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PubMed1996

A short autocomplementary sequence plays an essential role in avian sarcoma-leukosis virus RNA dimerization.

Retroviral genomes consist of two identical RNA molecules joined noncovalently near their 5'-ends. Recently, two models have been proposed for RNA dimer formation on the basis of results obtained in vitro with human immunodeficiency virus type 1 RNA and Moloney murine leukemia virus RNA. It was first proposed that viral RNA dimerizes by forming an interstrand quadruple helix with purine tetrads. The second model postulates that RNA dimerization is initiated by a loop-loop interaction between the two RNA molecules. In order to better characterize the dimerization process of retroviral genomic RNA, we analyzed the in vitro dimerization of avian sarcoma-leukosis virus (ASLV) RNA using different transcripts. We determined the requirements for heterodimer formation, the thermal dissociation of RNA dimers, and the influence of antisense DNA oligonucleotides on dimer formation. Our results strongly suggest that purine tetrads are not involved in dimer formation. Data show that an autocomplementary sequence located upstream from the splice donor site and within a major packaging signal plays a crucial role in ASLV RNA dimer formation in vitro. This sequence is able to form a stem-loop structure, and phylogenetic analysis reveals that it is conserved in 28 different avian sarcoma and leukosis viruses. These results suggest that dimerization of ASLV RNA is initiated by a loop-loop interaction between two RNA molecules and provide an additional argument for the ubiquity of the dimerization process via loop-loop interaction.

Alpharetrovirus↗
PubMed1998

Oxidative charge transfer To repair thymine dimers and damage guanine bases in DNA assemblies containing tethered metallointercalators.

Potent oxidants which intercalate in DNA serve as tools to probe DNA-mediated electron-transfer reactions. A photoexcited rhodium intercalator, Rh(phi)2DMB3+ (phi = 9,10-phenanthrenequinone diimine and DMB = 4,4'-dimethyl-2,2'-bipyridine), tethered to DNA, promotes both oxidative damage to 5'-GG-3' doublets in DNA and the repair of thymine dimers from a remote site on the DNA duplex. DNA-mediated repair of a thymine dimer lesion by charge transfer from the tethered rhodium intercalator is quantitative, albeit with low photoefficiency, occurs in an intraduplex reaction over long range (36 A), and requires that the intervening bases be paired. When both oxidative reactions, repair and oxidative damage, are monitored on the same duplex, competition is evident; the presence of both a 5'-GG-3' site and the thymine dimer diminished the dimer repair efficiency by 20-40% and decreased damage at the 5'-GG-3' sites 2-fold compared to similar sequences lacking either the guanine doublet or thymine dimer, respectively. In addition to damage at the 5'-G of 5'-GG-3' sites, we also observe oxidation at the 3'-G of the 5'-GT<>TG-3' tetrad only in the presence of thymine dimer. Overall, the yield of repaired thymine strand was at least 10 times higher than the yield of oxidized guanine in the same sequences. While the 5-GG-3' may represent the thermodynamically favored site for oxidative reaction, repair of the thymine dimer appears to be kinetically more favorable. Dipyridophenanzine (dppz) complexes of ruthenium(III), less potent oxidants which intercalate in DNA, oxidize 5'-GG-3' doublets efficiently but cannot trigger the repair of the thymine dimer lesion. Oxidative damage to DNA from a distance, mediated by the DNA base pair stack, can, however, be utilized to probe the disruption in the base stack generated by the thymine dimer. The presence of the dimer does not diminish oxidation by a Ru(III) intercalator at a distal guanine doublet, suggesting that the disruption caused by the dimer does not block charge transfer through the DNA duplex. DNA-mediated electron-transfer reactions of metallointercalators therefore serve to illustrate important aspects of radical migration and its consequence with respect to reactions at a distance through the DNA base pair stack.

Binding Sites↗
PubMed1998

Effect of N-terminal truncation and solution conditions on chemokine dimer stability: nuclear magnetic resonance structural analysis of macrophage inflammatory protein 1 beta mutants.

Chemokines (chemotactic cytokines) are a family of immune system proteins, several of which have been shown to block human immunodeficiency virus (HIV) infection in various cell types. While the solved structures of most chemokines reveal protein dimers, evidence has accumulated for the biological activity of individual chemokine monomers, and a debate has arisen regarding the biological role of the chemokine dimer. Concurrent with this debate, several N-terminal truncations and modifications in the CC subfamily of chemokines have been shown to have functional significance, in many cases antagonizing their respective receptors and in some cases retaining the ability to block HIV entry to the cell. As the dimer interface of CC chemokines is located at their N-terminus, a structural study of N-terminally truncated chemokines will address the effect that this type of mutation has on the dimer-monomer equilibrium. We have studied the structural consequences of N-terminal truncation in macrophage inflammatory protein 1 beta (MIP-1 beta), a CC chemokine that has been shown to block HIV infection. Examination of nuclear magnetic resonance (NMR) spectra of a series of N-terminally truncated MIP-1 beta variants reveals that these proteins possess a range of ability to dimerize. A mutant beginning at amino acid Asp6 [termed MIP(6)] has near wild-type dimer properties, while further truncation results in weakened dimer affinity. The mutant MIP(9) (beginning with amino acid Thr9) has been found to exist solely as a folded monomer. Relaxation measurements yield a rotational correlation time of 8.6 +/- 0.1 ns for wild-type MIP-1 beta and 4.5 +/- 0.1 ns for the MIP(9) mutant, consistent with a wild-type dimer and a fully monomeric MIP(9) variant. The presence of physiological salt concentration drastically changes the monomer-dimer equilibrium for both wild-type and most mutant proteins, heavily favoring the dimeric form of the protein. These results have implications for structure-function analysis of existing chemokine mutants as well as for the larger debate regarding the biological existence and activity of the chemokine dimer.

Amino Acid Sequence↗
PubMed2000

Reversible dissociation/association of D-amino acid transaminase subunits: properties of isolated active dimers and inactive monomers.

The crystal structure of dimeric D-amino acid transaminase shows that the two Trp-139 sites are located in a hydrophobic pocket at the interface between the subunits and that the two indole side chains face one another and are within 10 A of coenzyme. This enzyme prefers an aromatic character at position 139, as previously demonstrated by the finding that Phe-139 but no other substitution tested provides the maximum degree of thermostability and catalytic efficiency. Here we show that an equilibrium between active dimers and inactive monomers can be demonstrated with the W139F mutant enzyme, whereas with the wild-type enzyme the subunit interface is so tight that a study of this equilibrium is precluded. We show how the processes of dimerization of monomers and dissociation of dimers to monomers are controlled. Lower pH (5.0) favors monomer formation from dimers. Gel filtration and activity analysis show that at higher pH (7.0) the monomers combine to form active dimers with a K(d) of 0.17 microM. This assembly process is relatively slow and takes several hours for completion, thereby permitting accurate measurement of kinetics and equilibrium parameters. Absorption and circular dichroism spectra of dimers and monomers are significantly different, indicating that the environment around the cofactor is very likely altered between them. The circular dichroism peak of the W139F dimer at 418 nm is less negative than that of the wild-type enzyme in accordance with its lower visible absorbance; the circular dichroism peak of the W139F monomer at 418 nm is more negative than that of the wild-type enzyme. The dissociation of dimers to monomers has also been studied by taking advantage of these spectral differences, thus permitting the rates of the dissociation and the reassociation to be calculated and compared. 2-Mercaptoethanol assists in the conversion of monomers to dimers. The results here describe dissociation/reassociation in the dimeric enzyme under native conditions without denaturants.

Chromatography, Gel↗
PubMed1982

Ligation of oligonucleotides by pyrimidine dimers--a missing 'link' in the origin of life?

One of the principal photochemical reactions of DNA on exposure to UV is the formation of intrastrand cyclobutane-type pyrimidine dimers. The efficiency of this reaction depends on both the wavelength of the UV2 and the specific nucleotide sequence in the DNA. The formation of the pyrimidine dimer and its repair in living cells have been studied extensively. We have examined the possibility that pyrimidines at the ends of DNA strands may be adequately juxtaposed for dimer formation by the presence of a complementary strand, even when no phosphodiester linkage joins their sugars. In these conditions the formation of a dimer will 'ligate' two DNA strands end-to-end. We report here that thymidine oligonucleotides annealed to polydeoxyadenylate can be ligated end-to-end by UV irradiation, via thymine dimerization of the terminal nucleotides in adjacent oligonucleotides. The linkages are susceptible to direct photoreversal by 254 nm UV, as expected for cyclobutane-type thymine dimers, but they are not cleaved by the bacteriophage T4 endonuclease V, a dimer-specific DNA repair enzyme. We demonstrate that the ligating dimers are also resistant to photolyase from Escherichia coli. Although the phosphodiester backbone is not required for dimer formation, it is required for recognition of dimers by these DNA repair enzymes. We discuss the possibility that high molecular weight polynucleotides in primordial seas might have been generated from oligonucleotides by pyrimidine dimerization under the intense solar UV flux unattenuated by an ozone layer.

Endonucleases↗
PubMed2003

Intracellular formation of "undisruptable" dimers of inducible nitric oxide synthase.

Overproduction of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in the pathogenesis of many diseases. iNOS is active only as a homodimer. Dimerization of iNOS represents a potentially critical target for therapeutic intervention. In this study, we show that intracellular iNOS forms dimers that are "undisruptable" by boiling, denaturants, or reducing agents. Undisruptable (UD) dimers are clearly distinguishable from the easily dissociated dimers formed by iNOS in vitro. UD dimers do not form in Escherichia coli-expressed iNOS and could not be assembled in vitro, which suggests that an in vivo cellular process is required for their formation. iNOS UD dimers are not affected by intracellular depletion of H4B. However, the mutation of Cys-115 (critical for zinc binding) greatly affects the formation of UD dimers. This study reveals insight into the mechanisms of in vivo iNOS dimer formation. UD dimers represent a class of iNOS dimers that had not been suspected. This unanticipated finding revises our understanding of the mechanisms of iNOS dimerization and lays the groundwork for future studies aimed at modulating iNOS activity in vivo.

Animals↗
PubMed1998

The crystal structure of a 3D domain-swapped dimer of RNase A at a 2.1-A resolution.

The dimer of bovine pancreatic ribonuclease A (RNase A) discovered by Crestfield, Stein, and Moore in 1962 has been crystallized and its structure determined and refined to a 2.1-A resolution. The dimer is 3D domain-swapped. The N-terminal helix (residues 1-15) of each subunit is swapped into the major domain (residues 23-124) of the other subunit. The dimer of bull seminal ribonuclease (BS-RNase) is also known to be domain-swapped, but the relationship of the subunits within the two dimers is strikingly different. In the RNase A dimer, the 3-stranded beta sheets of the two subunits are hydrogen-bonded at their edges to form a continuous 6-stranded sheet across the dimer interface; in the BS-RNase dimer, it is instead the two helices that abut. Whereas the BS-RNase dimer has 2-fold molecular symmetry, the two subunits of the RNase A dimer are related by a rotation of approximately 160 degrees. Taken together, these structures show that intersubunit adhesion comes mainly from the swapped helical domain binding to the other subunit in the "closed interface" but that the overall architecture of the domain-swapped oligomer depends on the interactions in the second type of interface, the "open interface." The RNase A dimer crystals take up the dye Congo Red, but the structure of a Congo Red-stained crystal reveals no bound dye molecule. Dimer formation is inhibited by excess amounts of the swapped helical domain. The possible implications for amyloid formation are discussed.

Animals↗
PubMed2000

Direct demonstration that homotetrameric chaperone SecB undergoes a dynamic dimer-tetramer equilibrium.

We have shown here that the cytosolic bacterial chaperone SecB is a structural dimer of dimers that undergoes a dynamic equilibrium between dimer and tetramer in the native state. We demonstrated this equilibrium by mixing two tetrameric species of SecB that can be distinguished by size. We showed that the homotetrameric species exchanged dimers, because when the mixture was analyzed both by size exclusion chromatography and native polyacrylamide gel electrophoresis a third hybrid tetrameric species was detected. Furthermore, treatment of SecB with 5,5'-dithiobis-(2-nitrobenzoic acid), which modifies the sulfhydryl group on cysteines, caused irreversible dissociation to a dimer indicating that cysteine must be involved in the stabilizing interactions at the dimer interface. It is clear that the two dimer-dimer interfaces of the SecB tetramer are differentially stable. Dissociation at one interface allows for a dynamic dimer-tetramer equilibrium. Because only dimers were exchanged it is clear that the other interface between dimers is significantly more stable, otherwise oligomers should have formed with a random distribution of monomers.

Bacterial Proteins↗
PubMed2002

Dimerization and phosphorylation of thyrotropin-releasing hormone receptors are modulated by agonist stimulation.

Dimerization and phosphorylation of thyrotropin-releasing hormone (TRH) receptors was characterized using HEK293 and pituitary GHFT cells expressing epitope-tagged receptors. TRH receptors tagged with FLAG and hemagglutinin epitopes were co-precipitated only if they were co-expressed, and 10-30% of receptors were isolated as hemagglutinin/FLAG-receptor dimers under basal conditions. The abundance of receptor dimers was increased when cells had been stimulated by TRH, indicating that TRH either stabilizes pre-existing dimers or increases dimer formation. TRH increased receptor dimerization and phosphorylation within 1 min in a dose-dependent manner. TRH increased phosphorylation of both receptor monomers and dimers, documented by incorporation of (32)P and an upshift in receptor mobility reversed by phosphatase treatment. The ability of TRH to increase receptor phosphorylation and dimerization did not depend on signal transduction, because it was not inhibited by the phospholipase C inhibitor. Receptor phosphorylation required an agonist but was not blocked by the casein kinase II inhibitor apigenin, the protein kinase C inhibitor GF109203X, or expression of a dominant negative form of G protein-coupled receptor kinase 2. TRH receptors lacking most of the cytoplasmic carboxyl terminus formed dimers constitutively but failed to undergo agonist-induced dimerization and phosphorylation. TRH also increased phosphorylation and dimerization of TRH receptors expressed in GHFT pre-lactotroph cells.

Amino Acid Sequence↗
PubMed2003

Natriuretic peptide receptor A activation stabilizes a membrane-distal dimer interface.

We have shown previously (Rondeau, J.-J., McNicoll, N., Gagnon, J., Bouchard, N., Ong, H., and De Léan, A. (1995) Biochemistry 34, 2130-2136) that atrial natriuretic peptide (ANP) stabilizes a dimeric form of the natriuretic peptide receptor A (NPRA) by simultaneously interacting with both receptor subunits. However, the first crystallographic study of unliganded NPRA extracellular domain documented a V-shaped dimer involving a membrane-proximal dimer interface and separate binding sites for ANP on each monomer. We explored the possibility of an alternative A-shaped dimer involving a membrane-distal dimer interface by substituting an unpaired solvent-exposed cysteine for Trp(74) in the amino-terminal lobe of full-length NPRA. The predicted spacing between Trp(74) from both subunits drastically differs, depending on whether the V-shaped (84 A) or the A-shaped (8 A) dimer model is considered. In contrast with the expected results for the reported V-shaped dimer, the NPRA(W74C) mutant was constitutively covalently dimeric. Also, the subunits spontaneously reassociated following transient disulfide reduction by dithiothreitol and reoxidation. However, ANP could neither bind to nor activate NPRA(W74C). Permanent disulfide opening by reduction with dithiothreitol and alkylation with N-ethylmaleimide rescued ANP binding to NPRA(W74C). The NPRA mutant could be maintained as a covalent dimer while preserving its function by crosslinking with the bifunctional alkylating agent phenylenedimaleimides (PDM), the ortho-substituted oPDM being more efficient than mPDM or pPDM. These results indicate that the membrane-distal lobe of the NPRAM extracellular domains are dynamically interfacing in the unliganded state and that ANP binding stabilizes the receptor dimer with more stringent spacing at the dimer interface.

Atrial Natriuretic Factor↗
PubMed1998

Dimerization of the polymeric immunoglobulin receptor controls its transcytotic trafficking.

Binding of dimeric immunoglobulin (Ig)A to the polymeric Ig receptor (pIgR) stimulates transcytosis of pIgR across epithelial cells. Through the generation of a series of pIgR chimeric constructs, we have tested the ability of ligand to promote receptor dimerization and the subsequent role of receptor dimerization on its intracellular trafficking. Using the cytoplasmic domain of the T cell receptor-zeta chain as a sensitive indicator of receptor oligomerization, we show that a pIgR:zeta chimeric receptor expressed in Jurkat cells initiates a zeta-specific signal transduction cascade when exposed to dimeric or tetrameric IgA, but not when exposed to monomeric IgA. In addition, we replaced the pIgR's transmembrane domain with that of glycophorin A to force dimerization or with a mutant glycophorin transmembrane domain to prevent dimerization. Forcing dimerization stimulated transcytosis of the chimera, whereas preventing dimerization abolished ligand-stimulated transcytosis. We conclude that binding of dimeric IgA to the pIgR induces its dimerization and that this dimerization is necessary and sufficient to stimulate pIgR transcytosis.

Animals↗
PubMed2002

Dissociation of the tubulin dimer is extremely slow, thermodynamically very unfavorable, and reversible in the absence of an energy source.

The finding that exchange of tubulin subunits between tubulin dimers (alpha-beta + alpha'beta' <--> alpha'beta + alphabeta') does not occur in the absence of protein cofactors and GTP hydrolysis conflicts with the assumption that pure tubulin dimer and monomer are in rapid equilibrium. This assumption underlies the many physical chemical measurements of the K(d) for dimer dissociation. To resolve this discrepancy we used surface plasmon resonance to determine the rate constant for dimer dissociation. The half-time for dissociation was approximately 9.6 h with tubulin-GTP, 2.4 h with tubulin-GDP, and 1.3 h in the absence of nucleotide. A Kd equal to 10(-11) M was calculated from the measured rate for dissociation and an estimated rate for association. Dimer dissociation was found to be reversible, and dimer formation does not require GTP hydrolysis or folding information from protein cofactors, because 0.2 microM tubulin-GDP incubated for 20 h was eluted as dimer when analyzed by size exclusion chromatography. Because 20 h corresponds to eight half-times for dissociation, only monomer would be present if dissociation were an irreversible reaction and if dimer formation required GTP or protein cofactors. Additional evidence for a 10(-11) M K(d) was obtained from gel exclusion chromatography studies of 0.02-2 nM tubulin-GDP. The slow dissociation of the tubulin dimer suggests that protein tubulin cofactors function to catalyze dimer dissociation, rather than dimer assembly. Assuming N-site-GTP dissociation is from monomer, our results agree with the 16-h half-time for N-site GTP in vitro and 33 h half-life for tubulin N-site-GTP in CHO cells.

Animals↗
PubMed1992

PCNA-induced DNA synthesis past cis-syn and trans-syn-I thymine dimers by calf thymus DNA polymerase delta in vitro.

Calf thymus proliferating cell nuclear antigen (PCNA) promoted DNA synthesis past cis-syn and trans-syn-I cyclobutane thymine dimers by calf thymus DNA polymerase delta (Pol delta) in vitro. Templates containing site-specific cis-syn and trans-syn-I thymine dimers were prepared via a combination of solid phase synthesis with photoproduct building blocks and DNA ligation. Extension of a 15-mer primer on the UV dimer-containing templates by Pol delta produced termination and bypass products in a dNTP and PCNA dependent manner. In the absence of PCNA and at dNTP concentrations varying between 1 and 100 microM, Pol delta could not bypass the cis-syn dimer and terminated elongation one nucleotide prior to the 3'-T of the dimer. DNA synthesis past the trans-syn-I dimer was even less efficient. In the presence of PCNA, termination occurred primarily one nucleotide prior to the 3'-T of both dimers at 1 microM dNTPs but opposite the 5'-T of the dimers at 100 microM dNTPs. In addition, under the latter conditions, bypass of the dimers was observed, to the extent of about 30% of the products for the cis-syn dimer and about 15% for the trans-syn-I dimer.

Animals↗
PubMed2004

Splicing affects presentation of RNA dimerization signals in HIV-2 in vitro.

During retroviral replication, full-length viral RNAs are encapsidated into new virus particles, while spliced RNAs are excluded. The Retroviridae are unique among viruses in that infectious viral particles contain a dimer of two identical genomic RNA strands. A variety of experimental data has suggested that dimerization and encapsidation of full-length viral RNAs are linked processes, although whether dimerization is a prerequisite for encapsidation, or conversely, dimerization follows encapsidation, has not been firmly established. If dimerization was the sole determinant for encapsidation, then spliced viral RNAs might be expected to display a reduced capacity for dimerization, resulting in their exclusion from the dimerization pool. Here, we studied the in vitro dimerization properties of unspliced and spliced HIV-2 RNA. We find that the rate and yield of dimerization of Nef, Rev and Tat spliced RNAs exceeded those of unspliced RNA. Although these data do not support a simple correlation between dimerization efficiency and the presence of introns, they establish that splicing affects the presentation of dimerization signal(s), which we corroborate with structure probing. This change in RNA conformation likely affects the RNA's suitability for packaging. Furthermore, the presence of upstream and downstream elements that affect the conformation of the packaging signal represents a potentially efficient viral strategy for correctly sorting spliced versus unspliced RNAs.

Base Sequence↗
PubMed2004

Role of lipid in sulfite-dependent propofol dimerization.

BACKGROUND: During long-term intravenous infusions, sulfite in sulfite-containing propofol emulsions can cause the peroxidation of lipid and dimerization of propofol. This study evaluated the role of lipid in sulfite-dependent propofol dimerization by determining the effects of individual fatty acids in soybean oil emulsion and peroxidized lipids in a model system. METHODS: Individual fatty acids, stearic (18:0), oleic (18:1), linoleic (18:2), linolenic (18:3), and arachidonic (20:4), were added to sulfite-containing propofol emulsion and incubated for 90 min at 37 degrees C. Model systems containing soybean oil (100 microl), water (900 microl), propofol (10 mg/ml), and sulfite (0.25 mg/ml) composed of oils with different peroxide values were allowed to react for 60 min at room temperature. After the reactions, propofol dimer and propofol dimer quinone were analyzed by reversed-phase high-pressure liquid chromatography. RESULTS: Propofol did not dimerize when added to aqueous sulfite unless soybean oil was also included. The addition of the polyunsaturated fatty acids (linoleic, linolenic, arachidonic) to sulfite-containing propofol emulsion resulted in large increases of propofol dimerization compared with stearic or oleic acid. Using biphasic mixtures of soybean oil and aqueous sulfite, propofol dimerization increased with increasing peroxide content of the oil. In propofol emulsion, lipoxidase and ferrous iron in the absence of sulfite also caused the dimerization of propofol. CONCLUSIONS: These results show that lipid can play a significant role in sulfite-dependent propofol dimerization. The relation of dimerization to polyunsaturated fatty acid and soybean oil peroxide content suggests that sulfite reacts with unsaturated lipid or peroxide-modified lipid to facilitate propofol dimerization.

Dimerization↗
PubMed1992

Flavin-sensitized photochemically induced dynamic nuclear polarization detection of pyrimidine dimer radicals.

A photochemically induced dynamic nuclear polarization (photo-CIDNP) study of carboxymethyllumiflavin-sensitized splitting of pyrimidine dimers has been carried out. In aqueous solution at high pH, an emission signal (delta 3.9 ppm) was observed from the dimer C(6)- and C(6')-protons of an N(1), N(1')-trimethylene-bridged thymine dimer (1). The dimer photo-CIDNP signal was seen only above pD 11.6 and was most intense at pD 12.9. Also observed were weak enhanced absorption signals from the product of splitting, trimethylenebis(thymine) (delta 1.7 and 7.2 ppm). In contrast, cis, syn-thymine dimer (3) gave no photo-CIDNP signals from the dimer. An enhanced absorption at 1.8 ppm, however, due to the product of splitting (thymine) was observed. It was found that dimer 1 and, to a lesser extent, dimer 3 quenched flavin fluorescence. An N(3),N(3')-dimethylated derivative of 1, however, failed to quench flavin fluorescence. Comparison of the pD profile of the dimer photo-CIDNP signal to the pKa values for thymidine dimer suggested that principally the dideprotonated dimer undergoes electron abstraction by the excited flavin.

Flavins↗
PubMed1995

Photo-CIDNP study of pyrimidine dimer splitting. I: Reactions involving pyrimidine radical cation intermediates.

The light-induced splitting of pyrimidine dimers was studied using the electron acceptor anthraquinone-2-sulfonate (AQS) as a photosensitizer. To this end, photochemically induced dynamic nuclear polarization (photo-CIDNP) experiments were performed on a series of pyrimidine monomers and dimers. The CIDNP spectra demonstrate the existence of both the dimer radical cation, which is formed by electron transfer from the dimer to the photoexcited sensitizer AQS*, and its dissociation product, the monomer radical cation. In spectra of 1,1'-trimethylene bridged cis, syn pyrimidine dimers, polarization is observed that originates from a spin-sorting process in the dimer radical pair. This points to a relatively long lifetime of the dimer radical cation involved, which is presumably due to stabilization by the trimethylene bridge. Polarization originating from a dimer radical pair is detected in the spectrum of trans,anti (1,3-dimethyluracil) dimer as well. The spectra of the bridged pyrimidines also demonstrate the reversibility of the dissociation of dimer radical cation into monomer radical cation, which is concluded from the observation of polarization in the dimer as a result of spin sorting in the monomer radical pair.

Cations↗
PubMed2001

Role of stem B, loop B, and nucleotides next to the primer binding site and the kissing-loop domain in human immunodeficiency virus type 1 replication and genomic-RNA dimerization.

Stem-loop B is a 12-nucleotide [nt]-long completely conserved sequence postulated to form a 4-bp stem and a 4-nt internal loop under the kissing-loop hairpin (klh) (nt 248 to 270) of human immunodeficiency virus type 1 (HIV-1) genomic RNA. We investigated its role in viral replication, genomic RNA dimerization, and dimerization of partial HIV-1 RNA transcripts. The putative CUCG246-CGAG277 duplex was replaced by nine alternative complementary sequences, five likely to base pair only in short RNAs and four likely to base pair in long (approximately 500-nt) RNAs, as assessed by the algorithm mfold. Among the five former sequences, none preserved genome dimerization and all reduced viral replication by 98 to 99.9%. Among the four latter sequences, three (MB6, -9, and -10) preserved genome dimerization, one (MB7) did not significantly inhibit it, and two (MB9 and -10) preserved viral replication. We conclude that duplex formation by stem B nucleotides is necessary for viral infectivity and complete genome dimerization. Deleting the 5' or 3' side of loop B or of stem B had little impact on dimerization of partial RNA transcript and no impact on klh folding (and, for loop B mutations, on stem B folding), but each deletion inhibited genome dimerization almost as much as klh destruction. This suggests that loop B is required for complete genome dimerization and that loop B and stem B stimulate dimerization only in very long RNAs and/or in the presence of unidentified viral and cellular factors. Finally, we asked if nine deletions or nucleotide substitutions within nt 200 to 242 and/or nt 282 to 335 could influence genome dimerization. These mutations had intermediate inhibitory impacts consistent with their predicted influence on stem B, loop B, and klh formation. Two exceptions were Delta200-226 and Delta236-242 genomic RNAs, which dimerized relatively poorly despite having neutral or positive influences on stem B, loop B, and klh folding.

Animals↗