Enzymatic reaction with unnatural substrates: DNA photolyase (Escherichia coli) recognizes and reverses thymine [2+2] dimers in the DNA strand of a DNA/PNA hybrid duplex

Peptide nucleic acids (PNA) are mimics with normal bases connected to a pseudopeptide chain that obey Watson–Crick rules to form stable duplexes with itself and natural nucleic acids. This has focused attention on PNA as therapeutic or diagnostic reagents. Duplexes formed with PNA mirror some but not all properties of DNA. One fascinating aspect of PNA biochemistry is their reaction with enzymes. Here we show an enzyme reaction that operates effectively on a PNA/DNA hybrid duplex. A DNA oligonucleotide containing a cis, syn-thymine [2+2] dimer forms a stable duplex with PNA. The hybrid duplex is recognized by photolyase, and irradiation of the complex leads to the repair of the thymine dimer. This finding provides insight into the enzyme mechanism and provides a means for the selective repair of thymine photodimers.

Structural origins of the exonuclease resistance of a zwitterionic RNA

Nuclease resistance and RNA affinity are key criteria in the search for optimal antisense nucleic acid modifications, but the origins of the various levels of resistance to nuclease degradation conferred by chemical modification of DNA and RNA are currently not understood. The 2′-O-aminopropyl (AP)-RNA modification displays the highest nuclease resistance among all phosphodiester-based analogues and its RNA binding affinity surpasses that of phosphorothioate DNA by 1°C per modified residue. We found that oligodeoxynucleotides containing AP-RNA residues at their 3′ ends competitively inhibit the degradation of single-stranded DNA by the Escherichia coli Klenow fragment (KF) 3′-5′ exonuclease and snake venom phosphodiesterase. To shed light on the origins of nuclease resistance brought about by the AP modification, we determined the crystal structure of an A-form DNA duplex with AP-RNA modifications at 1.6-Å resolution. In addition, the crystal structures of complexes between short DNA fragments carrying AP-RNA modifications and wild-type KF were determined at resolutions between 2.2 and 3.0 Å and compared with the structure of the complex between oligo(dT) and the D355A/E357A KF mutant. The structural models suggest that interference of the positively charged 2′-O-substituent with the metal ion binding site B of the exonuclease allows AP-RNA to effectively slow down degradation.

Coilin Can Form a Complex with the U7 Small Nuclear Ribonucleoprotein

Coiled bodies (CBs) in the amphibian oocyte nucleus are spherical structures up to 10 μm or more in diameter, much larger than their somatic counterparts, which rarely exceed 1 μm. Oocyte CBs may have smaller granules attached to their surface or embedded within them, which are identical in structure and composition to the many hundreds of B-snurposomes found free in the nucleoplasm. The matrix of the CBs contains the diagnostic protein p80-coilin, which is colocalized with the U7 small nuclear ribonucleoprotein (snRNP), whereas the attached and embedded B-snurposomes contain splicing snRNPs. A few of the 50–100 CBs in the oocyte nucleus are attached to lampbrush chromosomes at the histone gene loci. By coimmunoprecipitation we show that coilin and the U7 snRNP can form a weak but specific complex in the nucleoplasm, which is dependent on the special U7 Sm-binding site. Under the same conditions coilin does not associate with the U1 and U2 snRNPs. Coilin is a nucleic acid-binding protein, as shown by its interaction with single-stranded DNA and with poly r(U) and poly r(G). We suggest that an important function of coilin is to form a transient complex with the U7 snRNP and accompany it to the CBs. In the case of CBs attached to chromosomes at the histone gene loci, the U7 snRNP is thus brought close to the actual site of histone pre-mRNA transcription.

In vitro cloning of complex mixtures of DNA on microbeads: Physical separation of differentially expressed cDNAs

We describe a method for cloning nucleic acid molecules onto the surfaces of 5-μm microbeads rather than in biological hosts. A unique tag sequence is attached to each molecule, and the tagged library is amplified. Unique tagging of the molecules is achieved by sampling a small fraction (1%) of a very large repertoire of tag sequences. The resulting library is hybridized to microbeads that each carry ≈106 strands complementary to one of the tags. About 105 copies of each molecule are collected on each microbead. Because such clones are segregated on microbeads, they can be operated on simultaneously and then assayed separately. To demonstrate the utility of this approach, we show how to label and extract microbeads bearing clones differentially expressed between two libraries by using a fluorescence-activated cell sorter (FACS). Because no prior information about the cloned molecules is required, this process is obviously useful where sequence databases are incomplete or nonexistent. More importantly, the process also permits the isolation of clones that are expressed only in given tissues or that are differentially expressed between normal and diseased states. Such clones then may be spotted on much more cost-effective, tissue- or disease-directed, low-density planar microarrays.

Multiplex detection of four pathogenic retroviruses using molecular beacons

We describe a multiplex nucleic acid assay that identifies and determines the abundance of four different pathogenic retroviruses (HIV-1, HIV-2, and human T-lymphotrophic virus types I and II). Retroviral DNA sequences are amplified in a single, sealed tube by simultaneous PCR assays, and the resulting amplicons are detected in real time by the hybridization of four differently colored, amplicon-specific molecular beacons. The color of the fluorescence generated in the course of amplification identifies which retroviruses are present, and the number of thermal cycles required for the intensity of each color to rise significantly above background provides an accurate measure of the number of copies of each retroviral sequence that were present originally in the sample. Fewer than 10 retroviral genomes can be detected. Moreover, 10 copies of a rare retrovirus can be detected in the presence of 100,000 copies of an abundant retrovirus. Ninety-six samples can be analyzed in 3 hr on a single plate, and the use of a closed-tube format eliminates crossover contamination. Utilizing previously well characterized clinical samples, we demonstrate that each of the pathogenic retroviruses can be identified correctly and no false positives occur. This assay enables the rapid and reliable screening of donated blood and transplantable tissues.

whmD is an essential mycobacterial gene required for proper septation and cell division

A study of potential mycobacterial regulatory genes led to the isolation of the Mycobacterium smegmatis whmD gene, which encodes a homologue of WhiB, a Streptomyces coelicolor protein required for sporulation. Unlike its Streptomyces homologue, WhmD is essential in M. smegmatis. The whmD gene could be disrupted only in the presence of a plasmid supplying whmD in trans. A plasmid that allowed chemically regulated expression of the WhmD protein was used to generate a conditional whmD mutant. On withdrawal of the inducer, the conditional whmD mutant exhibited irreversible, filamentous, branched growth with diminished septum formation and aberrant septal placement, whereas WhmD overexpression resulted in growth retardation and hyperseptation. Nucleic acid synthesis and levels of the essential cell division protein FtsZ were unaltered by WhmD deficiency. Together, these phenotypes indicate a role for WhmD in mycobacterial septum formation and cell division.

Role of the proteasome and NF-κB in streptococcal cell wall-induced polyarthritis

The transcription factor NF-κB activates a number of genes whose protein products are proinflammatory. In quiescent cells, NF-κB exists in a latent form and is activated via a signal-dependent proteolytic mechanism in which the inhibitory protein IκB is degraded by the ubiquitin–proteasome pathway. Consequently, inhibition of the proteasome suppresses activation of NF-κB. This suppression should therefore decrease transcription of many genes encoding proinflammatory proteins and should ultimately have an anti-inflammatory effect. To this end, a series of peptide boronic acid inhibitors of the proteasome, exemplified herein by PS-341, were developed. The proteasome is the large multimeric protease that catalyzes the final proteolytic step of the ubiquitin–proteasome pathway. PS-341, a potent, competitive inhibitor of the proteasome, readily entered cells and inhibited the activation of NF-κB and the subsequent transcription of genes that are regulated by NF-κB. Significantly, PS-341 displayed similar effects in vivo. Oral administration of PS-341 had anti-inflammatory effects in a model of Streptococcal cell wall-induced polyarthritis and liver inflammation in rats. The attenuation of inflammation in this model was associated with an inhibition of IκBα degradation and NF-κB-dependent gene expression. These experiments clearly demonstrate that the ubiquitin–proteasome pathway and NF-κB play important roles in regulating chronic inflammation and that, as predicted, proteasome inhibition has an anti-inflammatory effect.

Studies on the enzymatic and transcriptional activity of the dimerization cofactor for hepatocyte nuclear factor 1

The relationship between the enzymatic and the transcriptional activity of the bifunctional protein pterin-4a-carbinolamine dehydratase/dimerization cofactor for hepatocyte nuclear factor 1 (DCoH) has been elucidated by site-directed mutagenesis. DCoH dimers harbor a binding site for hepatocyte nuclear factor 1 (HNF1), two active centers that bind pterins, and a saddle-shaped surface that resembles nucleic acid binding domains. Two domains of the protein have been selectively targeted to determine if a change in one activity affects the other. No strong correlation has been found, supporting the idea that carbinolamine dehydratase activity is not required for HNF1 binding in vitro or transcriptional coactivation in vivo. Double mutations in the active center, however, influence the in vivo transcriptional activity but not HNF1 binding. This finding suggests that some active center residues also are used during transcription, possibly for binding of another (macro)molecule. Several mutations in the saddle led to a surprising increase in transcription, therefore linking this domain to transcriptional regulation as well. The transcriptional function of DCoH therefore is composed of two parts, HNF1 binding and another contributing effect that involves the active site and, indirectly, the saddle.

Hybridization of single-stranded DNA targets to immobilized complementary DNA probes: comparison of hairpin versus linear capture probes

A microtiter-based assay system is described in which DNA hairpin probes with dangling ends and single-stranded, linear DNA probes were immobilized and compared based on their ability to capture single-strand target DNA. Hairpin probes consisted of a 16 bp duplex stem, linked by a T2-biotin·dT-T2 loop. The third base was a biotinylated uracil (UB) necessary for coupling to avidin coated microtiter wells. The capture region of the hairpin was a 3′ dangling end composed of either 16 or 32 bases. Fundamental parameters of the system, such as probe density and avidin adsorption capacity of the plates were characterized. The target DNA consisted of 65 bases whose 3′ end was complementary to the dangling end of the hairpin or to the linear probe sequence. The assay system was employed to measure the time dependence and thermodynamic stability of target hybridization with hairpin and linear probes. Target molecules were labeled with either a 5′-FITC, or radiolabeled with [γ-33P]ATP and captured by either linear or hairpin probes affixed to the solid support. Over the range of target concentrations from 10 to 640 pmol hybridization rates increased with increasing target concentration, but varied for the different probes examined. Hairpin probes displayed higher rates of hybridization and larger equilibrium amounts of captured targets than linear probes. At 25 and 45°C, rates of hybridization were better than twice as great for the hairpin compared with the linear capture probes. Hairpin–target complexes were also more thermodynamically stable. Binding free energies were evaluated from the observed equilibrium constants for complex formation. Results showed the order of stability of the probes to be: hairpins with 32 base dangling ends > hairpin probes with l6 base dangling ends > 16 base linear probes > 32 base linear probes. The physical characteristics of hairpins could offer substantial advantages as nucleic acid capture moieties in solid support based hybridization systems.

Protein–RNA interactions: a structural analysis

A detailed computational analysis of 32 protein–RNA complexes is presented. A number of physical and chemical properties of the intermolecular interfaces are calculated and compared with those observed in protein–double-stranded DNA and protein–single-stranded DNA complexes. The interface properties of the protein–RNA complexes reveal the diverse nature of the binding sites. van der Waals contacts played a more prevalent role than hydrogen bond contacts, and preferential binding to guanine and uracil was observed. The positively charged residue, arginine, and the single aromatic residues, phenylalanine and tyrosine, all played key roles in the RNA binding sites. A comparison between protein–RNA and protein–DNA complexes showed that whilst base and backbone contacts (both hydrogen bonding and van der Waals) were observed with equal frequency in the protein–RNA complexes, backbone contacts were more dominant in the protein–DNA complexes. Although similar modes of secondary structure interactions have been observed in RNA and DNA binding proteins, the current analysis emphasises the differences that exist between the two types of nucleic acid binding protein at the atomic contact level.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Detection of alkali metal ions in DNA crystals using state-of-the-art X-ray diffraction experiments

The observation of light metal ions in nucleic acids crystals is generally a fortuitous event. Sodium ions in particular are notoriously difficult to detect because their X-ray scattering contributions are virtually identical to those of water and Na+…O distances are only slightly shorter than strong hydrogen bonds between well-ordered water molecules. We demonstrate here that replacement of Na+ by K+, Rb+ or Cs+ and precise measurements of anomalous differences in intensities provide a particularly sensitive method for detecting alkali metal ion-binding sites in nucleic acid crystals. Not only can alkali metal ions be readily located in such structures, but the presence of Rb+ or Cs+ also allows structure determination by the single wavelength anomalous diffraction technique. Besides allowing identification of high occupancy binding sites, the combination of high resolution and anomalous diffraction data established here can also pinpoint binding sites that feature only partial occupancy. Conversely, high resolution of the data alone does not necessarily allow differentiation between water and partially ordered metal ions, as demonstrated with the crystal structure of a DNA duplex determined to a resolution of 0.6 Å.

LGICdb: the ligand-gated ion channel database

Ligand-Gated Ion Channels (LGIC) are polymeric transmembrane proteins involved in the fast response to numerous neurotransmitters. All these receptors are formed by homologous subunits and the last two decades revealed an unexpected wealth of genes coding for these subunits. The Ligand-Gated Ion Channel database (LGICdb) has been developed to handle this increasing amount of data. The database aims to provide only one entry for each gene, containing annotated nucleic acid and protein sequences. The repository is carefully structured and the entries can be retrieved by various criteria. In addition to the sequences, the LGICdb provides multiple sequence alignments, phylogenetic analyses and atomic coordinates when available. The database is accessible via the World Wide Web (http://www.pasteur.fr/recherche/banques/LGIC/LGIC.html), where it is continuously updated. The version 16 (September 2000) available for download contained 333 entries covering 34 species.

Database resources of the National Center for Biotechnology Information

In addition to maintaining the GenBank® nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI’s Web site. NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser. Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, GeneMap’99, Human–Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, Cancer Genome Anatomy Project (CGAP), SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheri­tance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD). Augmenting many of the Web applications are custom implementations of the BLAST program optimized to search specialized data sets. All of the resources can be accessed through the NCBI home page at: http://www.ncbi.nlm.nih.gov.

The non-coding RNAs as riboregulators

The non-coding RNAs database (http://biobases.ibch.poznan.pl/ncRNA/) contains currently available data on RNAs, which do not have long open reading frames and act as riboregulators. Non-coding RNAs are involved in the specific recognition of cellular nucleic acid targets through complementary base pairing to control cell growth and differentiation. Some of them are connected with several well known developmental and neuro­behavioral disorders. We have divided them into four groups. This paper is a short introduction to the database and presents its latest, updated edition.