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PubMed1979

Preparation of a disulfide-linked dimer of human placental lactogen fragment 1--134 with immunologic and biologic activity.

In order to determine the chemical features of human placental lactogen (hPL) necessary for its biologic activity we prepared the following fragments from the plasmin-cleaved hormone: reduced and alkylated 1--134, reduced and alkylated 141--191, and a 1--134 dimer joined through the single cysteinyl residue at position 53. In a radioimmunoassay using antibodies against native hPL, the two reduced and alkylated fragments produced nonparallel displacement and had less than 1% of the activity of hPL. The ability of reduced and alkylated 1--134 to bind to mammary gland receptors was less than 5% of that of hPL; reduced and alkylated 141--191 showed no detectable activity in the same assay. The 1--134 dimer, in contrast, had 20% of the immunologic activity and 30% of the ability to bind to lactogenic receptors relative to the native hormone. In an in vitro bioassay the lactogenic activity of the 1--134 dimer was equivalent to that of the native hormone. The circular dichroic spectra of hPL, reduced and alkylated 1--134, and 1--134 dimer indicated that the dimer had regained much of the helical content of the native hormone. Antibodies produced to reduced and alkylated 1--134 did not significantly crossreact with either native hPL or 1--134 dimer. From these data we conclude that the information for the lactogenic activity of hPL is contained in the first 134 amino acid residues and that the proper conformation is necessary for its biologic expression.

Amino Acids↗
PubMed2025

Mechanisms of enhanced or impaired DNA target selectivity driven by protein dimerization.

Successful DNA transcription demands coordination between proteins that bind DNA while simultaneously binding to one another to form dimers or higher-order complexes. For proteins with numerous DNA targets throughout the genome, measurements that report on their dwell time or occupancy thus represent a convolution over a population interacting with specific DNA, nonspecific DNA, or protein partners on DNA. Dimerization is known to add contacts that can help a single protein to stably bind DNA. However, we show here that dimerization can also impair measured dwell times and occupancy on target sequences because the population redistributes across DNA. We combine mass-action kinetic models of pairwise reversible reactions between proteins and DNA with theory and spatial stochastic simulations to isolate the role of dimerization on observed DNA dwell times, occupancy, and spatial distribution of proteins on DNA. Three key themes emerge: (i) Protein-protein interactions, in addition to protein-DNA interactions, can localize a protein to DNA, and relative binding rates can thus widely tune dwell times. (ii) Dimensional reduction achieved through nonspecific binding and subsequent 1D diffusion controls the order-of-magnitude of enhancements despite nucleosome barriers. (iii) Dimerization enhances selectivity for locally clustered targets and often impairs binding to widely-spaced targets by sequestration. Compared with ChIP-seq data, our model explains how the distribution of the essential GAF protein throughout the genome is highly selective for clustered targets due to protein interactions. This model framework predicts when even weak dimerization can redistribute and stabilize proteins on DNA as a necessary part of transcription.

DNA binding↗
PubMed1977

Structure and formation of circular dimers of simian virus 40 DNA.

Most of the viral DNA extracted from simian virus 40 (SV40)-infected African green monkey kidney cells consists of circular molecules about 5.3 kilobases in contour length. However, about 1% of the viral DNA was found to occur as closed circular dimers that appeared to be formed, preferentially, late in infection. The monomeric units of dimers were organized in a head-to-tail, tandem arrangement; moreover, the monomeric units were not defective; i.e., they lacked deletions or other rearrangements. After infections with dimer DNA, nondefective monomers were formed. These findings suggest that dimers are not intermediates in the production of defective SV40 genomes. The majority of the dimers formed in mixed infections with two mutants were homodimers, but about 5% of the circular dimers were heterodimers and must have arisen by intermolecular recombination.

Cell Line↗
PubMed1978

High-molecular-weight RNAs of AKR, NZB, and wild mouse viruses and avian reticuloendotheliosis virus all have similar dimer structures.

Several 50 to 70S tumor viral RNAs have previously been shown by electron microscopy to be dimers, with the two monomer subunits joined near their 5' ends. Five additional naturally occurring type C RNA tumor viruses have now been examined: AKR, and endogenous murine ecotropic virus; NZB, an endogenous murine xenotropic virus; and ecotropic and an amphotropic virus isolated from a wild mouse; and the avian reticuloendotheliosis virus (REV). All five 50 to 70S RNAs have similar 5'-to-5' dimer structures. Therefore, the observations support the hypothesis that the dimer linkage is a structural feature common to all type C mammalian viruses. REV is the first example of an avian virus with a clear 5'-to 5' dimer linkage. All of the mammalian viral RNAs, but not REV, showed symmetrically placed loops in each subunit of the dimer. Possible molecular structures and biological functions of the dimer linkages and loops are discussed.

AKR murine leukemia virus↗
PubMed1975

The use of hybrid molecules in a study of the equilibrium between nerve growth factor monomers and dimers.

The major protein in beta nerve growth factor preparations, beta1NGF, is a dimer in which both peptide chains have COOH-terminal arginine residues. Digestion of beta1NGF with carboxypeptidase B produced a dimer, beta3NGF, in which both chains lack these terminal arginine residues. Exposure of mixtures of beta1 and beta3NGF dimers to 8 M urea to produce monomers, followed by removal of urea to allow recombination, resulted in the formation of the hybrid beta2NGF, comprising one arginine-containing and one arginine-less chain, as well as the parent dimers. The amount of the three dimers formed was close to that expected from random association of monomers. Hybrid beta2NGF was also formed from mixtures of beta1 and beta3NGF where incubated at pH 2.6 to 4.5. The formation of beta2NGF has a half-time of 6 h at pH 4.0 and 4 degrees C. Its rate of formation decreased above pH 4.5, becoming minimal between pH 9.5 and pH 10.5, and increased with increasing temperature. The amount of beta2NGF formed was determined by the lowest pH to which the parent mixture was exposed, irrespective of its prior history. These data suggest that the hybrid is formed by the same mechanism in the absence and presence of the urea step. An approximate value for Kd, the equilibrium dissociation constant of the dimer equilibrium monomer equilibrium was derived. Its value was 3 - 10(-10) M at pH 4.0 and 4 degrees C. The alpha-subunit of 7S NGF decreased the rate of formation of beta2NGF not only at pHs where an alphabeta complex is stable, but also at an acid pH where no complex formation is observed by sedimentation analysis, suggesting that the present methodology offers a more sensitive probe of subunit interactions. In contrast, the gamma subunit and a number of indifferent proteins had little or no effect on the appearance of beta2NGF at the pHs studied.

Animals↗
PubMed1979

Role of secretory component, a secreted glycoprotein, in the specific uptake of IgA dimer by epithelial cells.

The binding of secretory component (SC) to epithelial cells and its role in the specific uptake of immunoglobulin A (IgA) dimer has been studied in rabbit mammary gland and liver. SC, Mr approximately 80,000, secreted by epithelial cells of the mammary gland was found associated with the cell surface of mammary cells in intact tissue. Dispersed mammary cells and plasma membrane-enriched fractions obtained from mammary glands of midpregnant rabbits bound 125I-labeled SC in a saturable time- and temperature-dependent process. The association rate followed a second order reversible reaction (k+1 approximately equal to 2.7 x 10(6) M-1 min-1 at 4 degrees C) and equilibrium was reached in about 4 h at 4 degrees C. The dissociation rate for membranes was first order (k-1 approximately equal to 1.7 x 10(-2) min-1 at 4 degrees C), whereas displacement from cells was incomplete. The apparent affinity constant was similar for membranes and cells (Ka approximately equal to 5 x 10(8) M-1) with one class of binding sites. The number of binding sites varied from one animal to another (260 to 7,000 sites/mammary cell) in relation to endogenous occupancy by SC, which was assessed by immunocytochemistry and complement-mediated cytotoxicity. Rabbit liver and heart membranes did not bind SC, and serum proteins present in rabbit milk failed to interact with mammary cells or membranes. Mammary membranes or cells and liver membranes bound 125I-labeled IgA dimer in a saturable, reversible time- and temperature-dependent process. Association and dissociation rate constants at 4 degrees C (k+1 approximately equal to 5 x 10(6) M-1 min-1 and k-1 approximately equal to 5 x 10(-3) min-1, respectively) and the apparent affinity constant (Ka approximately equal to 10(9) M-1) were similar for liver and mammary membranes; these parameters differed, however, from those reported for free SC-IgA dimer interaction. The binding capacity of membranes for IgA dimer was directly related to the amount of free SC bound to membranes. Interaction of IgA dimer with mammary or liver membranes or cells was abrogated by excess of free SC and was prevented by preincubation of membranes or cells with Fab antibody fragments directed against SC. These data indicate that the first step in the translocation process of polymeric immunoglobulins across epithelia consists of binding of SC to the surface of epithelial cells which in turn acts as a receptor for the specific uptake of IgA dimer.

Animals↗
PubMed1975

Photodissociable dimer reduction products of 2-thiopyrimidine derivatives.

Both 4,6-dimethyl-2-thipyrimidine and its 1-methyl derivative undergo polarographic reduction in aqueous medium, via a 1e/1H+ reduction to a free radical which rapidly dimerizes to products isolates and identified as 4,4'-bis-(4,6-dimethyl-3,4-dihydropyrimidin-2-thione) and the corresponding 1-methyl dimer. The dimers may be oxidized electrolytically to regenerate the parent monomers. Both dimers also undergo photodissociation to quantitatively regenerate the parent monomers, in high quantum yield, 0.23 and 0.35 M/Einstein. The correlation between electrochemical and photochemical reductions of 2-thiopyrimidines are discussed, as well as the significance of the dimer photodissociation reactions in relation to nucleic acid photochemistry.

Electrolysis↗
PubMed1979

Anti-V region framework antibodies affect the ligand binding of VL dimer.

The effect of antibodies to the light chain variable region (VL) of protein MOPC-315 (alpha, lambda 2), on the binding of hapten by VL315 dimer or Fv315 (VL + VH) was studied by equilibrium dialysis. Anti-VL did not change the binding properties of Fv but affected the binding properties of VL dimer. At pH 5, the binding properties of VL in the presence or absence of anti-VL were the same, whereas at pH 8, anti-VL reduced the number of ligands bound to VL from two to one. It has previously been shown that VL dimer binds one ligand at pH 5 and two ligands at pH 8, and that VL conformation at pH 5 is tighter. Hence, our results suggest that anti-VL tightens the conformation of VL dimer at pH 8.0 such that it can bind only one ligend. Since Fv is not affected by anti-VL, the results indicate that a combining site made of two identical chains (VL dimer) can undergo a conformational change upon interaction with its antibody. Such conformational change can indirectly affect the binding properties.

Animals↗
PubMed1978

Low pH dimerization of chymotrypsin in solution.

The mechanism of the acid dimerization of alpha-chymotrypsin in solution was reexamined using a number of chemical derivatives. Blocking of the carboxyl of Tyr-146, while that of ASP-64 remained free, eliminated completely the ability of alpha-chymotrypsin to dimerize, as did methylation of His-57. O-Acetylation of Tyr-146 reduced the dimerization constant to that of gamma-chymotrypsin, and deacetylation of the other accessible tyrosines did not affect the dimerization. It is concluded that the mechanism proposed by Aune and Timasheff [Aune, K.C., and Timasheff, S.N.(1971) Biochemistry 10, 1609-1617] for the solution dimerization which involves the electrostatic interaction between the His-57 imidazolium ring and the terminal carboxyl of Tyr-146 is still most consistent with all the experimental observations. The interactions in dilute solution may differ somewhat from those observed in crystals. In particular, the two intermolecular bridges formed by sulfate ions in crystals cannot be present in solution.

Acetylation↗
PubMed1975

Evolutionary variation between a monomer and a dimer arginine kinase. Purification of the enzyme from Holothuria forskali and a comparison of some properties with that from Homarus vulgaris.

1. A purification procedure for the dimeric arginine kinase of the sea cucumber Holothuria forskali is described. 2. The enzyme has a mean molecular weight of 77250 and is composed of two equal, dissociable subunits. 3. It also shows co-operativity between substrate binding at one catalytic site to a much greater extent than the nomomeric lobster arginine kinase for which such co-operativity could not be detected unambiguously. The constants for substrate binding are reported assuming that the enzyme follows rapid-equilibrium random kinetics. From a comparison with other species, the development of co-operativity between the nucleotide- and guanidine-binding sites on one subunit is suggested to have occurred more than once in the evolution of the phosphagen kinases and is not dependent on subunit aggregation. 4. Both enzymes show similar pH profiles for thermal inactivation at 22 degrees C and have very similar stabilities. Above 40 degrees C the dimeric enzyme is much more stable than the monomer. Rate constants for heat inactivation and Arrhenius activation energies are reported. 5. The dimeric enzyme is also more stable to urea inactivation. Substrates and argininic acid all improve the stability of both enzymes. The effects of individual substrates are more distincitive with the dimeric enzymes and increase its stability to an extent that makes it about as stable as dogfish creatine kinase. In the physiological range dimerization does not seem to confer any particular advantage with respect to stability over the monomer form.

Animals↗
PubMed2025

FoxP3 forms a head-to-head dimer in vivo and stabilizes its multimerization on adjacent microsatellites.

FoxP3, the master regulator of Tregs, employs two DNA-binding modes to recognize diverse DNA sequences. It multimerizes on long TnG repeats (n = 2-5) to bridge DNA segments and stabilize chromatin loops, and it forms head-to-head (H-H) dimers on inverted repeat forkhead motifs (IR-FKHM) without bridging DNA. Although genomic data confirm its multimeric role, in vivo evidence for H-H dimerization has been elusive. Here, unbiased pull-down sequencing uncovers a range of relaxed motifs that drive H-H dimerization, enabling systematic genome-wide analysis. We demonstrate that FoxP3 binds genomic DNA as both H-H dimers and multimers in Tregs, with H-H binding often seeding and stabilizing multimerization on adjacent TnG repeats-especially on shorter, suboptimal repeats. While multimerization is conserved across FoxP family members, H-H dimerization is unique to FoxP3 orthologs, conferred by its divergent accessory loop. This dual-mode strategy broadens FoxP3's sequence repertoire and enhances its architectural function in chromatin looping.

Journal Article↗
PubMed1978

Dimer state of protein L7/L12 and EF-G-dependent reactions of ribosomes.

A number of different monomer and dimer derivatives of protein L7/L12 has been studied in EF-G-dependent reactions on the ribosome. It has been shown that only dimer derivatives of protein L7/L12 are able to interact with the ribosome. This means that it is the dimer forms of protein L7/L12 that are present in the functionally active ribosome. It is likely that the N-terminal sequence of protein L7/L12 is responsible for dimerization of the protein in solution and at the same time contributes mainly to the interaction of the protein L7/L12 dimer with the ribosome. The results obtained suggest that there are four copies of protein L7/L12 in the translating ribosome.

Escherichia coli↗
PubMed2026

A novel dimerization site in non-structural protein 5A of hepatitis C virus regulates viral replication fitness.

We previously found that high genome replication fitness of the hepatitis C virus (HCV) was associated with severe disease in immunocompromised patients. Elevated replication fitness was mediated by accumulation of mutations in the replication enhancing domain (ReED) within domain (D) 2 of non-structural protein (NS) 5A. NS5A is a partially unstructured phosphoprotein lacking enzymatic activity but fulfilling a key role in HCV replication due to interacting with various cellular and viral proteins. It can exist in a variety of dimeric and oligomeric conformations mediated by NS5A D1 with clinically approved NS5A inhibitors proposed to exert their antiviral function by fixing these dimers in distinct conformations. In this study, we aimed at elucidating the ReED's mode of action. AlphaFold modelling indicated a so far unrecognized NS5A dimerization site in the ReED. Indeed, split nano luciferase assays revealed a significantly stronger NS5A dimerization of high replicator ReED variants, suggesting that high replication fitness is mediated by enforcement of NS5A self-interaction. This hypothesis was supported by the effect of low dose (1 pM) NS5A inhibitor treatment, increasing replication fitness and phenocopying the effects of ReED mutations. Furthermore, we found that HCV isolate JFH1, replicating with very high efficiency, is completely resistant to the regulatory function of the ReED. Chimeric replicons composed of ReED resistant JFH1 and the ReED sensitive isolate J6 identified NS3 helicase and NS5B polymerase as critical genetic elements mediating ReED sensitivity/resistance. Our data overall suggest that the ReED in NS5A is a negative regulator of HCV replication fitness with dimerization releasing the inhibitory interaction with helicase and/or polymerase, thereby likely facilitating initiation of RNA synthesis.

Viral Nonstructural Proteins↗
PubMed1979

Small angle X-ray scattering of dimeric yeast hexokinase in solution.

Small angle x-ray scattering measurements on dimeric yeast hexokinase B at pH 5.5 in acetate buffer yield a radius of gyration of 31.28 +/- 0.23 angstrom. This measured value is comparable to the radius of gyration of 31.5 angstrom calculated from the refined coordinates of the dimer in the BII crystal form. The hexokinase dimer found in the BI crystal form has a radius of gyration of 42 angstrom calculated from the atomic coordinates. Thus, the measured radius of gyration is consistent with the BII dimer being the predominant species in solution and rules out the existence of the BI dimer as a major species under these conditions.

Hexokinase↗
PubMed1977

Repair of pyrimidine dimers in radiation-sensitive mutants rad3, rad4, rad6 and rad9 of Saccharomyces cerevisiae.

The ability to remove ultraviolet (UV)-induced pyrimidine dimers was examined in four radiation-sensitive mutants of Saccharomyces cerevisiae. The susceptibility of DNA from irradiated cells to nicking by either the T4 UV-endonuclease or an endonuclease activity found in crude extracts of Micrococcus luteus was used to measure the presence of dimers in DNA. The rad3 and rad4 mutants are shown to be defective in dimer excision whereas the rad6 and rad9 mutants are proficient in dimer excision.

Coliphages↗
PubMed1978

Removal of pyrimidine dimers from Saccharomyces cerevisiae nuclear DNA under nongrowth conditions as detected by a sensitive, enzymatic assay.

A sensitive and quantitative procedure for the detection of pyrimidine dimers in yesast nuclear DNA is described. The assay employs dimer-specific, endonuclease activities from Micrococcus luteus together with DNA sedimentation through calibrated, alkaline sucrose gradients to detect endonuclease-induced, single-strand breaks. Breaks were induced in a dose-dependent manner from 0 to 80 J m-2 at 254 nm and in numbers equivalent to the numbers of dimers induced by similar doses (Unrau et al., Biochim. Biophys. Acta, 312 (1973) 626--632). This procedure also allows the use of [6-3H] uridine to label cellular nucleic acids, but dose not require extensive DNA purification to eliminate concomitantly labeled RNA. Endonuclease-sensitive sites in the wild-type, haploid strain S288C, after irradiation with 5 J m-2 (254 nm), were removed in less than 5 min when cells were incubated in buffer (pH 7.0) at 28 degrees C. After irradiation with doses from 30 to 100 Jm-2 site removal in S288C required longer postirradiation incubations and was about 90% complete. In a radiation-sensitive strain carrying the mutant allele rad4-3 the number of endonuclease-sensitive sites remained constant for 6 h after irradiation with 5 Jm-2. The retention of sites in this strain indicates that it is defective in the excision of pyrimidine dimers.

Centrifugation, Density Gradient↗
PubMed1977

Differences in removal of acetylaminofluorene and pyrimidine dimers from the DNA of cultured mammalian cells.

The rate and extent of disappearance of two DNA lesions (pyrimidine dimers and covalently bound acetylaminofluorene), both thought to be removed by the so-called wide-patch (approximately 100 nucleotides) repair process, were studied in a variety of cultured mammalian cells. With the exception of mouse cells, dimers were removed more rapidly and extensively than covalently bound acetylaminofluorene. In human cells, for example, about 50% of the dimers were excised from DNA in 1 hr while only 25-50% of the chemically induced lesions were excised from DNA after 48 hr. Surprisingly mouse cells, which remove few dimers, were about as competent as control human fibroblasts at removing acetylaminofluorene lesions; however, xeroderma pigmentosum cells (group D) removed fewer N-acetoxy-2-acetylaminofluorene-induced lesions than control human cells. Our data raise the possibility of separate repair processes for these two types of lesions and suggest that their expression may be under similar genetic control in human cells.

2-Acetylaminofluorene↗
PubMed1979

The relationship between pyrimidine dimers and replicating DNA in UV-irradiated human fibroblasts.

The relationship between pyrimidine dimers (measured as endonuclease-sensitive sites) and newly-synthesized DNA has been examined in several different ways, with the following results:- 1. After UV-irradiation of normal human fibroblasts the frequency of pyrimidine dimer sites in sections of DNA which have been synthesized immediately before the UV-irradiation is similar to that in the bulk DNA. 2. The frequency of pyrimidine dimer sites in the parental strands of replicating DNA in UV-irradiated normal human fibroblasts is similar to that in the bulk DNA. 3. In UV-irradiated XP variant cells the size of DNA synthesized in the presence of caffeine immediately after UV irradiation accurately corresponds with the average interdimer distance in the parental DNA. This suggests that in this experimental situation each pyrimidine dimer gives rise to a disocntinuity or a termination site in the daughter strand.

Cells, Cultured↗