Terminal Dinucleotide Research Articles

Inhibition of HIV-1 integrase by flavones, caffeic acid phenethyl ester (CAPE) and related compounds

The inhibition of HIV-1 integrase by flavones and related compounds was investigated biochemically and by means of structure-activity relationships. Purified enzyme and synthetic oligonucleotides were used to assay for three reactions catalysed by integrase: (1) processing of 3' termini by cleavage of the terminal dinucleotide; (2) strand transfer, which models the integration step; and (3) “disintegration,” which models the reversal of the strand transfer reaction. Inhibitions of all three reactions by flavones generally occurred in parallel, but caffeic acid phenethyl ester (CAPE) appeared to inhibit reaction 2 selectively. CAPE, however, inhibited reactions 1 and 3 effectively when preincubated with the enzyme, suggesting that this compound differs from the flavones primarily in requiring more time to block the enzyme. The core integrase fragment consisting of amino acids 50–212 retained the ability to catalyse reaction 3, and flavones and CAPE retained the ability to inhibit. Hence, the putative zinc-finger region that is deleted in this fragment is probably not the target of inhibition. Inhibition by flavones usually required the presence of at least one ortho pair of phenolic hydroxyl groups and at least one or two additional hydroxyl groups. Potency was enhanced by the presence of additional hydroxyl groups, especially when present in ortho pairs or in adjacent groups of three. Inhibitory activity was reduced or eliminated by methoxy or glycosidic substitutions or by saturation of the 2,3 double bond. These structure-activity findings for flavones were generally concordant with those previously reported for reverse transcriptase and topoisomerase II. These findings are discussed in the context of a review of the effects of flavones on various enzymes, the possible mechanisms of inhibition, and the potential for building upon a general pharmacophore to generate target specificity.

A stable complex between integrase and viral DNA ends mediates human immunodeficiency virus integration in vitro.

Retroviral replication depends on integration of the viral genome into a chromosome of the host cell. The steps in this process are orchestrated in vivo by a large nucleoprotein complex and are catalyzed by the retroviral enzyme integrase. Several biochemical properties of the in vivo nucleoprotein complex were reproduced in vitro with purified integrase of human immunodeficiency virus type 1 and model viral DNA substrates. A stable complex between integrase and viral DNA was detected as an early intermediate in the integration reaction. After formation of this initial complex, the enzyme processively catalyzed the 3' end processing and strand transfer steps in the reaction. Complexes containing only purified integrase and the model viral DNA end were stable under a variety of conditions and efficiently used nonviral DNA molecules as integration targets. These complexes required a divalent cation for their formation, and their stability was highly dependent on the 5'-terminal dinucleotide of the viral DNA, for which no functional role has previously been defined. Thus, interactions between integrase and the extreme ends of the viral DNA molecule may be sufficient to account for the stability of the in vivo integration complex.

Molecular pathways of pain: Fos/Jun-mediated activation of a noncanonical AP-1 site in the prodynorphin gene

Noxious stimulation provokes the activation of genes that are thought to play a crucial role in the phenomena of stress and pain. Among these is the prodynorphin gene. By double-labeling in situ hybridization/immunohistochemistry, we show that increased prodynorphin gene expression is preceded, in the same neurons, by an early induction of c- fos. Inspection of the prodynorphin promoter region revealed the presence of several AP-1-like sequences. We demonstrate that only one of these sites is a functional AP-1 element. It is constituted by the noncanonical TGACAAACA sequence, in which the palindromic structure is partly conserved by the 3′ terminal CA dinucleotide. Transfection experiments in NCB20 neuroblastoma cells indicated that this site is a target of Fos/Jun trans-activation. Our results suggest that Fos/Jun oncoproteins may function as third messengers in the signal transduction mechanisms of stress/pain processes.

Structure of RNAs replicated by the DNA-dependent T7 RNA polymerase

The DNA-dependent RNA polymerase of bacteriophage T7 efficiently and specifically replicates two structurally related RNAs, termed X and Y RNAs. Replication of both RNAs involves synthesis of complementary strands initiated with pppC and pppG. RNAs transcribed from DNA template containing the established sequences of X and Y RNAs were efficiently replicated by T7 RNA polymerase. Both RNAs possess palindromic sequences with a dual axis of symmetry, permitting formation of hairpin-, dumbbell-, or cloverleaf-type structures. The template must consist of RNA and not DNA sequence, and the terminal unpaired dinucleotides of the RNA are necessary for replication. Nucleotidyl transferase activity of E. coli adenylates the unpaired CC OH dinucleotide at the 3′ end of a C strand of X RNA. This feature, as well as the length (64 nucleotides) and compact structure of X and Y RNAs, suggests that they may resemble tRNA molecules and tRNA-like structures at the 3′ termini of many plant viral RNA genomes.

Characterization of the staphylococcal beta-lactamase transposon Tn552.

The staphylococcal beta-lactamase transposon Tn552 is a member of a novel group of transposable elements. The organization of genes in Tn552 resembles that of members of the Tn21 sub-group of Tn3 family transposons, which transpose replicatively by cointegrate formation and resolution. Thus, a possible resolution site ('resL') and a resolvase gene (tnpR or 'binL') have been identified. However, consistent with the fact that Tn552 generates 6 bp (rather than 5 bp) flanking direct repeats of target DNA, neither the putative transposase protein, nor the terminal inverted repeats of Tn552 are homologous to those of Tn3 elements. Tn552, like phage Mu and retroelements, is defined by the terminal dinucleotides 5' TG .. CA 3'. A naturally occurring staphylococcal plasmid, pI9789, contains a Tn552-derived resolution system ('resR-binR') that acts as a 'hotspot' for Tn552 transposition; insertion creates a segment of DNA flanked by inversely repeated resolution sites, one (resR) on pI9789 and the other (resL) on Tn552. The putative Tn552 resolvase, the most closely related of known resolvases to the homologous DNA invertases, initially was identified as a DNA invertase ('Bin') as a result of its ability to mediate efficient inversion of this segment in vivo.

An aberrant avian leukosis virus provirus inserted downstream from the chicken c-myc coding sequence in a bursal lymphoma results from intrachromosomal recombination between two proviruses and deletion of cellular DNA.

A chicken bursal lymphoma, LL6, contains avian leukosis virus DNA integrated 3' of the c-myc coding sequences, unlike all other examined bursal lymphomas, which have integrations 5' to c-myc. To better understand this unusual mutation, we examined a molecular clone containing the LL6 c-myc gene and determined the structure of the proviral insertion by DNA sequencing. Viral DNA begins 575 base pairs downstream of the c-myc coding sequences within the untranslated region, disrupting the use of the normal polyadenylation signal. An internal deletion of the provirus extends from within U3 in the 5' long terminal repeat to within the gp37-coding region of the env gene, disabling virus replication and protein synthesis. Both host-virus boundaries appear normal with respect to the site in viral DNA which is joined to host DNA; both long terminal repeats lack the terminal dinucleotide found in unintegrated DNA. However, in contrast to normal integrations, the six bases of cellular sequence at the 5' junction are not repeated at the 3' junction. The DNA sequences immediately downstream of the LL6 recombinant provirus are not part of the c-myc gene; they originate from the same chromosome as c-myc, but at least 15 kilobases (kb) away. In addition, DNA sequences normally residing 3' of c-myc are deleted in LL6. In summary, these results imply that the LL6 provirus is the result of recombination between two proviruses; that both proviruses were originally downstream of c-myc in the same orientation and separated by at least 15 kb; and that the recombination event was preceded, accompanied, or followed by an internal proviral deletion. No transcript could be detected within a 20-kb region downstream of the LL6 provirus, leaving unresolved the question of whether the additional chromosomal alterations make a specific contribution to LL6 tumorigenesis.

Nucleotide sequence of an alfalfa glutamine synthetase gene

One of the members of the small gene family encoding glutamine synthetase in alfalfa (Medicago sativa L.) has been cloned and its nucleotide sequence determined. Approximately full length cDNAs corresponding to the gene have also been cloned and their sequences determined. The gene is about 4 kb long, and consists of twelve exons (size range 37 to 159 bp) and eleven introns (size range 90 to 715 bp). The messenger RNA contains a 120 nucleotide 5′-untranslated region, a 1,071 nucleotide coding region, and a 210 nucleotide 3′-untranslated region. Several eukaryotic consensus transcription and processing signals are present in this plant gene: (1) a TATA box (TATAAATA) 25 bp 5′ to the putative transcription start site; (2) a potential polyadenylation signal (AATAATAA) 20 bp 5′ to the polyadenylation site; and (3) the terminal dinucleotides of the introns (5′-GT...AG-3′). Also the dinucleotide, CpG, is significantly underrepresented throughout the gene. The derived amino acid sequence predicts a relatively hydrophilic protein with a molecular weight of 39,215. Several regions of amino acid homology are apparent when this sequence is compared to the Anabaena and Chinese hamster glutamine synthetase sequences. Using data derived from the gene structure, the minimum size of a glutamine synthetase gene amplification unit in an l-phosphinothricin resistant alfalfa tissue culture line has been determined to be 35 kb.

Evidence that discontinuous DNA replication in Escherichia coli is primed by approximately 10 to 12 residues of RNA starting with a purine

Intact primer RNA for discontinuous DNA replication of Escherichia coli has been detected by specific labeling in vitro of its 5′-terminal tri- (or di-) phosphate group with vaccinia guanylyltransferase and [α- 32P]GTP. A mutant defective either in RNase H or in both RNase H and DNA polymerase I accumulated about 10 or 30 times more intact primer RNA, respectively, than wild-type cells. The primers started with purine in an A to G ratio of 5 and the most abundant 5′-terminal dinucleotide sequence was (p)ppA-Pu. The chain length of the intact primer RNA was approximately 10 to 12 nucleotide residues. The structural properties of the E. coli primer RNA resemble those of the eukaryotic primer RNA.

Anomalous hairpin formation in an oligodeoxyribonucleotide.

An accurate method for deriving molar absorptivity-temperature profiles applied to a set of single-stranded oligodeoxyribonucleotides shows that the undecamer CGAGTTTGACGp exists in a hairpin conformation involving Watson-Crick base pairing between the two terminal CG dinucleotides. The hairpin, which has a transition midpoint of 40 degrees C in 0.115 M Na+, is unusually stable in comparison with previously reported hairpins. A non-linear least squares analysis of the undecamer's profile in terms of a two-state equilibrium model indicates that the hairpin-to-coil transition occurs with an enthalpy change about twice that expected if only combinations of Watson-Crick base-paired stacking interactions are considered. The analogous hairpin structure (containing an identical CG/CG stem) assignable to the complementary strand CGTCAAACTCGp does not form above 0 degrees C. Measurements on the two undecamers indicate that variation in non Watson-Crick interactions within the loops of two similar hairpins can produce a difference in stability of at least 2.2 kcal/mol (25 degrees C, 0.115 M Na+), roughly equal to the amount contributed to a double helix by a 5'-CG-3'/5'-CG-3' base-paired stacking interaction.

Sequence-specific insertion of the Drosophila transposable genetic element 17.6.

As in the case of retrovirus proviruses, most of the Drosophila copia-like transposable elements so far examined are bounded by 5'TG...CA3' and inserted into the chromosome without obvious site-specificity. In the other copia-like elements, 297, HMS Beagle and 17.6 (refs 5-7), terminal dinucleotides (5'TG...CA3') are completely absent and, instead, 5'(A)GT is present at least at one of the termini. One important feature of these three elements may be frequent insertion into 'TATA' boxes, since the three of four insertion sites so far examined were TATA boxes, two for H3 histone genes and one for a cuticle gene. Because of the importance of this type of insertion, we extensively analysed site-specificity using 17.6 as a model. Our results, described here, suggest that insertion of 17.6 takes place in a site-specific fashion, using a target 5'ATAT corresponding to the major portion of the consensus TATA box, TATAATAAT.

Initiation of adenovirus DNA replication: detection of covalent complexes between nucleotide and the 80-kilodalton terminal protein.

We have previously shown that the 5'-terminal deoxycytidine residue of each nascent adenovirus 5 DNA strand synthesized in vitro is covalently linked to the 80-kilodalton (kd) terminal protein precursor via a phosphodiester bond to a serine residue in the protein. When extracts prepared from adenovirus 5-infected cells are incubated with [alpha-33P]dCTP as the only added deoxynucleoside triphosphate, complexes consisting of nucleotide covalently linked to the 80-kd protein can be detected. The nucleotide moieties present in such complexes include d(pC) and d(pCpA), the 5'-terminal nucleotide and dinucleotide of adenovirus 5 DNA, respectively, as well as some longer oligonucleotides. The formation of these complexes requires the presence of adenovirus DNA containing the attached 55-kd terminal protein and ATP. Extracts from H5ts125-infected cells which are defective in DNA replication catalyze complex formation to the same extent as extracts prepared from wild-type infected cells; thus, the presence of the adenovirus-coded 72-kd DNA-binding protein is apparently not required. Most, if not all, of the 80-kd protein-nucleotide complexes that are formed are noncovalently bound to the input viral DNA. These observations are consistent with the protein-priming model for the initiation of adenovirus DNA replication.

Terminal sequences of the genome and replicatioe-form RNA of the flavivirus west nile virus: absence of poly(A) and possible role in RNA replication

The structures of the infectious 42 S genome RNA of the flavivirus West Nile (WN) virus and of the replicative-form (RF) RNA containing 42 S RNA of positive and negative polarity have been investigated. The RF RNA has been labeled in vitro at the 3′ and 5′ termini and the terminal sequences have been determined by the mobility shift method. The results obtained indicate that both RNA molecules are exact complements of each other and that the 3′ terminus of the 42 S plus-strand RNA component of the RF RNA does not contain a poly(A) sequence but terminates with a heteropolymeric AACACAGGAUCU OH sequence. The 3′ terminus of the 42 S minus-strand RNA has the sequence CUCACACAGGCGAACUACU OH. Comparison of these sequences shows that both molecules contain the 3′-terminal dinucleotide CUOH and the heptanucleotide ACACAGG which is separated from the 3′-terminal dinucleotide by two and seven nucleotides in 42 S plus- and minus-strand RNA, respectively. The 42 S viral genome RNA also does not contain a 3′-terminal poly(A) sequence but terminates with the 3′-terminal sequence identified in the 42 S plus-strand RNA of the RF. Analysis of the nucleotides adjacent to the cap at the 5′ terminus of the viral genome RNA together with the 3′-terminal sequence analysis indicates that the nucleotide sequence of the viral genome RNA is identical to that of the 42 S plus-strand RNA component of the virus-specific RF RNA.

Structure of the RNA portion of the RNA-linked DNA pieces in bacteriophage T4-infected Escherichia coli cells

A method for the isolation of the RNA portion of RNA-linked DNA fragments has been developed. The method capitalizes on the selective degradation of DNA by the 3′ to 5′ exonuclease associated with bacteriophage T4 DNA polymerase. After hydrolysis of the DNA portion, the RNA of RNA-linked DNA is recovered mostly as RNA tipped with a deoxyribomononucleotide and a small fraction as pure RNA. On the other hand, the 5′ ends of RNA-free DNA are recovered mostly as dinucleotides and a small fraction as mononucleotides. Using this method, we have isolated the primer RNA for T4 phage DNA synthesis. Nascent short DNA pieces were isolated from T4 phage-infected Escherichia coli cells and the 5′ ends of the pieces were dephosphorylated and then phosphorylated with polynucleotide kinase and [γ- 32P]ATP. After selective degradation of the DNA portions, [5′- 32P]oligoribonucleotides (up to pentanucleotide) were obtained with covalently bound deoxymononucleotides at their 3′ ends. More than 40% of the oligoribonucleotides isolated were pentanucleotides with pApC at the 5′-terminal dinucleotide. The 5′-terminal nucleotide of the tetraribonucleotides was AMP, but that of the shorter chains was not unique. The pentanucleotide could represent the intact primer RNA for T4 phage DNA synthesis.

Wheat embryo ribonucleates. XIII. Methyl-substituted nucleoside constituents and 5'-terminal dinucleotide sequences in bulk poly(AR)-rich RNA from imbibing wheat embryos.

(1) N6-Methyladenosine, not previously detected as a component of plant mRNA, is shown to be present in the poly(A)-rich RNA which is isotopically labeled when wheat embryos imbibe water in a medium which contains [methyl-3H]methionine.(2) N6-Methyladenosine and 7-methylguanosine 'caps' can constitute more than 85% of the radioactivity when specimens of poly(A)-rich [methyl-3H]RNA are freed of most of the radioactivity contributed by contaminating rRNA, N6-methyladenosine accounting for 75–80% and 7-methylguanosine accounting for 20–25% of this radioactivity.(3) There are three principal 5′-terminal dinucleotide sequences (type 0 'cap' structures) in the poly(A)-rich RNA from imbibing wheat embryos: m7GpppA, m7GpppG, and m7GpppC.(4) The proportional distribution of radioactivity among the 5′-terminal dinucleotide sequences varies with the time of labeling of the imbibing wheat embryos (30 min and 24 h), m7GpppA being the most 'rapidly labeled' sequence and m7GpppC being the most 'slowly labeled' sequence.(5) Poly(A)-rich mRNA from imbibing wheat embryos is distinct from the corresponding RNA from any other organism yet studied in terms of its component methyl-substituted nucleosides and 5′-terminal dinucleotide sequences. The possible significance of these differences, as well as signal similarities between the poly(A)-rich mRNA of imbibing wheat embryos and other organisms, are subjects of discussion.

RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. II. Primary structure of the RNA portion

Short DNA chains were purified from phage T7 infected E. coli cells and 5' ends were labeled with 32P. By an alkali-treatment, pNp's rich in pAp and pCp were liberated from the T7 short DNA chains. After digestion of the [5'-32P] short DNA with the 3' to 5' exonuclease of T4 DNA polymerase, [5'-32P] mono- to pentaribonucleotides tipped with a deoxyribonucleotide residue at their 3' ends were isolated. 5' terminal ribonucleotides were; exclusively AMP in the penta- and the tetraribonucleotides, mostly CMP in the triribonucleotide and mainly CMP and AMP in di- and monoribonucleotides. The 5' terminal dinucleotide of the penta- and the tetraribonucleotides was pApC. The nucleotide sequence of the tetraribonucleotide was mainly pApCpCpN and some pApCpApN, where N was mainly A and C. These results indicate that oligoribonucleotides shorter than trinucleotide may result from in vivo degradation of the tetra- and pentaribonucleotides. A possibility that the tetra- and pentaribonucleotides with a 5' triphosphate terminus are the intact primers for the discontinuous T7 DNA replication is discussed.

RNA-linked nascent DNA pieces in phage T7-infected Escherichia coli. III. Detection of intact primer RNA

RNA-linked DNA fragments of T7-infected Escherichiacoli were labeled with [(32)P]orthophosphate invivo. The RNA segments of the labeled fragments were isolated by degrading the DNA portion with the 3'--> 5' exonuclease intrinsic to bacteriophage T4 DNA polymerase and fractionated according to net charge by a DEAE-Sephadex A-25 column chromatography in the presence of 7 M urea. Tri-, tetra- and pentanucleotides were obtained which have ATP residues at their 5' ends. Most of the pentanucleotides had a single deoxynucleotide at the 3' end but a minor portion was totally an oligoribonucleotide. In the light of prior results, the former is a cooligomer of an intact tetraribonucleotide primer and a monodeoxynucleotide and the latter is an intact pentaribonucleotide primer. Tri- and tetraribonucleotides with ATP at the 5' ends had no deoxynucleotide at the 3' ends, therefore it is not clear if intact triribonucleotide primers are present. The 5'-terminal dinucleotides of the tetra- and pentanucleotides were mostly pppApC and a trace amount of pppApA was present.Images

Novel properties of Escherichia coli exonuclease III.

The specificity of hydrolysis of polynucleotide termini by Escherichia coli exonuclease III was studied with the use of oligothymidylate annealed to polydeoxyadenylate. The size of the products after 3' leads to 5'-hydrolysis of 5'-labeled substrate is temperature-dependent. At 25 degrees the enzyme can hydrolyze a polynucleotide chain up to the last 5'-terminal dinucleotide. A gradation of higher 5'-terminal oligonucleotides of defined chain lengths is produced after limit digestion by the enzyme when the temperature is raised between 25 degrees to 60 degrees. When the oligothymidylate was labeled at the 3'-ends with ribonucleotides, it was observed that exonuclease III can cleave a single or two consecutive ribonucleotides regardless of whether the ribonucleotides are base-paired or mismatched.

Nucleotide clusters in deoxyribonucleic acids XIII. Sequence analysis of the longer unique pyrimidine oligonucleotides of bacteriophage S13 DNA by a method using unlabeled starting oligonucleotides

A method has been designed for sequence analysis of unlabeled oligodeoxynucleotides of chain length up to 20 nucleotides with no restriction on base composition. The unlabeled oligonucleotide preparation is partially degraded with spleen exonuclease to give a series of products each differing in size by one nucleotide. The oligonucleotides in the digest are 5′- 32P terminally labeled with [γ- 32P]ATP and T4 polynucleotide kinase, the excess ATP removed by chromatography on Sephadex G-25 then the oligonucleotides fractionated according to chain length on DEAE-Sephadex. Each isostich fraction is analyzed for base composition and the nucleotide at the 5′ terminus determined by its 32P label, resulting in direct read off of the sequence up to the penultimate 3′-terminal nucleotide. The 3′-terminal dinucleotide is analyzed by DEAE-cellulose chromatography of the Sephadex G-25 dinucleotide fraction. The method has been demonstrated by sequence analysis of the unique longer pyrimidine oligonucleotides C 5T 6, C 2T 8, C 6T 4 and C 6T 3 from S13 DNA. The sequences have extensive internal sequence homology.

Preparation and specificity of endonuclease IV induced by bacteriophage T4.

Bacteriophage-T4-induced endonuclease IV suitable for DNA sequence analysis has been prepared by a modified and easily reproducible method. The specificity of T4-induced endonuclease IV has been investigated in order to verify whether this enzyme exhibits a single nucleotide recognition or a short sequence recognition. The 5'-terminal dinucleotides and 3'-terminal nucleotides of oligonucleotides released by T4-induced endonuclease IV from three single-stranded DNAs (from bacteriophages phiX174, fd, M 13) have been analysed. In different DNAs, 74-82% of the 5'-terminal dinucleotides end in 5'-deoxycytidylic acid; small but significant levels of several dinucleotides ending in 5'-deoxyadenylic acid, 5'-thymidylic acid and 5'-deoxyguanylic acid are also found. As far as 3'-terminal nucleotides are concerned all nucleotides are present with a large predominance of thymidylic acid. It is concluded that T4-induced endonuclease IV recognizes short nucleotide sequences like all other DNases investigated so far. The spectrum of such sequences is, however, very narrow.

The involvement of the anticodon adjacent modified nucleoside N-[9-( -D-ribofuranosyl) purine-6-ylcarbamoyl]-threonine in the biological function of E.coli tRNAile

tRNAile was isolated from E. coli Cp 79 (leu-, arg-, thr-, his-, thiamin-, RCrel) which had been grown on a sub-optimal concentration of thr and was found to contain an average of 50% less N-[9-(beta-D-ribofuranosyl)- purin-6-ylcarbamoyl]threonine, t6Ado, than tRNAile from cells grown on an optimum concentration of thr and containing a normal complement of t6Ado. The two tRNA's were identical in their ability to be aminoacylated, to accept the 3'-terminal dinucleotide, and to form an ile-tRNAile-Tu-GTP complex. In contrast, the t6Ado-deficient-tRNA was significantly less efficient in binding to ribosomes compared to the normal tRNA. This difference was seen in the binding of deacylated tRNA and in the nonenzymatic and enzymatic binding of ile-tRNA, all in response to poly AUC. The t6Ado-deficient ile-tRNA demonstrated no binding at Mg2+ concentrations less than or equal to 10 mM, while the normal ile-tRNA bound at low Mg2+ concentrations. Tetracycline had the same effect on the normal as on the t6Ado-deficient ile-tRNA binding. As a control, the binding of phe-tRNA (which does not contain t6Ado) from normal and thr-starved cells in response to poly U was identical. It was concluded that t6Ado is required for proper codon-anticodon interaction.