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PubMed1992

DNA-induced dimerization of the Escherichia coli rep helicase. Allosteric effects of single-stranded and duplex DNA.

The Escherichia coli Rep helicase is a stable monomer (Mr = 72,802) in the absence of DNA; however, binding of single-stranded (ss) or duplex (ds) DNA induces Rep monomers to dimerize. Furthermore, a chemically cross-linked Rep dimer retains both its DNA-dependent ATPase and helicase activities, suggesting that the functionally active Rep helicase is a dimer (Chao, K., and Lohman, T. M. (1991) J. Mol. Biol. 221, 1165-1181). Using a modified "double-filter" nitrocellulose filter binding assay, we have examined quantitatively the equilibrium binding of Rep to a series of ss-oligodeoxynucleotides, d(pN)n (8 less than or equal to n less than or equal to 20) and two 16-base pair duplex oligodeoxynucleotides, which are short enough so that only a single Rep monomer can bind to each oligonucleotide. This strategy has enabled us to examine the linkage between DNA binding and dimerization. We also present a statistical thermodynamic model to describe the DNA-induced Rep dimerization in the presence of ss- and/or ds-oligodeoxynucleotides. We observe quantitative agreement between this model and the experimental binding isotherms and have analyzed these isotherms to obtain the seven independent interaction constants that describe Rep-DNA binding and Rep dimerization. We find that Rep monomers (P) can bind either ss-DNA (S) or ds-DNA (D) to form PS or PD, respectively, which can then dimerize to form P2S or P2D. Furthermore, both protomers of the DNA-induced Rep dimer can bind DNA to form either P2S2, P2D2 or the mixed dimer species P2SD and ss- and ds-DNA compete for the same sites on the Rep protein. When bound to DNA, the Rep dimerization constants are approximately 1-2 x 10(8) M-1 (6 mM NaCl, pH 7.5, 4 degrees C), which are greater than the dimerization constant for free Rep monomers by at least 10(4)-fold. The Rep-ss-DNA interaction constants are independent of base composition and sequence, consistent with its role as a nonspecific DNA-binding protein. Allosteric effects are associated with ss- and ds-DNA binding to the half-saturated Rep dimers, i.e. the affinity of either ss- or ds-DNA to the free promoter of a half-saturated Rep dimer is clearly influenced by the conformation of DNA bound to the first protomer. These allosteric effects further support the proposal that the Rep dimer is functionally important and that the Rep-DNA species P2S2 and P2SD may serve as useful models for intermediates that occur during DNA unwinding.(ABSTRACT TRUNCATED AT 400 WORDS)

Adenosine Triphosphatases↗

PubMed1983

Dimers of porcine skeletal muscle lactate dehydrogenase produced by limited proteolysis during reassociation are enzymatically active in the presence of stabilizing salt.

Lactate dehydrogenase from pig skeletal muscle is known to be a "dimer of dimers" stabilized in its tetrameric state by an N-terminal sequence of 20 aminoacid residues. Limited proteolysis of dimeric intermediates of association with thermolysin prevents association of the enzyme to its tetrameric structure. The resulting stable "dimers" are still capable of binding to a Procion Green dinucleotide affinity column. This may indicate that the dimeric intermediates contain the dinucleotide fold of the native enzyme. Since this structural feature is preserved after thermolysin treatment, affinity chromatography may be applied to separate the "dimer" on a preparative scale. Endgroup analysis and peptide mapping of the proteolytic dimer indicates that the reaction product consists of intact chains lacking the "N-terminal arm", apart from "nicked subunits" with fragments of 18 000 and 12 000 molecular weight. According to its hydrodynamic and conformational characteristics (sedimentation velocity and circular dichroism), the "dimer" does not differ significantly from the native enzyme in the backbone structure of its subunits. Its sedimentation properties resemble those of other dimeric dehydrogenases. The spectral data are similar to those observed for the dimeric intermediates that form during reconstitution after acid denaturation. Under standard test conditions, the proteolytic "dimer" (as well as the dimeric intermediate on the pathway of folding) do not show enzymatic activity. However, in the presence of "structure-making ions" like 2 M ammonium sulfate, about 40% of the native catalytic function is restored in the dimeric state. As shown by activity transport measurements in the ultracentrifuge, both the native tetramer and the proteolytic "dimer" maintain their quaternary structure in this solvent.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1976

Tetramer-dimer dissociation in homoglobin and the Bohr effect.

The pH dependence of the apparent tetramer to dimer dissociation constant has been determined at 20 degrees for both oxy- and deoxyhemoglobins A and Kansas. These measurements were made by three different procedures: gel chromatography, sedimentation velocity, and kinetic methods in either of three buffer systems: 0.05 M cacodylate, Tris, or glycine with 1 mM EDTA and 0.1 M NaCl between pH 6.5 and 11. The tetramer-dimer dissociation constant of human oxyhemoglobin A decreases from about 3.2 X 10(-6) M at pH 6.0 to about 3.2 X 10(-8) M at pH 8.5. The slope of this line indicates that the dissociation of tetramer to dimer is accompanied by the uptake of about 0.6 protons per mol of tetramer in this region. The corresponding dissociation constant for deoxyhemoglobin in the same pH region increases apparently almost linearly from 1.0 x 10(-12) M at pH 6.5 to about 1.0 x 10(-5) M at pH 11. To dimer is associated with the release of about 1.6 protons per mol of tetramer. Comparison of these data with the known proton release accompanying the oxygenation of tetramers confirms that the pH dependence of oxygen binding by dimers must be very small. The present data predict that the overall proton release or uptake per oxygen bound by dimer should be less than 0.1. The tetramer-dimer dissociation equilibria of oxy- and deoxyhemoglobins above pH 8.5 have identical pH dependences. In this range the dissociation constant of deoxy-Hb is about one-tenth that of oxyhemoglobin. Human oxyhemoglobin Kansas is known to have an enhanced tetramer-dimer dissociation compared with that of hemoglobin A. Below pH 8.5 the tetramer-dimer dissociation constant of Hb Kansas is about 400 times greater than that of HbA in the absence of phosphate buffers. In contrast, the tetramer-dimer dissociation constants of deoxyhemoglobins A and Kansas appear to be identical. These findings are consistent with previous structural observations on these hemoglobins. The data on the tetramer-dimer dissociation of human hemoglobin were used to calculate the total free energy of binding of oxygen to the tetramer and the median oxygen pressure on the basis of fundamental linkage relations and a pH-independent estimate of the total free energy of binding oxygen to dimer. Simulated oxygen binding curves were generated with the equations of Ackers and Halvorson (Ackers, G. K., and Halvorson, H. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 4312-4316) by making two assumptions: (a) that the dimers are noncooperative and pH-independent in O2 binding and (b) that the distribution of cooperative energy in the oxygenation of tetramers is independent of pH. We have compared these simulations with experimental data obtained at low protein concentrations (30 to 124 muM heme) to show that the variation in oxygen affinity with pH can be described in terms of the subunit equilibria. We conclude that an accurate analysis of the contributions of individual oxygen binding steps to the Bohr effect cannot be made without considering the contributions of the dimers to oxygen binding...

Hemoglobins↗

PubMedDate not supplied

The trans-syn-I thymine dimer bends DNA by approximately 22 degrees and unwinds DNA by approximately 15 degrees.

Irradiation of DNA with ultraviolet light leads to the formation of two classes of cyclobutane dimers at adjacent thymines sites, of which the cis-syn is the major class and the trans-syn is the minor class. While the structure and properties of DNA containing cis-syn thymine dimers have been extensively studied, virtually nothing is known about DNA containing trans-syn thymine dimers. To investigate the bending and unwinding of DNA induced by the trans-syn-I thymine dimer, the electrophoretic properties of oligomers of trans-syn-I dimer-containing DNA duplexes were studied. Oligonucleotides 10, 11, and 12 bp in length containing a centrally located trans-syn-I thymine dimer were synthesized, polymerized, and analyzed by polyacrylamide gel electrophoresis. In contrast to the small bending angle (approximately 7 degrees) induced by the cis-syn thymine dimer, we found that trans-syn-I thymine dimer bends DNA significantly (approximately 22 degrees). Both dimers, however, are found to unwind DNA by the same amount (approximately 15 degrees). On the basis of previous NMR studies, it appears that the bend of the trans-syn-I dimer is localized at the 5'-side of the dimer. Gel electrophoretic analysis of multimers of two 11-mers containing a cis-syn thymine dimer at the 5'-end and at the center of a dT6.dA6 tract confirmed our previous estimates for the bending angles of thymine dimer-containing T6 tracts. The substrates reported may be useful in determining how general repair enzymes recognize DNA damage.

Autoradiography↗

PubMed1991

Site-specific effect of thymine dimer formation on dAn.dTn tract bending and its biological implications.

dAn.dTn sequences, otherwise known as A tracts, are hotspots for cis-syn thymine dimer formation and deletion mutations induced by UV light. Such A tracts are also known to bend DNA, suggesting that some biological effects of UV light might be related to the distinctive structure and properties of cis-syn dimer-containing A tracts. To investigate the effect of thymine dimer formation on A-tract bending multimers of all possible dimer monoadducts of a dA6.dT6-containing decamer known to bend DNA were prepared along with multimers of a dimer-containing 21-mer of heterogeneous sequence. The characteristic anomalous electrophoretic behavior of the phased A-tract multimers was essentially abolished by dimer formation at the center of the A tract and was only slightly reduced by dimer formation at the ends. These effects are attributed to disruption of the A-tract structure at the site of the dimer, resulting in intact A tracts of reduced length and, hence, reduced bending. This model was suggested by the ability to formulate the estimated bend angles of the dimer-containing A tracts as approximately equal to the sum of the bend angles induced by the dimer and the remaining intact portion of the A tract. Contrary to a previous experimental study that concluded that the thymine dimer bends DNA by approximately 30 degrees, the dimer was determined to bend DNA by only approximately 7 degrees. Reduction of the bending of a DNA sequence by dimer formation may have a number of unpredicted and important biological consequences.

Base Composition↗

PubMed1990

A study of the dimer formation of Rous sarcoma virus RNA and of its effect on viral protein synthesis in vitro.

The genetic material of all retroviruses examined so far is an RNA dimer where two identical RNA subunits are joined at their 5' ends by a structure named dimer linkage structure (DLS). Since the precise location and structure of the DLS as well as the mechanism and role(s) of RNA dimerization remain unclear, we analysed the dimerization process of Rous sarcoma virus (RSV) RNA. For this purpose we set up an in vitro model for RSV RNA dimerization. Using this model RSV RNA was shown to form dimeric molecules and this dimerization process was greatly activated by nucleocapsid protein (NCp12) of RSV. Furthermore, RSV RNA dimerization was performed in the presence of complementary 5'32P-DNA oligomers in order to probe the monomer and dimer forms of RSV RNA. Data indicated that the DLS of RSV RNA probably maps between positions 544-564 from the 5' end. In an attempt to define sequences needed for the dimerization of RSV RNA, deletion mutageneses were generated in the 5' 600 nt. The results showed that the dimer promoting sequences probably are located within positions 208-270 and 400-600 from the 5' end and hence possibly encompassing the cis-acting elements needed for the specific encapsidation of RSV genomic RNA. Also it is reported that synthesis of the polyprotein precursor Pr76gag is inhibited upon dimerization of RSV RNA. These results suggest that dimerization and encapsidation of genome length RSV RNA might be linked in the course of virion formation since they appear to be under the control of the same cis elements, E and DLS, and the trans-acting factor nucleocapsid protein NCp12.

Avian Sarcoma Viruses↗

PubMed1992

Analytical study of avian reticuloendotheliosis virus dimeric RNA generated in vivo and in vitro.

The retroviral genome consists of two identical RNA molecules associated at their 5' ends by a stable structure called the dimer linkage structure. The dimer linkage structure, while maintaining the dimer state of the retroviral genome, might also be involved in packaging and reverse transcription, as well as recombination during proviral DNA synthesis. To study the dimer structure of the retroviral genome and the mechanism of dimerization, we analyzed features of the dimeric genome of reticuloendotheliosis virus (REV) type A and identified elements required for its dimerization. Here we report that the REV dimeric genome extracted from virions and infected cells, as well as that synthesized in vitro, is more resistant to heat denaturation than avian sarcoma and leukemia virus, murine leukemia virus, or human immunodeficiency virus type 1 dimeric RNA. The minimal domain required to form a stable REV RNA dimer in vitro was found to map between positions 268 and 452 (KpnI and SalI sites), thus corresponding to the E encapsidation sequence (J. E. Embretson and H. M. Temin, J. Virol. 61:2675-2683, 1987). In addition, both the 5' and 3' halves of E are necessary in cis for RNA dimerization and the extent of RNA dimerization is influenced by viral sequences flanking E. Rapid and efficient dimerization of REV RNA containing gag sequences in addition to the E sequences and annealing of replication primer tRNA(Pro) to the primer-binding site necessitate the nucleocapsid protein.

3T3 Cells↗

PubMed1990

D-dimer and TAT measurement in patients with deep venous thrombosis: utility in diagnosis and judgement of anticoagulant treatment effectiveness.

The plasma levels of thrombin-antithrombin III-complexes (TAT) and the fibrin split product D-Dimer were measured in 39 patients with phlebographically proven acute DVT: 34 patients had proximal DVT, 5 had calf DVT. The sensitivity of D-Dimer and TAT measurements in the diagnosis of proximal DVT was found to be dependent on the duration of symptoms: 0 to 7 days (n = 27): elevated D-Dimer levels (greater than 120 ng/ml) = 1, D-Dimer Latex test positive (greater than 500 ng/ml) = 1, elevated TAT levels (greater than 6 ng/ml) = 0.88. Eight to 14 days (n = 7): elevated D-Dimer levels = 1, D-Dimer Latex test positive = 0.33, elevated TAT levels = 0.66; specificity: elevated D-Dimer: 0.48, D-Dimer Latex test: 1, elevated TAT: 0.76. Calf DVT patients (n = 5) had elevated D-Dimer levels, negative Latex tests and 3 of them had normal TAT values. Hemostatic and fibrinolytic parameters were also determined in 13 patients during heparin treatment of proximal DVT. Elevated D-Dimer and TAT levels rapidly decreased after initiation of anticoagulant therapy. In 2 of 13 patients a marked increase in D-Dimer and TAT levels was observed in periods of ineffective heparinization, documented by normal or only slightly prolonged thrombin clotting times. We conclude from our results that 1) D-Dimer EIA measurement, in contrast to TAT measurement, shows a very high sensitivity in the diagnosis of DVT, 2) due to low specificity this test can only be used to exclude thrombosis in patients with suspected DVT, and 3) the determination of the plasma levels of D-Dimer and TAT may be useful for judging the effect of anticoagulant treatment on thrombotic processes.

Adult↗

PubMed1990

Chloranil-photosensitized monomerization of dimethylthymine cyclobutane dimers and effect of magnesium perchlorate.

The photosensitized monomerization of the cyclobutane dimers of 1,3-dimethylthymine by p-chloranil was investigated by means of steady-state irradiation and laser-flash photolysis. Quantum yields for the monomerization are 0.34 for the cis,syn dimer, 0.39 for the trans,syn dimer, and much less than 10(-2) for the cis,anti isomer. Formation of the chloranil anion radical associated with quenching of triplet chloranil by the dimers demonstrates that electron transfer from dimers to triplet chloranil occurs to initiate the monomerization. Kinetic analysis suggested that the syn-dimer cation radicals undergo the ring cleavage at greater than or equal to 10(9) s-1 before escaping from the solvent cage, while the reactivity of the anti-dimer cation radical is very low. The different reactivities of the syn and anti dimer cation radicals are discussed in terms of through-bond coupling between the n orbitals of N(1) and N(1') involving the cyclobutane-ring sigma orbitals. In the cases of the syn-dimers, the sensitizer-dimer ion-radical pairs undergo the rapid geminate recombination that works as a major energy dissipating channel responsible for the lower-than-unity quantum yields. It has been found that the presence of Mg(ClO4)2 at 0.1 M enhances approximately 1.5 times either the monomerization of the syn dimers or the formation of the chloranil anion radical. A laser-flash photolysis study shows that Mg2+ forms a complex with either the triplet or the anion radical of chloranil. The net salt effects are attributed to the retardation of the rapid geminate recombination by the participation of Mg2+ in the sensitizer-dimer ion-radical pairs.

Chloranil↗

PubMed1990

Photo-CIDNP detection of pyrimidine dimer radical cations in anthraquinonesulfonate-sensitized splitting.

Anthraquinone-2-sulfonate (AQS) photosensitizes pyrimidine dimer splitting. Electron abstraction from the dimer is thought to induce dimer splitting, but direct evidence for the existence and intermediacy of dimer radical cations has been lacking. By employing photochemically induced dynamic nuclear polarization, we have found emission signals in the NMR spectra of dimers upon photolysis of dimers in the presence of anthraquinone-2-sulfonate. The two dimers employed were cis, syn-thymine dimer in which the N(1)-positions were linked by a three-carbon bridge and the N(3), N(3')-dimethyl derivative of that compound. The anthraquinone-2-sulfonate sensitized photochemically induced dynamic nuclear polarization spectrum of the methylated derivative exhibited an emission signal from the dimer-C(6) hydrogens. This result implied the existence of a dimer radical cation (mD+.) formed by electron abstraction by excited anthraquinone-2-sulfonate and nuclear spin sorting within a solvent caged radical ion pair [mD+. AQS-.]. Product pyrimidine photochemically induced dynamic nuclear polarization signals were also seen [enhanced absorption by C(6)-hydrogens and emission by C(5)-methyl groups]. Nuclear spin polarization in the product resulted from spin sorting in one or more of its precursors, including mD+. The results support the conclusion that dimer radical cations not only exist but are intermediates in the photosensitized splitting of pyrimidine dimers by anthraquinonesulfonate.

Anthraquinones↗

PubMed1992

A new class of bradykinin antagonists: synthesis and in vitro activity of bissuccinimidoalkane peptide dimers.

A systematic study on the dimerization of the bradykinin (BK) antagonist D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Phe 7-Leu8-Arg9 has been performed. The first part of this study involved compounds wherein dimerization was carried out by sequentially replacing each amino acid with cysteine and cross-linking with bismaleimidohexane. The second part of this study utilized a series of bissuccinimidoalkane dimers wherein the intervening methylene chain was varied systematically from n = 2 to n = 12 while the point of dimerization was held constant at position 6. The biological activities of these dimers were then evaluated on BK-induced smooth muscle contraction in two different isolated tissue preparations: guinea pig ileum (GPI) and rat uterus (RU). Several of the dimeric BK antagonists displayed remarkable activities and long durations of action. In addition, dimerization at position 4, 7, 8, or 9 produced dimeric analogues with markedly reduced potency. Rank order of antagonist potency as a function of dimerization position is as follows: rat uterus, 6 greater than 5 greater than 0 greater than 2 greater than 1 greater than 3 much greater than 4, 7, 8, 9; guinea pig ileum, 6 greater than 5 greater than 3 greater than 2 greater than 1 greater than 0 much greater than 4, 7, 8, 9. Evaluation of the linker length as represented by the number of methylene units indicated an optimal distance between the two monomeric peptides of six to eight methylene moieties. These studies also revealed that the carbon-chain length significantly affected the duration of action in vitro and resulted in partial agonism effects when n greater than 8. The optimum activity in vitro was achieved with dimerization at position 6 and n = 6 (designated herein as compound 25; alternatively, CP-0127). Similar effects in potency were also seen when the monomeric antagonist D-Arg0-Arg1-Pro2-Hyp3-Gly4-Phe5-Ser6-D-Phe 7-Phe8-Arg9 (NPC-567) was dimerized using similar chemistry. These results suggest that the development of BK antagonists of significant therapeutic potential may be possible using a dimerization strategy that can overcome the heretofore limiting problems of potency and in vivo duration of action found with many of the BK antagonists in the literature.

Amino Acid Sequence↗

PubMed1995

Functional differences between dimeric and octameric mitochondrial creatine kinase.

Mitochondrial creatine kinase (Mi-CK) consists of octameric and dimeric molecules that are interconvertible. In the present study, the kinetic properties of purified chicken heart Mi-CK (Mib-CK) dimers and octamers were investigated separately under highly controlled conditions. Gel-permeation chromatography was performed before and after kinetic measurements in order to clearly define the proportions of octamers and dimers. 'Dimeric' Mi-CK solutions consisted of > or = 90% dimers throughout the experiment whereas 'octameric' Mi-CK solutions consisted in the beginning of 90% octamers, but upon measuring with the highest concentrations of creatine (Cr) and ATP approximately one-third of the octamers dissociated into dimers. These proper controls enabled us to pinpoint the observed kinetic differences between dimers and octamers solely to the oligomeric state of Mib-CK. Both dimeric and octameric Mi-CK displayed synergism in substrate binding (Kd values are higher than Km values), meaning that binding of the first substrate facilities subsequent binding of the second substrate. Most interestingly, Km(Cr) and Kd(Cr) values are both 2-3 times higher for octameric than for dimeric Mi-CK. Thus, at low Cr concentrations, the dimer is kinetically favoured for the forward direction of the reaction (phosphorylcreatine synthesis) compared with the octamer. The possible physiological significance of the lower Kd(Cr) value of dimeric versus octameric Mib-CK, as well as the apparent negative cooperativity of ATP binding at higher [Cr], are discussed within the context of a possible functional role for dimeric Mib-CK in vivo.

Animals↗

PubMed1987

Measurement of plasma fibrin D-dimer levels with the use of a monoclonal antibody coupled to latex beads.

Recently, monoclonal antibody (DD-3B6) to fibrin D-dimer was prepared and coupled to latex beads to provide a specific test (Dimertest) for fibrinolysis. The purpose of this study was to evaluate the Dimertest assay as a clinical laboratory test for the measurement of plasma fibrin D-dimer derivatives. The Dimer-test assay specifically detected 2 micrograms/mL of purified fibrin D-dimer or fibrin D-dimer/fragment E complex added to afibrinogenemic plasma but did not detect 500 micrograms/mL of either fibrinogen fragments X, D, E, or 160 micrograms/mL cross-linked fibrinogen. The fibrin(ogen) degradation product (FDP) assays of American Dade or Wellcome Diagnostics detected 5.0 micrograms/mL of fibrin D-dimer and from 1 to 10 micrograms/mL of the other FDPs. Twenty-eight percent of 150 random plasma samples assayed from hospitalized patients were positive for fibrin D-dimer derivatives. Plasma samples from 152 patients suspected of having disseminated intravascular coagulation (DIC) were assayed for serum FDP (Wellcome Diagnostics) and plasma fibrin D-dimer derivatives. Samples from 69% of patients with serum FDP levels less than 10 micrograms/mL, and more than 90% of those with serum FDP levels greater than 10 micrograms/mL, were positive for fibrin D-dimer derivatives. Dimertest results were not modified by heparin, streptokinase, freeze-thawing, or clotting plasma. Serum fibrinogen-related antigens were immunoadsorbed from Dimer-test positive sera by anti-fibrinogen antibody and formalin-fixed Cowan I strain Staphylococcus aureus. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and protein blotting with the use of monoclonal antibody DD-3B6 demonstrated a protein band with similar mobility to purified D-dimer. The measurement of plasma fibrin D-dimer derivatives by the Dimertest assay is a rapid, sensitive, and specific laboratory test for fibrinolysis. The Dimertest assay has proven to be a useful addition to the clinical laboratory and should be helpful in the diagnosis and management of patients with diseases associated with fibrinolysis.

Antibodies, Monoclonal↗

PubMed1991

Dimerization of human immunodeficiency virus (type 1) RNA: stimulation by cations and possible mechanism.

The retroviral genome consists of two identical RNA molecules joined close to their 5' ends by the dimer linkage structure. Recent findings indicated that retroviral RNA dimerization and encapsidation are probably related events during virion assembly. We studied the cation-induced dimerization of HIV-1 RNA and results indicate that all in vitro generated HIV-1 RNAs containing a 100 nucleotide domain downstream from the 5' splice site are able to dimerize. RNA dimerization depends on the concentration of RNA, mono- and multivalent cations, the size of the monovalent cation, temperature, and pH. Up to 75% of HIV-1 RNA is dimeric in the presence of spermidine. HIV-1 RNA dimer is fairly resistant to denaturing agents and unaffected by intercalating drugs. Antisense HIV-1 RNA does not dimerize but heterodimers can be formed between HIV-1 RNA and either MoMuLV or RSV RNA. Therefore retroviral RNA dimerization probably does not simply proceed through mechanisms involving Watson-Crick base-pairing. Neither adenine and cytosine protonation, nor quartets containing only guanines appear to determine the stability of the HIV-1 RNA dimer, while quartets involving both adenine(s) and guanine(s) could account for our results. A consensus sequence PuGGAPuA found in the putative dimerization-encapsidation region of all retroviral genomes examined may participate in the dimerization process.

Avian Sarcoma Viruses↗

PubMed1996

Nucleocapsid protein 10 activates dimerization of the RNA of Moloney murine leukaemia virus in vitro.

Short RNA species that encompass the psi domain of the retroviral genome spontaneously form dimers in vitro, and the retroviral nucleocapsid protein activates this dimerization in vitro. Addition of gag RNA sequences downstream of the 3' end of the psi domain decreases the level of spontaneous dimerization. Here, we report the effects of RNA length on dimerization in vitro, studied with RNA fragments from Moloney murine leukaemia virus that contain the psi domain and all or part of the gag sequence. Extension of the RNA leads to progressive inhibition of the in vitro dimerization process. Sequences located downstream of the 3' end of the psi domain seem to stabilize the monomeric structures. This stabilization participates in dimerization of the RNA sequences involved in the recognition of two RNA molecules. We studied the ability of nucleocapsid protein 10 to promote dimerization of such long RNA fragments, and found that the protein greatly enhances their dimerization in vitro. We propose that nucleocapsid protein 10 stimulates the overall dimerization process by reduction of the energy barrier that must be overcome to allow dimer formation. Our results show that dimerization of RNA form Moloney murine leukaemia virus in vitro is enhanced by nucleocapsid protein 10. This finding is in agreement with the involvement of the nucleocapsid protein in RNA dimerization in vivo.

Base Sequence↗

PubMed1990

Beta gamma dimers of G proteins inhibit atrial muscarinic K+ channels.

It has been proposed that beta gamma dimers of signal-transducing G proteins mediate muscarinic activation of atrial K+ channels. We examined this hypothesis by testing the effects of beta gamma dimers from four sources (human erythrocytes, human placenta, bovine brain, and bovine retina) on single channel muscarinic K+ (K+[acetylcholine (ACh)]) currents in inside-out membrane patches of adult guinea pig atria. None of the four beta gamma dimer preparations stimulated K+[ACh] currents; on the contrary, each inhibited the currents whether the currents were activated with GTP alone (agonist-independent activity) or with GTP plus a muscarinic agonist (agonist-dependent activity). Detergents at concentrations used to suspend erythrocyte, brain, and placental beta gamma dimers had no effect by themselves, and detergents were not used with the retinal beta gamma dimers. We conclude that beta gamma dimers do not mediate stimulatory effects of the endogenous G protein that regulates the K+ channels. In fact beta gamma dimers appear to inhibit activation by the endogenous G alpha subunits. Further insight into the role of beta gamma dimers came from the observation that agonist-independent GTP-activated K+[ACh] currents were inhibited by beta gamma dimers at about one-tenth the concentration required to inhibit agonist-dependent activation. One possibility is that dimeric beta gamma may have a higher affinity for free alpha subunits than for alpha subunits associated with agonist-occupied receptors. Thus, in addition to the known requirement of beta gamma dimers for the interaction of alpha subunits with receptors, beta gamma dimers may also improve the signal-to-noise ratio for agonists by reducing agonist-independent background activities.

Acetylcholine↗

PubMed1990

A view of the human idiotypic repertoire. Electron microscopic and immunologic analyses of spontaneous idiotype-anti-idiotype dimers in pooled human IgG.

It has previously been reported that up to 40% of the molecules of human IgG from pooled plasma (100,000 donations) spontaneously dimerize, whereas IgG prepared from a single donor contains only trace quantities of dimer. We have conducted immunoelectron microscopic analyses on such samples and have verified that the majority of dimers are composed of complexes in which two arms of each molecule are bound in a reciprocal fashion at or near the distal tips of their respective arms as previously seen in bona fide Id-anti-Id complexes. A significant role for Fc was ruled out by showing the formation of dimeric ring structures in purified F(ab')2 samples. Spontaneous dimerization was also observed in pooled bovine or mouse IgG, but not in that from single animals. Radiolabeled monomeric, single donor human IgG was used as a probe to investigate dimer formation; this material readily codimerized with IgG from other donors or from pooled plasma (either human or bovine), but did not dimerize with IgG from the same donor. Subclass analysis of multiple donor human IgG revealed that the most flexible subclass (IgG3) was overrepresented in the dimer fraction, whereas one of the less flexible subclasses (IgG2) was underrepresented. Although IgG2 could dimerize to some extent with IgG of other subclasses, it could not self-dimerize. These data suggest that structural constraints (hinge flexibility) may play a role in limiting dimerization. This suspicion was confirmed by showing that an additional 15.5% of the monomeric IgG fraction from a multidonor sample could dimerize after a chemically induced increase in hinge flexibility. Our results are interpreted to show that, although the number of functionally distinct Id produced by a species is immense, it is nontheless finite. Moreover, the Id repertoire of an individual is much smaller than that of the species. Pooling the IgG from a number of individuals increases the Id diversity, which increases the chance that any given Id-bearing molecular will encounter a complementary partner.

Alkylation↗

PubMed1991

Enhanced pyrimidine dimer removal in repair-proficient murine fibroblasts transformed with the denV gene of bacteriophage T4.

The denV gene of bacteriophage T4, which encodes the pyrimidine dimer-specific repair enzyme endonuclease V, was introduced into murine fibroblasts with normal rodent pyrimidine dimer repair capabilities. Endonuclease V recognizes ultraviolet radiation (UVR)-induced pyrimidine dimers and produces single-strand breaks adjacent to the dimers. These nicks may serve as substrates to initiate excision repair of pyrimidine dimers by endogenous enzymes. In the present study, murine fibroblasts stably transfected with denV were able to remove 50-80% of UVR-induced pyrimidine dimers, while control cells removed only about 20% of dimers under the same conditions of pyrimidine dimer induction and repair. For both control and denV-transfected cells, repair continued for at least 24 h after exposure. When removal of UVR-induced photoproducts was initiated by endogenous excision repair mechanisms, an average of 38 nucleotides were replaced per dimer removed, as determined by bromouracil photolysis; denV-initiated excision repair, on the other hand, resulted in removal of an average of 6 nucleotides per dimer repaired. The enhanced pyrimidine dimer repair capabilities conferred by denV gene expression did not appear to improve post-UVR survival.

Animals↗