study of the expressed proteins. Although proteomics approach is largely applied on experimental models such as rodents, other animal models such as medaka fish (Oryzias latipes) also attracts considerable interest in proteomics field. Medaka is one of the most studied animal model in r eproductive or developmental biology. Although, its genome has been sequenced, only a few proteins are available in the databases. We pr esent the procedure used in one experiment as a model of identificatio n of proteins, in this case from liver of medaka treated by a hepatotoxic cyanotoxin, the microcystin. Because O. latipes is not listed in mascot search engine, the identification of proteins (selected because modified by the treatment) was done on closed related species. First, 15 spots were analyzed using peptide mass fingerprinting (PMF). However none could be reliably identified using mascot engine. The reason for low sequence coverage is due to the fact that the outcome of the research depends on the completness of the protein database obtained from NCBInr and the availability of cross-species for protein identification. In order to improve the identification, the PMF search was combined with a search in a medaka nucleotide specific database and confirmed by MS/MS ion searches which revealed successful. This identification procedure has been successfully applied in different experiments with the medaka model.

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. A proteomic approach was applied to the medaka fish (Oryzias latipes) to obtain an overview of the effects of MC-LR on the liver. As membrane and organelle proteins are major structural and functional components of several cell signalling pathways, we decided to investigate here the membrane and organelle-enriched fractions from the livers of control and MC-LR treated medaka fish. Seventeen proteins were identified by proteomic analysis as being modulated in response to MC-LR treatment. This is the first time for eight of them to be reported as being involved in MC-LR effects: prohibitin, fumarylacetoacetase, protein disulfide isomerase A4 and A6, glucose regulated protein 78kDa, 40S ribosomal protein SA, cytochrome b5, and ATP synthase mitochondrial d subunit. These proteins are involved in protein maturation or in the response to oxidative stress highlighting the role of organelles in protein processing and the complex cooperation associated with oxidative stress.

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. When the nucleic acid-binding protein is known, the nucleic acid can be isolated and identified by sensitive techniques such as polymerase chain reaction followed by DNA sequencing or hybridization on chips. The reverse identification procedure is less straightforward in part because interesting nucleic acid-binding proteins are generally of low abundance and there are no methods to amplify amino acid sequences. In this article, we will review the strategies that have been developed to identify nucleic acid-binding proteins. We will focus on methods permitting the identification of these proteins without a priori knowledge of protein candidates.

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. Herein, we report a toxicological study conducted on aquatic animal models such as the medaka fish. To date, the detailed mechanisms underlying the toxicity of microcystins are unknown. MC-leucine-arginine (MC-LR) is the most toxic and the most commonly encountered variant of MCs in aquatic environment. It has been used for toxicological investigations on the liver of intoxicated medaka. We performed differential proteome analyses of MC-LR-treated and untreated medaka fish to investigate the mechanisms of establishment of early responses to the toxin. The identification of proteins involved in these early responses might constitute candidates of biomarkers of MC-LR exposure. Cytosolic proteins from livers of exposed or nonexposed medaka were resolved by 2D electrophoresis and detected using stains specific for phosphoproteins and for whole protein content. Overall, 15 spots were found to vary significantly on the proteomic 2D maps or on the phosphoproteomic 2D maps. Of these 15 proteins, only two could not be identified by mass spectrometry. Among the other proteins that were identified, phenylalanine hydroxylase and keratin 18 (type I) showed variations in phoshoryl content in agreement with inhibition of PP2A activity after exposure of the fish to MC-LR. The other identified proteins exhibited variations in their expression level. The identified proteins appear to be involved in cytoskeleton assembly, cell signalling, oxidative stress, and apoptosis. The functional implications of responses to MC-LR exposure of these proteins are discussed. The methodology described in this report should be widely used to a number of tissues and organisms, thus helping in the search for biomarkers of MC-LR contamination.

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. In this work, a photocleavable linker connected the target oligonucleotidic sequence to the chromatographic beads so as to allow the irradiation-based release of the purified DNA–protein complexes off the beads. Our bioanalytical conditions were validated by purifying the tetracycline repressor protein onto a specific oligonucleotide. The purification factor was unprecedented, with a single contaminant. The robustness of our method was challenged by applying it to the purification of multiprotein assemblies forming onto DNA damage-mimicking oligonucleotides. The purified components were identified as well-known DNA repair proteins, and were shown to retain their enzymatic activities, as seen by monitoring DNA ligation products. Remarkably, kinase activities, also monitored, were found to be distinct on the beads and on the purified DNA–protein complexes, showing the benefits to uncouple the DNA–protein assemblies from the beads for a proper understanding of biochemical regulatory mechanisms involved in the DNA–protein assemblies.

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. The most toxic and most commonly encountered variant in aquatic environment is MC-LR (MC Leucine-Arginine). It has been used for toxicological investigations on the liver of intoxicated medaka. Differential proteome as well as differential phosphoproteome analyses have been performed for providing new information on early responses to the toxin. The experiments are also aiming at selecting biomarkers of MC-LR exposure. In the 2D electrophoresis gel protein maps from cytosol of liver cells of animals exposed or non-exposed to the cyanotoxin, 15 spots showed a significant increase or decrease of their stain signal either in specific phosphoprotein stain or total protein stain. Thirteen of these proteins have been identified by mass spectrometry. Among them, phenylalanine hydroxylase (PAH) and keratin 18 type I showed variations in phosphorylation stain in possible agreement with inhibition of PP2A activity. The other identified proteins exhibited variations in their expression level. The identified proteins appear to be involved in cytoskeleton assembly, cell signalling, oxidative stress and apoptosis. Such results confirm that proteomics and phosphoproteomics approaches may become valuable tools to identify signalling pathways implied in MC-LR effects. From accumulated data, specific pools of biomarkers could possibly be selected as specific for toxin exposure.

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. Beside their role in RNA metabolism, the precise cellular functions of these multifunctional proteins are not yet fully elucidated. We previously showed that both TLS/FUS and PSF display activities able to pair homologous DNA on membrane in an in vitro assay. In the present study, we address the question whether EWS and hTAF(II)68 also display pairing on membrane activities, and to a larger extent whether other proteins also exhibit such activity. We applied the pairing on membrane assay to 2-DE coupled to MS analysis for a global screening of DNA pairing on membrane activities. In addition to TLS/FUS and PSF, this test allowed us to identify EWS and hTAF(II)68, but no other proteins, indicating a feature specific to a protein family whose members share extensive structural similarities. This common activity suggests a role for TET proteins and PSF in genome plasticity control.

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. In this review, the contributions of mass spectrometry (MS) to the deciphering of these intricate networked interactions are described along with the numerous applications in which it has proven useful. Such applications include, for example, the identification of the partners involved in NA-P or P-P complexes, the identification of post-translational modifications that (may) regulate such complexes' activities, or even the precise molecular mapping of the interaction sites in the NA-P complex. From a biological standpoint, we felt that it was worth the reader's time to be as informative as possible about the functional significance of the analytical methods reviewed herein. From a technical standpoint, because mass spectrometry without proper sample preparation would serve no purpose, each application described in this review is detailed by duly emphasizing the sample preparation-whenever this step is considered innovative-that led to significant analytical achievements. (C) 2003 Wiley Periodicals, Inc.

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. In this review, the contributions of mass spectrometry (MS) to the deciphering of these intricate networked interactions are described along with the numerous applications in which it has proven useful. Such applications include, for example, the identification of the partners involved in NA-P or P-P complexes, the identification of post-translational modifications that (may) regulate such complexes' activities, or even the precise molecular mapping of the interaction sites in the NA-P complex. From a biological standpoint, we felt that it was worth the reader's time to be as informative as possible about the functional significance of the analytical methods reviewed herein. From a technical standpoint, because mass spectrometry without proper sample preparation would serve no purpose, each application described in this review is detailed by duly emphasizing the sample preparation-whenever this step is considered innovative-that led to significant analytical achievements.

Oligopurine-oligopyrimidine sequences, when they exhibit a mirror symmetry can fold under conditions of negative superhelicity into triple-helical structures and adopt the so-called H-DNA form characterized by the presence of both a triple helix and a single-strand DNA structure (see Chapter 15). The over-representation of oligopurine- oligopyrimidine stretches in eukaryotic genomes constitutes an argument in favor of the role of these structures in vivo (1). These intramolecular DNA triplexes may have a function in regulation of gene expression, and have been postulated to play a role in replication, transcription, and recombination, and also in chromatin structure and condensation. Proteins could be involved in the biological functions of these sequences by promoting and stabilizing altered DNA structures. A number of non-B-DNA-structure binding proteins have been identified, arguing for the implication of such altered structures in transcriptional activity (2). In this review we will discuss the potential role of H-DNA binding proteins.

Two 10-mer oligopyrimidine peptide nucleic acids (PNAs) were designed to interfere with IL-2Rα promoter expression by binding to the regulatory sequences overlapping SRF and NF-κB 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. On a double-stranded target, these complexes only formed at low ionic strength and were slowly dissociated at physiological ionic strength (pH 6.5) with a t12 of 6.5–7 h. The salt-dependent instability of preformed complexes on a plasmid target was probably the critical factor responsible for their lack of significant sequence-specific effect on IL-2Rα promoter activity inside living cells.

Porphyrins linked to oligonucleotides produce various types of photodamage on a complementary target DNA. The observed reactions include oxidation of guanine bases and cross-linking reactions of the oligonucleotide to its target sequence. Guanines located close to the porphyrin macrocycle were the most altered as compared to more remote guanines on the target sequence. No specific reaction was observed when the complexes were dissociated at temperatures above the melting temperature of the oligonucleotide-target hybrid. Both cross-linking and oxidation reactions accounted for ca. 60% modification of the target chains in the complex. Our results show that oligonucleotides covalently linked to porphyrins are efficient systems for inducing irreversible sequence-specific photodamage on a target DNA.

Paf‐acether (platelet‐activating factor) is a phospholipid initially described as a potent platelet‐aggregating compound. It is produced by numerous cell types and is now considered as an important mediator of cell‐cell interactions. The effect of paf‐acether on the expression of CD2 and CD3, two human T cell surface glycoproteins, was investigated by indirect immunofluorescence and flow cytometry. Paf‐acether partially down‐regulated, in a time‐ and dose‐dependent manner, CD2 and CD3 but not HLA class I antigen expression on peripheral human T cells and Jurkat cells. Lysophosphatidylcholine, a phospholipid closely related to paf‐acether, had no detectable modulatory effect on CD2 and CD3 expression. In addition to CD2/CD3 modulation, pafacether markedly inhibited T cell proliferative response not only to phytohemagglutinin or concanavalin A but also to anti‐CD3 or a stimulatory combination of anti‐CD2 monoclonal antibodies. These data demonstrate for the first time that lipid mediators such as paf‐acether might be involved in the regulation of the expression of cell surface glycoproteins that are essential in the execution of T cell function.