Search PubMed⌕ Search

SEARCH · Search PubMed

Results for “Dimerization”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 793 records · Page 44Linked to original sources
PubMed2002

A dimeric mutant of the homotetrameric single-stranded DNA binding protein from Escherichia coli.

A single amino acid substitution (Y78R) at the dimer-dimer interface of homotetrameric single stranded DNA binding protein from E. coli (EcoSSB) renders the protein a stable dimer. This dimer can bind single-stranded DNA albeit with greatly reduced affinity. In vivo this dimeric SSB cannot replace homotetrameric EcoSSB. Amino acid changes at the rim of the dimer-dimer interface nearby (Q76K, Q76E) show an electrostatic interaction between a charged amino acid at position 76 and bound nucleic acid. In conclusion, nucleic acid binding to homotetrameric SSB must take place across both dimers to achieve functionally correct binding.

Amino Acid Substitution↗
PubMed2005

Backbone dynamics of a symmetric calmodulin dimer in complex with the calmodulin-binding domain of the basic-helix-loop-helix transcription factor SEF2-1/E2-2: a highly dynamic complex.

Calmodulin (CaM) interacts specifically as a dimer with some dimeric basic-Helix-Loop-Helix (bHLH) transcription factors via a novel high affinity binding mode. Here we report a study of the backbone dynamics by (15)N-spin relaxation on the CaM dimer in complex with a dimeric peptide that mimics the CaM binding region of the bHLH transcription factor SEF2-1. The relaxation data were measured at multiple magnetic fields, and analyzed in a model-free manner using in-house written software designed to detect nanosecond internal motion. Besides picosecond motions, all residues also experience internal motion with an effective correlation time of approximately 2.5 ns with squared order parameter (S(2)) of approximately 0.75. Hydrodynamic calculations suggest that this can be attributed to motions of the N- and C-terminal domains of the CaM dimer in the complex. Moreover, residues with significant exchange broadening are found. They are clustered in the CaM:SEF2-1mp binding interface, the CaM:CaM dimer interface, and in the flexible helix connecting the CaM N- and C-terminal domains, and have similar exchange times (approximately 50 micros), suggesting a cooperative mechanism probably caused by protein:protein interactions. The dynamic features presented here support the conclusion that the conformationally heterogeneous bHLH mimicking peptide trapped inside the CaM dimer exchanges between different binding sites on both nanosecond and microsecond timescales. Nature has thus found a way to specifically recognize a relatively ill-fitting target. This novel mode of target-specific binding, which neither belongs to lock-and-key nor induced-fit binding, is characterized by dimerization and continuous exchange between multiple flexible binding alternatives.

Binding Sites↗
PubMed2006

Triggering of T cell activation via CD4 dimers.

The onset of activation in Th cells is triggered by localized co-engagement of TCRs and the coreceptor CD4. A CD4 crystal suggested that CD4 may form dimers in some circumstances. In this study, we use live-cell fluorescence resonance energy transfer imaging to demonstrate that CD4 dimers are present at a basal level on the cell surface and accumulate at the synapse. Mechanistically, we reveal two conditions under which dimers are highly relevant. First, CD4 dimers are more proficient in mediating prolonged cell contacts with APCs in the presence or absence of Ag. This is consistent with a model whereby the dimer functions to increase T-APC avidity. Second, we show that dimer mutations result in an increased level of an inactive lckTyr(505) bound to the CD4 molecule relative to dimer-competent CD4. We also find a consistent defect in signaling onset in these cells. This supports a role for CD4 dimerization in maintaining active signaling machinery. We suggest that modulation of the dimer/monomer ratio may permit tuning of activation thresholds during initial engagement.

Animals↗
PubMed1988

Distribution of thymine dimers induced in mouse skin by ultraviolet radiation.

The cellular and tissue distributions of thymine dimers induced in mouse skin by UV radiation were compared in 3 haired mouse strains: Sencar, BALB/c, and C3H. These strains differ markedly in their overall susceptibility to photocarcinogenesis, and they develop UV-induced skin tumors of different histological types and in different anatomical locations. The purpose of this study was to determine whether the differences in strain and site susceptibility are due to inequalities in the distribution of UV radiation-induced DNA damage in the target tissues. An indirect immuno-fluorescence assay was used to examine the location of thymine dimers induced by 254-nm UV radiation (UVC) or 280-400 nm UV radiation (UVAB) in ears and dorsal skin. No differences in the distribution of dimers in skin cells or tissues were found among mouse strains or between anatomical sites (ear vs. dorsum). Thus, these results do not support the hypothesis that differences in the optical properties of skin, which govern penetration and absorption of UV radiation, contribute to site or strain susceptibility in photocarcinogenesis. However, two distinct differences in the cellular and tissue distribution of dimers were found when UVC and UVAB-treated tissues were compared: a) UVAB produced dimers in both dermal and epidermal cells, whereas UVC induced a detectable number of dimers only in epidermal cells; b) UVAB-induced dimers were observed in both the nuclei and cytoplasm of affected cells, whereas dimers induced by UVC were confined to nuclei.

Animals↗
PubMed1983

Immunological detection of two types of cyclobutane pyrimidine dimers in DNA.

A hyperimmune rabbit antiserum to ultraviolet-irradiated DNA was observed to contain two activities directed against irradiated DNA. Radioimmunoassay studies demonstrated that both antigenic determinants were pyrimidine dimers as evidenced by the reduction in antibody binding to enzymatically photoreactivated irradiated DNA. Enzymatic photoreactivation specifically monomerizes pyrimidine dimers in DNA. Under conditions in which antiserum binding was measured with native DNA, binding was observed with DNA that contained both thymine-thymine and cytosine-containing dimers but not against native DNA that contained only thymine-thymine dimers. Under these assay conditions, competitive binding to irradiated DNA increased as a linear function of fluence of ultraviolet radiation. Under a second set of conditions in which binding was measured with single-stranded DNA, antibodies were bound to DNA which contained only thymine-thymine dimers. The fluence response for the increase in binding to thymine-thymine dimers was nonlinear and increased as a function of fluence squared. Binding to thymine dimers appeared to require bivalent attachment to two dimers that occurred in close proximity.

Animals↗
PubMed1996

Dimerization of lactase-phlorizin hydrolase occurs in the endoplasmic reticulum, involves the putative membrane spanning domain and is required for an efficient transport of the enzyme to the cell surface.

Analysis of the quaternary structure of human intestinal lactase-phlorizin hydrolase (LPH) by chemical cross-linking and sucrose-gradient centrifugation reveals that the brush border form of LPH (LPH beta; 160-kDa) is a homodimeric molecule. Dimerization ensures in the ER when LPH is still exclusively found as an uncleaved mannoserich precursor (pro-LPHb; 215-kDa). This is supported by the following observations. (i) Biosynthetically labeled intestinal biopsy specimens as well as transfected COS-1 cells expressing pro-LPH contain monomeric and dimeric forms of pro-LPHb; the complex glycosylated pro-LPH (pro-LPHc; 230-kDa) as well as the cleaved mature LPH beta species in intestinal biopsy samples are discerned exclusively as dimers. (ii) Dimeric forms of pro-LPHh could be also detected when cells were biosynthetically labeled at 15 degrees C, at which temperature the egress of pro-LPH from the ER is blocked. Dimerization is essential for the transport competence of pro-LPH and is strongly associated with the presence of an intact transmembrane domain. Mutant pro-LPH-mact lacking the complete transmembrane domain persists as a monomeric, mannose-rich and transport-incompetent molecule that is not secreted into the exterior milieu, accumulates most likely in the ER and is ultimately degraded. Further, deletion of the cytoplasmic tail in the pro-LPH-ct mutant leads to marked reduction in the proportion of dimeric as well as complex glycosylated pro-LPH-ct. Finally, dimerization is linked to the acquisition of LPH to its biological function, since only dimers of wild type pro-LPH or pro-LPH-ct are enzymatically active, while their monomeric counterparts as well as pro-LPH-mact are not.

Animals↗
PubMed1998

Analysis of c-kit receptor dimerization by fluorescence resonance energy transfer.

Stem cell factor (SCF) binding to the c-kit receptor triggers homodimerization and intermolecular tyrosine phosphorylation of the c-kit receptor, thus initiating signal transduction. Receptor dimerization is a critical early step in this process. Prior biochemical studies of c-kit receptor dimerization have mainly used affinity cross-linking techniques, which are beset with problems including low efficiency of cross-linking and the usual requirement for radiolabeled SCF to detect the cross-linked complex. We used the fluorescence resonance energy transfer (FRET) technique to examine the effects of SCF and other hematopoietic cytokines on c-kit receptor dimerization. The nonneutralizing anti-c-kit receptor monoclonal antibody 104D2 was directly conjugated to fluorescein isothiocyanate (FITC) or to the carbocyanine dye Cy3 and used to label cytokine-responsive human hematopoietic cell lines. The ability of SCF to induce c-kit receptor dimerization was assessed by flow cytometric analysis of FRET between the donor fluorochrome FITC and the acceptor fluorochrome Cy3. SCF induced a dose-dependent increase in c-kit receptor dimerization that correlated well with the concentrations of SCF required to stimulate cell proliferation. Receptor dimerization was detectable within 3 minutes after the addition of SCF and was maximal 30 minutes after the addition of SCF. Confocal microscopy showed redistribution of the c-kit receptor (from a diffuse distribution on the cell surface to "caps" at one end of the cell) within 3 minutes after SCF addition, followed by receptor internalization. Reappearance of the c-kit receptor on the cell surface required new protein synthesis, suggesting that the c-kit receptor is not recycled to the cell surface after internalization. Finally, erythropoietin (Epo), but not the structurally and functionally related cytokine thrombopoietin (Tpo), stimulated c-kit receptor dimerization detectable by FRET, and tyrosine phosphorylation of the c-kit receptor. These results suggest that exposure to Epo can activate the c-kit receptor and provide further evidence for cross-talk between the Epo and c-kit receptors in human hematopoietic cell lines. Studies with progeny of burst-forming unit-erythroid (BFU-E) suggest that the FRET technique is sufficiently sensitive to detect c-kit receptor dimerization on normal human hematopoietic cells.

Carbocyanines↗
PubMed1999

Normal D-dimer levels in patients with pulmonary embolism.

BACKGROUND: Pulmonary embolism (PE) is frequently evaluated in acute care settings. Despite this, the clinical diagnosis of PE is difficult. Results of ventilation-perfusion (V/Q) scans may be inconclusive, and pulmonary angiograms (PAGs) are cumbersome, involve risk, and are often unavailable. Using PAG as the standard criterion, we evaluated the relationship between PE, V/Q scans, and semiquantitative latex agglutination (LA) D-dimer levels. METHODS: Ninety-eight patients who underwent V/Q scanning for suspected PE were enrolled; based on the results of the scans, the patients were scheduled for PAG. Blood samples were drawn for LA D-dimer assays during the PAGs at Saint Joseph Hospital, Denver, Colo, from January 1, 1996, to February 1, 1997. A detailed medical record review was performed for all enrollees. RESULTS: The mean+/-SEM patient age was 56.6+/-1.9 years; 52 (53%) were men, 13 (13%) had cancer, 23 (23%) had undergone surgery within 30 days of their PAG, and 13 (13%) were receiving warfarin sodium. There were no differences in warfarin therapy, hypercoaguable state, or cancer prevalence between patients with negative and positive PAGs (P = .53). Ventilation-perfusion scan results were available for all study patients. Eight (27%) of 30 patients who had positive angiogram results had LA D-dimer levels less than 250 ng/mL. Patients with positive PAGs (n = 30) had the following V/Q scan results: normal, 0; low probability, 7; intermediate or indeterminate probability, 22; and high probability, 1. In patients with low-probability V/Q scan results (n = 34), a positive D-dimer result for PE (>250 ng/mL) had a sensitivity of 71.4% (95% confidence interval, 0.29-0.97) and a negative predictive value of 87.5% (95% confidence interval, 0.62-0.98). We found a significant difference in D-dimer levels in patients with an abnormal angiogram result (mean, 750 ng/mL) compared with patients with a normal angiogram result (mean, 250 ng/mL) (P= .01, chi2 test). CONCLUSIONS: Eight patients had normal D-dimer levels with angiographic evidence of PE. Algorithms in acute care settings have been proposed; they exclude PE with normal D-dimer levels using the enzyme-linked immunosorbent assay technique. These cannot be extrapolated to the more widely used LA assays. A normal LA D-dimer level alone or with V/Q scan results is not recommended to preclude the treatment of PE.

Angiography↗
PubMed2003

D-dimer levels and risk of recurrent venous thromboembolism.

CONTEXT: Widespread screening of patients with venous thromboembolism (VTE) for thrombophilic risk factors has become common clinical practice. Because of the increasing number of risk factors, assessing the risk of recurrence in an individual patient is intricate; therefore, a laboratory method that measures multifactorial thrombophilia is required. OBJECTIVE: To prospectively study the relationship between the risk of recurrent VTE and D-dimer, a global marker of coagulation activation and fibrinolysis. DESIGN, SETTING, AND PARTICIPANTS: Prospective cohort study of 610 patients older than 18 years who were treated with oral anticoagulants for at least 3 months with a first spontaneous VTE, in whom D-dimer levels were measured shortly after discontinuation of oral anticoagulation. The study was conducted at the Department of Internal Medicine I, University Hospital, Vienna, Austria. Patients entered the study at time of discontinuation of oral anticoagulants and were observed at 3-month intervals during the first year and every 6 months thereafter from July 1992 to October 2002. MAIN OUTCOME MEASURE: Objectively documented symptomatic recurrent VTE. RESULTS: A total of 79 (13%) of 610 patients had recurrent VTE with a mean observation time of 38 months. Patients with recurrence had significantly higher D-dimer levels compared with those without recurrence (553 ng/mL vs 427 ng/mL, P =.01). Compared with patients with D-dimer levels of 750 ng/mL or higher, the relative risk (RR) of recurrence was 0.6 (95% confidence interval [CI], 0.3-1.4), 0.6 (95% CI, 0.3-1.2), and 0.3 (95% CI, 0.1-0.6) in patients with D-dimer levels of 500 to 749 ng/mL, 250 to 499 ng/mL, and less than 250 ng/mL, respectively. The cumulative probability of recurrent VTE at 2 years was 3.7% (95% CI, 0.9%-6.5%) among patients with D-dimer levels of less than 250 ng/mL compared with 11.5% (95% CI, 8.0%-15.0%) among patients with higher levels (P =.001). Patients with D-dimer levels of less than 250 ng/mL had a 60% lower RR of recurrence compared with patients with higher levels (RR, 0.4; 95% CI, 0.2-0.8). CONCLUSION: Patients with a first spontaneous VTE and a D-dimer level of less than 250 ng/mL after withdrawal of oral anticoagulation have a low risk of VTE recurrence.

Adult↗
PubMed2000

Dimerization of formic acid--an example of a "noncovalent" reaction mechanism

The pulse deposition technique allows selectively the isolation of monomeric or dimeric formic acid in argon matrices at 7 K. Warming of matrices containing the monomer M from 7 K to 40 K results in the decrease of M and formation of a dimer B. This dimer is also labile, and further warming finally produces a second dimer A. By comparison with density functional theory (DFT) calculations and gas phase IR spectra taken from the literature, the latter dimer A was identified as the C2h-symmetrical cyclic dimer. The unstable dimer B was identified as the acyclic Cs-symmetrical dimer. An activation energy of 2.3 kcal mol(-1) was calculated for the B --> A rearrangement at the B3LYP/ 6-311 ++ G(d,p) level of theory, which is in qualitative agreement with the experimental finding of a slow thermal reaction under the conditions of matrix isolation.

Journal Article↗
PubMed2004

Comparative dimerization of 1-butene with a variety of metal catalysts, and the investigation of a new catalyst for C=H bond activation.

Catalytic dimerization of 1-butene by a variety of catalysts is carried out, and the products are analyzed by gas chromatography and mass spectrometry. Catalysts based on cobalt and iron can produce highly linear dimers, with the cobalt-based dimers exceeding 97 % linearity. Catalysts based on vanadium and aluminum prefer to make branched dimers, which are most often methyl-heptenes in the case of vanadium and almost exclusively 2-ethyl-1-butene in the case of aluminum. The vanadium catalyst also produces substantial amounts of dienes and alkanes, suggesting a competing hydrogenation/dehydrogenation pathway that appears to involve vinyl Cbond;H bond activation. Nickel catalysts are generally less selective than those based on iron or cobalt for making linear dimers, but they can make dimers with 60 % linearity. The major by-products for the nickel systems are trisubstituted internal olefins. An important side reaction that must be considered for dimerization reactions is 1-butene isomerization to 2-butene, which makes recycling the butene difficult for a linear dimerization process. Aluminum, iron, and vanadium systems promote very little isomerization, but nickel and cobalt systems tend to isomerize the undimerized substrate heavily.

Journal Article↗
PubMed1997

Monomeric variants of IL-8: effects of side chain substitutions and solution conditions upon dimer formation.

IL-8 dimers have been observed in NMR and X-ray structures of the protein. We have engineered IL-8 monomers by mutations of residues throughout the dimer interface, which introduce hindrance determinants to dimerization. These IL-8 variants are shown by NMR to have wild-type monomer folding, but by ultracentrifugation to have a range of dimerization constants from microM to mM, as compared with a dimerization constant of about 10 microM for wild-type IL-8, under physiological salt and temperature conditions. The monomeric variants of IL-8 bind the erythrocyte chemokine receptor DARC, as well as the neutrophil IL-8 receptors CXCR1 and CXCR2 with affinities similar to that of wild-type IL-8. In addition, the monomeric variants were shown to have agonist activity, with similar potency to wild-type, in both Ca(2+)-flux assays on CXCR1 and CXCR2 transfected cells, and in chemotaxis assays on neutrophils. Thus, these variants confirm that monomeric IL-8 is functionally equivalent to wild-type in vitro assays. We have also investigated the effects of various solution conditions upon IL-8 dimer formation using analytical ultracentrifugation. At salt concentrations, temperatures, and pH conditions lower than physiological, the dimerization affinity of IL-8 is greatly enhanced. This suggests that, under some conditions, IL-8 dimer formation may occur at concentrations of IL-8 considerably lower than 10 microM, with consequences in vivo that are yet to be determined.

Amino Acid Sequence↗
PubMed1993

Covalent dimerization of recombinant human interferon-gamma.

An apparently nonreducible covalent dimer has been consistently observed as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These studies endeavor to understand the nature of this covalent dimerization. Amino acid sequence analysis of a recombinant human interferon-gamma stored for 2 years showed a major sequence starting with the intact N-terminal methionine and a minor sequence corresponding to the C-terminal seven amino acids of the intact protein. Moreover, when the same material was analyzed by gel filtration in the presence of 8 M urea, a minor peak corresponding to the dimer was observed prior to the monomer peak. Reducing and nonreducing SDS-PAGE also showed a minor band corresponding to a dimer. These results suggest that the reactions of C-terminal processing and dimerization have occurred during storage. Reverse-phase chromatography of stored, unfractionated material showed three peaks. Mass spectral analysis of the first, second, and third peaks gave molecular weights of 16,900, 16,100, and 33,000. Since no major cleavage was observed in the N-terminal region of the protein, the observed masses suggest that the first peak corresponds to residues 1 to 144 (a full-length molecule), the second peak to residues 1 to 137 (des 7 interferon-gamma), and the third peak to a dimer. Calculation of theoretical molecular weight from the amino acid sequence suggests that this dimer corresponds to some combination of the intact protein and des 7 protein. Tryptic peptide maps in conjunction with sequence and mass analyses identified a new tryptic peptide in the map of the dimer corresponding to residue 133 to 137 followed by residues 1 to 7. The conclusion is recombinant methionyl human interferon-gamma undergoes a specific cleavage at the C-terminal side of residue 137 phenylalanine, and a conventional peptide bond was formed between residues 137 and 1 methionine.

Amino Acid Sequence↗
PubMed1996

Protein radicals in myoglobin dimerization and myoglobin-catalyzed styrene epoxidation.

Two mechanisms have been identified for the H2O2-dependent epoxidation of styrenes by sperm whale myoglobin (Mb) [S. Rao, A. Wilks, and P. R. Ortiz de Montellano, J. Biol. Chem. 268, 803-908 (1993)]: (a) ferryl (FeIV = O) oxygen transfer with retention of stereochemistry and incorporation of an oxygen from H2O2, and (b) protein peroxy radical cooxidation with loss of stereochemistry and incorporation of an oxygen from O2. As shown here, cis-beta-methylstyrene is preferentially oxidized to the trans-epoxide when the H2O2:Mb ratio is <0.5 but increasingly to the cis-isomer as the ratio increases to and above 1. At a high (4:1) H2O2:Mb ratio, both the absolute yield and the cis:trans-epoxide ratio increase in proportion to the cis-beta-methylstyrene concentration. A protein radical formed in the Mb-H2O2 reaction also causes dimer and trimer formation, maximum dimer formation (approximately 30%) being obtained with 1 equivalent of H2O2. At low H2O2:Mb ratios, the oxidation equivalents utilized for protein oligomerization and styrene oxidation account for the available H2O2. Previous studies have shown that His-64 is important for protein-mediated olefin cooxidation and Tyr-151/Tyr-103 for Mb dimerization. The W7F, W14F, and W7F/W14F Mb mutants have now been prepared and the W14F, but not W7F, mutation shown to modestly decrease cooxidation of cis-beta-methylstyrene to the trans-epoxide. Neither tryptophan mutation alters dimer formation. Dimer formation is modestly increased rather than decreased by styrene, suggesting that styrene cooxidation and dimerization do not compete. The results indicate that (a) cis-beta-methylstyrene cooxidation and protein dimerization, both of which are mediated by protein radicals, are favored at low H2O2:Mb ratios, (b) as the H2O2:Mb ratio increases, the ferryl epoxidation pathway surpasses the cooxidation mechanism, (c) Trp-14 but not Trp-7 influences olefin cooxidation, and (d) different, possibly nonequilibrating, radicals mediate olefin cooxidation and protein dimerization.

Animals↗
PubMed1996

The dimerization domain upstream binding factor contains multiple helical structures.

The upstream binding factor, UBF, is an RNA polymerase I transcription factor which contains multiple DNA binding domains and a novel protein dimerization domain. Active UBF forms homodimers in vivo through the intramolecular interactions of its dimerization domain, which spans a hundred amino-terminal residues. In the presence of both UBF dimerization domain and its immediately adjacent lysine-rich basic DNA binding domain, the E. coli expressed recombinant polypeptide, dbUBF (dimerization plus basic motifs of UBF), forms homodimers in vitro and binds to double-stranded DNA nonselectively. In gel retardation assay, dbUBF dimers make multiple shift-ladders corresponding to numerous protein dimer-DNA complexes. The UBF dimerization domain contains multiple helical structures, as predicted by EMBO-PHD program. Most of hydrophobic residues in the dimerization domain are confined in the hydrophobic phase of these hypothetic helices. Mutating these hydrophobic residues to glutamate prohibits dbUBF association and gives a different shift pattern in gel retardation assay. The results we present here argue that UBF association is largely exerted by the hydrophobic interactions between the multiple helices to bring two molecules together.

Amino Acid Sequence↗
PubMed1995

Solution structure of mithramycin dimers bound to partially overlapping sites on DNA.

Mithramycin (MTH) is a DNA-binding antitumor agent containing A-B disaccharide and C-D-E trisaccharide segments projecting from opposite ends of an aglycone chromophore. We have previously reported on the solution structure of the MTH-DNA 6-mer complex based on a combined NMR and molecular dynamics study. This study established that the Mg(2+)-coordinated mithramycin dimer bound to a widened minor groove centered about the sequence-specific (G-C).(G-C) site and that the C-D-E trisaccharide segments from individual monomers were directed towards opposite ends of the helix spanning a six base-pair segment. This research is now extended to the binding of mithramycin dimers to partially overlapping sites on the self-complementary d(T-A-G-C-T-A-G-C-T-A) 10-mer duplex. The six base-pair mithramycin dimer footprint centered about (G-C).(G-C) steps should result in a potential steric clash in the center of the helix involving the inwardly pointing E-sugars of the pair of mithramycin dimers bound to the DNA 10-mer duplex. The MTH-d(T-A-G-C-T-A-G-C-T-A) complex (two MTH dimers per duplex) yields narrow and well-resolved NMR spectra, which have been assigned to identify intramolecular and intermolecular nuclear Overhauser enhancement (NOE) connectivities in the complex. The solution structure of the MTH-DNA 10-mer complex based on distance-restrained molecular dynamics calculations has defined the conformation of the drug and the DNA necessary for accommodation of the pair of mithramycin dimers on the DNA 10-mer helix. Specifically, the inwardly pointing E-sugars retain their face-down alignment towards the floor of the minor groove and occupy adjacent binding sites in the center of the duplex. This is achieved, in part, through torsion angle differences in the glycosidic linkage bonds along the length of the inwardly pointing aglycone-C-D-E trisaccharide segment relative to its outwardly pointing aglycone-C-D-E trisaccharide counterpart in the complex. In addition, a pronounced kink at the central (T-A).(T-A) step opens the minor groove and generates additional space to accommodate the inwardly pointing E-sugars at adjacent sites in the MTH-DNA 10-mer complex. These studies establish conformational plasticity in the C-D-E trisaccharide segment of the mithramycin dimer and deformability of the DNA helix allowing mithramycin dimers to bind to partially overlapping minor groove sites on the DNA helix.

Base Composition↗
PubMed1994

In vitro resolution of the dimer bridge of the minute virus of mice (MVM) genome supports the modified rolling hairpin model for MVM replication.

Previous characterization of the terminal sequences of the minute virus of mice (MVM) genome demonstrated that the right hand palindrome contains two sequences, each the inverted complement of the other. However, the left hand palindrome was shown to exist as a unique sequence [Astell et al., J. Virol. 54: 179-185 (1985)]. The modified rolling hairpin (MRH) model for MVM replication provided an explanation of how the right hand palindrome could undergo hairpin transfer to generate two sequences, while the left end palindrome within the dimer bridge could undergo asymmetric resolution and retain the unique left end sequence. This report describes in vitro resolution of the wild-type dimer bridge sequence of MVM using recombinant (baculovirus) expressed NS-1 and a replication extract from LA9 cells. The resolution products are consistent with those predicted by the MRH model, providing support for this replication mechanism. In addition, mutant dimer bridge clones were constructed and used in the resolution assay. The mutant structures included removal of the asymmetry in the hairpin stem, inversion of the sequence at the initiating nick site, and a 2-bp deletion within one stem of the dimer bridge. In all cases, the mutant dimer bridge structures are resolved; however, the resolution pattern observed with the mutant dimer bridge compared with the wild-type dimer bridge is shifted toward symmetrical resolution. These results suggest that sequences within the left hand hairpin (and hence dimer bridge sequence) are responsible for asymmetric resolution and conservation of the unique sequence within the left hand palindrome of the MVM genome.

Animals↗
PubMed1995

Generation of defective interfering RNA dimers of cymbidium ringspot tombusvirus.

Inoculation of Nicotiana clevelandii and N. benthamiana plants with in vitro transcripts of both genomic and defective interfering (DI) RNAs of cymbidium ringspot tombusvirus resulted in a rapid accumulation of new DI-like RNA species which were demonstrated by cloning and sequencing to be head-to-tail dimers of unit length DI RNAs. The junction regions of dimers were represented by sequences derived precisely from the 5' and 3' termini of DI RNAs. Only infection with DI RNAs of smaller size (DI-2 and DI-3, 402 and 482 nt, respectively) produced detectable amount of dimers; in contrast, infection with the largest DI RNA (DI-13, 679 nt) was unable to accumulate dimers during viral infection. Analysis of mutant DI RNAs containing deletions or insertions revealed that the size of the monomer molecule is a major factor in the accumulation of dimers. Monomeric DI RNAs were formed in both plants and protoplasts inoculated with in vitro-transcribed dimers. No heterodimers were found in plants inoculated simultaneously with DI-2 and DI-3 RNA molecules, which may indicate that replicase is not released from the template during synthesis of dimer molecules. However, the occurrence of a recombinant DI RNA dimer molecule derived from the two DI RNAs suggests that simultaneous infection of the same cells with two DI RNAs did indeed take place and that absence of heterodimers did not depend on compartmentalization.

Base Sequence↗