Linear Multimers Research Articles

An elastic siderophore synthetase and rubbery substrates assemble multimeric linear and macrocyclic hydroxamic acid metal chelators.

The trihydroxamic acid bacterial siderophore desferrioxamine B (DFOB, 1) produced by the DesABCD biosynthetic cluster coordinates metals beyond Fe(iii), which identifies potential to modify this chelator type to broaden metal sequestration and/or delivery applications. Rather than producing discrete chelators by total chemical synthesis from native monomers including N-hydroxy-N-succinyl-cadaverine (HSC, 2), the recombinant siderophore synthetase from Salinispora tropica CNB-440 (StDesD) was used with different substrate combinations to produce biocombinatorial mixtures of hydroxamic acid chelators. The mixtures were screened with Ga(iii) or Zr(iv) as surrogates of immunological positron emission tomography (PET) imaging radiometals 68Ga(iii) or 89Zr(iv) to inform known or new coordination chemistry. The last-in-line enzyme DesD forms amide bonds between two equivalents of 2 and N-hydroxy-N-acetyl-cadaverine to produce trimeric 1. Although hexadentate 1 is the terminal product evolved for Fe(iii) complexation, it was conceived amine-containing 1 might remain a viable DesD substrate for further iteration with 2 to generate higher-order hydroxamic acid multimers. Incubation of StDesD, cofactors ATP and Mg(ii), and 1 and 2, generated the octadentate hydroxamic acid DFOB-HSC (3) (previously characterised and named DFO*), decadentate DFOB-(HSC)2 (4), dodecadentate DFOB-(HSC)3 (5) and tetradecadentate DFOB-(HSC)4 (6). The system with StDesD and 2 alone generated a set of linear multimers containing flanking amine and carboxylic acid groups (HSC) x -L (x = 2 (7), x = 3 (8), x = 4 (9), x = 5 (10)) and a subset of the cognate ring-closed macrocycles (HSC) x -MC (x = 3 (12), x = 4 (13), x = 5 (14), with x = 2 (11) not detected). Liquid chromatography-mass spectrometry metal screening experiments detected 1 : 1 complexes of Ga(iii) or Zr(iv) and 1, 3-5, 8-10, and 12-14. Complexes of 2 : 1 stoichiometry were formed between Ga(iii) and the high-denticity, high-cavity-volume chelators 4-6, and 14. A processive intra-cavity assembly mechanism has been posited for this flexible siderophore synthetase in delivering a large set of multimeric chelators.

Unravelling hydrogen bond network in methanol-propanol mixtures via molecular dynamics simulation and experimental techniques

ABSTRACT Hydrogen-bonded networks in 1-propanol-methanol binary mixture are studied using molecular dynamics simulation along with experimental techniques (FTIR, dielectric spectroscopy and refractive index measurements) for the entire concentration range. The structure of hydrogen-bonded networks is studied through radial distribution function, hydrogen bond statistics and graph theoretical analysis from molecular dynamics. It is observed that the probability of hydrogen bonding is maximum in methanol molecules followed by hydrogen bonding between methanol and 1-propanol molecules and minimum among 1-propanol molecules, as the concentration changes. The graph theory results show the formation of linear chain hydrogen bond networks, with no caged structures, which is validated from the experimental results. Further, the deconvolution of the FTIR OH peak suggests the presence of linear multimers in all concentrations of the binary system. Average degree of the hydrogen-bonded networks from graph theory indicates a complex networks among pure alcohols and dimers between methanol and 1-propanol in their binary mixtures at all concentrations. The negative values of excess permittivity indicate that the components of the mixture interact in a manner such that the net dipole polarisation decreases.

ADAMTS13: what it does, how it works, and why it's important

ADAMTS13: what it does, how it works, and why it's important

Cutting edge: C3d functions as a molecular adjuvant in the absence of CD21/35 expression.

Complement component C3 covalently attaches to Ags following activation, where the C3d cleavage fragment can function as a molecular adjuvant to augment humoral immune responses. C3d is proposed to exert its adjuvant-like activities by targeting Ags to the C3d receptor (CD21/35) expressed by B cells and follicular dendritic cells. To directly assess the importance of CD21/35 in mediating the immunostimulatory effects of C3d, CD21/35-deficient (CD21/35(-/-)) mice were immunized with streptavidin (SA), SA-C3dg tetramers, recombinant HIV gp120 (gp120), or gp120 fused with linear multimers of C3d. Remarkably, SA- and gp120-specific Ab responses were significantly augmented in CD21/35(-/-) mice when these Ags were complexed with C3d in comparison to Ag alone. In fact, primary and secondary Ab responses and Ab-forming cell responses of CD21/35(-/-) mice approached those of wild-type mice immunized with SA-C3dg and gp120-C3d. Thus, C3d can function as a molecular adjuvant in the absence of CD21/35 expression.

Histone stoichiometry and DNA circularization in archaeal nucleosomes.

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.

Small Circles of Helical DNA Obtained on the Basis of Transcriptional Pause Sites Sequence

The 21-base pair synthetic DNA duplexes with basic ‘pause-motif’ site (‘CATGC’) were ligated head-to-tail to produce linear and circular multimers. This also was done from other closely related sequences. Electrophoretic mobilities of the linear multimers in Polyacrylamide gels were determined under the standard and modified conditions. We revealed that small linear multimers (∼ 90 bp) were characterized by comparable value of gel retardation relative to the well known curved DNA, while longer multimers (130–170 bp) had only slightly expressed mobility anomaly. Nevertheless these multimers containing nontruncated ‘pause-motif were capable of cyclization, in particular, formation of unusually small circles while truncated ones were not. We conclude that basic ‘pause-motif site increases the closure ability while the multimers based on truncated ‘pause motif fail to curve into the small circles. We tend to explain this situation as a result of intrinsic bending as well as the influence of the thermal fluctuations of DNA, the latter most probably can be associated with ‘pause motif. We have estimated the equilibrial and maximal bend angles per 10.5 bp to be 12°∼16° and 32° accordingly under experimental conditions of our study.

A role for p53 in DNA end rejoining by human cell extracts.

The tumor suppressor p53 is a major regulator in the response of human cells to DNA damage. In this study we assessed the role of p53 in the repair of DNA double-strand breaks in plasmid DNA using cell extracts from three human lymphoblastoid cell lines derived from the same donor. TK6, WI-L2-NS and TK6-E6-5e cells express wild-type, mutated and essentially no p53 protein, respectively. Total cellular extracts from TK6, WI-L2-NS and TK6-E6-5e cells were incubated with EcoRI linearized pUC19 DNA. Southern blot analysis of end-rejoined DNA indicated that the major products formed were linear multimers. There was approximately 2-fold greater end rejoining in WI-L2-NS and TK6-E6-5e extracts compared with TK6 extracts. Total DNA from end-rejoining reactions was purified and used to transform bacteria. Using the lacZ reporter gene as a measure of repair fidelity we found that misrepair, as indicated by white colonies, occurred at 4.1% to 6.5% of transformants, with no significant difference between the three cell lines. Gel analysis revealed that misrepair involved only deletions. Sequence analysis of 11 misrepaired products from each cell line showed 12 different deletions from 4 to 48 bp in length, but each cell line yielded similar product types. These results indicate that total cellular extracts from human lymphoblastoid cells lacking p53 or expressing mutated p53 have increased end-rejoining activity as compared with extracts from cells expressing wild-type p53. However, the p53 status does not influence the ratio of misrepair:correct repair, or the type of misrepair events.

The structure of the isolated, central hairpin of the HDV antigenomic ribozyme: novel structural features and similarity of the loop in the ribozyme and free in solution.

The structure of an RNA hairpin containing a seven-nucleotide loop that is present in the self-cleaving sequence of hepatitis delta virus antigenomic RNA was determined by high resolution NMR spectroscopy. The loop, which is composed of only one purine and six pyrimidines, has a suprisingly stable structure, mainly supported by sugar hydroxyl hydrogen bonds and base-base and base-phosphate stacking interactions. Compared with the structurally well-determined, seven-membered anticodon loop in tRNA, the sharp turn which affects the required 180 degrees change in direction of the sugar-phosphate backbone in the loop is shifted one nucleotide in the 3' direction. This change in direction can be characterized as a reversed U-turn. It is expected that the reversed U-turn may be found frequently in other molecules as well. There is evidence for a new non-Watson-Crick UC base pair formed between the first and the last residue in the loop, while most of the other bases in the loop are pointing outwards making them accessible to solvent. From chemical modification, mutational and photocrosslinking studies, a similar picture develops for the structure of the hairpin in the active ribozyme indicating that the loop structure in the isolated hairpin and in the ribozyme is very similar.

Determination of Self-Association Equilibrium Constants of Ethanol by FTIR Spectroscopy

The self-association of ethanol diluted in CCl4 has been studied by IR spectroscopy. Three OH stretching bands were assigned to monomers, linear dimers, and linear multimers. The self-association was described with two equilibrium constants, one for the formation of dimers, K2, the other for the formation of multimers, KA. These equilibrium constants were determined for ethanol concentration ranging from 0.19 to 0.76 mol L-1 by using a method developed by Coggeshall and Saier. At T = 25 °C, K2 was found to be equal to 1.08 L mol-1 in terms of molar concentration or 18.4 in terms of molar fraction; KA was found to be equal to 3.52 L mol-1 in terms of molar concentration or 60.0 in terms of molar fraction. The enthalpy of formation for K2 was found to be equal to −4.61 kcal mol-1 and to −4.15 kcal mol-1 for KA.

A new phenotype for sbcB mutations in Escherichia coli: RecA-dependent increase in plasmid-borne gene expression.

A new chromosomal mutation (cpeA), that causes increased expression of plasmid-borne genes in Escherichia coli, was identified and mapped to the sbcB locus. The effect of the mutation on plasmid transcription was non-specific with respect to the various promoters that were studied, but was more pronounced for an IncW low-copy-number plasmid than for ColE1- or p15A-based, high-copy-number plasmids. The mutant phenotype was observed even in recB+C+D+ strains, but not in recA mutants. The increased-expression phenotype was also observed in sbcB15 but not in xonA1 (another sbcB allele) mutants, suggesting that the expression of this phenotype is mediated by genes of the so-called RecF pathway family. Consistent with this interpretation was the observation that the cpeA mutant phenotype was less pronounced in recF, recJ and recO mutants. The increased-expression phenotype was also correlated with increased recovery of plasmid DNA from the cpeA/sbcB mutant strains, but there was no evidence for the occurrence of linear plasmid multimers in these strains.

Role of Erf recombinase in P22-mediated plasmid transduction.

In the absence of host RecA function, plasmid transduction by bacteriophage P22 can be mediated by Erf recombinase. Erf is not carried on the infecting particle but synthesized upon infection. In the recipient cell, Erf can promote both generalized plasmid transduction (which requires the circularization of plasmids transduced as linear multimers) and specialized plasmid transduction (which requires the release of plasmid DNA from linear plasmid-phage cointegrates). Both processes of Erf-mediated plasmid transduction require host RecBCD function. In contrast, RecBCD is not required for Erf-mediated circularization of P22 DNA.

Increase of Satellite Tobacco Ringspot Virus RNA Initiated by Inoculating Circular RNA

A small satellite RNA of tobacco ringspot virus (sTRSV RNA) generates circular and linear molecules of unit length and repetitive sequence, linear multimers during replication. The phosphodiester junction joining the unit satellite RNA sequences in multimeric and circular RNA resisted base-catalyzed cleavage in circles but not in linear dimers. We postulate that junctions of multimeric satellite RNA form during synthesis of the polyribonucleotide chain, whereas those of circular RNA result from a ligation reaction that introduces a group blocking the junction 2′-hydroxyl. To test the relative effectiveness of linear and circular satellite RNAs in initiating replication, we inoculated onto bean (Phaseolus vulgaris cv Black Valentine) the four possible pairs of satellite RNA molecules, one member of each pair having the wild-type sTRSV RNA sequence and the other that of the replicating mutant 51AG/212CU, with each sequence provided as the unit circular or linear form. The relative amounts of wild-type and mutant satellite RNA sequence recovered from progeny virions reflected their relative abundances in the inoculum without regard to whether the sequence was supplied as a linear or a circular molecule. These results are consistent with models for the replication of the satellite RNA in which a circular form of the satellite RNA is a template for rolling circle transcription or is otherwise a replication intermediate or is readily converted to an intermediate. We also show that a circular form of a nonaccumulating satellite RNA mutant induced an increase in a satellite RNA that is endogenous to some tobacco ringspot virus virion preparations, as demonstrated previously for the linear form.

Molecular association of pentanols in n-heptane IV: a photochromic reaction probe.

A photochromic reaction of a fulgide, Aberchrome 540 dye has been used to probe the molecular association of 1-,2-,and t-, pentanol in n-heptane. The kinetics of the photobleaching of the red dye (C-form) to the amber product (E-form) has been investigated spectrophotometrically over a low concentration range of < 0.3 mole fraction (F), at the temperature span of 303 to 323 K. Analyses of the results show that the photobleaching of the red dye follows a pseudo-first-order reaction, allowing for the extraction of the apparent rate constants (k) and the apparent activation energies (E a). Factors affecting the reaction are discussed. These factors are the solvent polarity effect, the solvent viscosity effect and the hydrogen bonding effect, deriving from the intermolecular hydrogen bond formation between the dye and the solvent molecules. The latter effect has been partially supported by the IR study of the dye in a number of solvents. Plots of E a against concentration (F) show extreme value for each pentanol. This result has been used to show the plausible presence of dominant multimers in the solutions. It appears that linear multimers are important for the 1-pentanol and 2-pentanol solutions. For the case of t-pentanol, E a of the reaction reaches a maximum at F=0.05. This is the same concentration at which the activation energy of viscous flow, E v was found to be maximum in the low concentration range. An explanation of the observed maximum E a is offered and it has led to the suggestion that the cyclic multimers may be the dominant species. Such a view is consistent with previous results from this laboratory i.e., the Kerr effect, viscometric and the NMR studies.

Transformation of Bacillus subtilis by DNA bound on clay in non-sterile soil

Chromosomal DNA from Bacillus subtilis and different forms of plasmid pHV14 (covalently closed circular (CCC), linear monomer (M), and linear multimer (LM)) were adsorbed and bound on the clay mineral montmorillonite. After extensive washing of the clay-DNA complexes with DNA buffer (pH 7.5), approx. 25% of the chromosomal DNA, and approx. 30, 90, and 5%, respectively, of the CCC, M and LM form remained bound. Chromosomal and plasmid DNA bound on clay were capable of transforming competent cells, with different specific activities. The clay-DNA complexes persisted in non-sterile soil and retained transforming ability up to 15 days after their addition to the soil. DNA bound on montmortillonite was protected from the activity of EcoRI, supporting the evidence that DNA adsorbed on soil components was resistant to degradation by nucleases.

Formation of linear plasmid multimers promoted by the phage lambda Red-system in lon mutants of Escherichia coli

We report here the formation of plasmid linear multimers promoted by the Red-system of phage lambda using a multicopy plasmid comprised of lambda red alpha and red beta genes, under the control of the lambda cI857 repressor. Our observations have revealed that the multimerization of plasmid DNA is dependent on the red beta and recA genes, suggesting a concerted role for these functions in the formation of plasmid multimers. The formation of multimers occurred in a recBCD+ sbcB+ xthA+ lon genetic background at a higher frequency than in the isogenic lon+ host cells. The multimers comprised tandem repeats of monomer plasmid DNA. Treatment of purified plasmid DNA with exonuclease III revealed the presence of free double-chain ends in the molecules. Determination of the size of multimeric DNA, by pulse field gel electrophoresis, revealed that the bulk of the DNA was in the range 50-240 kb, representing approximately 5-24 unit lengths of monomeric plasmid DNA. We provide a conceptual framework for Red-system-promoted formation and enhanced accumulation of plasmid linear multimers in lon mutants of E. coli.

The origin of greater-than-unit-length plasmids generated during bacterial conjugation.

In Gram-negative bacteria, the general mechanism of conjugal plasmid transfer, which is probably similar for many different groups of plasmids, involves the transfer of a single plasmid DNA strand with 5' to 3' polarity. Transfer is initiated by nicking of the duplex DNA at a particular site, i.e. the origin of transfer (oriT). We constructed plasmids containing two directly repeated copies of oriT, derived from the broad-host-range plasmid R1162 and flanking the lac operator. The number of lacO copies in the plasmid after transfer could be determined from the colour of transconjugant colonies on medium containing X-Gal. When the oriTs were mutated to prevent initiation and termination of transfer at the same oriT, almost all of the transconjugant cells contained greater-than-unit-length plasmids with two copies of lacO and three copies of oriT. We show that these molecules were generated by an intramolecular, conjugation-dependent mechanism unlikely to depend solely on a pre-existing population of circular or linear multimers in donor cells. We propose that the greater-than-unit-length molecules were instead generated by a rolling-circle mechanism of DNA replication.

Chi sequence protects against RecBCD degradation of DNA in vivo.

RecBCD is a multifunctional enzyme involved in DNA degradation and homologous recombination. It also produces an endonucleolytic cleavage near properly oriented chi sites (5'-GCTGGTGG-3'). Plasmids are not known to be affected by either RecBCD enzyme or the presence of a chi site. We report here that plasmids that replicate by a rolling circle mechanism accumulate large amounts of high molecular weight linear multimers (HMW), either if they contain a chi site or if RecBCD is absent. An in vivo inducible system for rolling circle replication was constructed to study RecBCD and its interactions with chi. Results show that (i) HMW accumulation is chi orientation dependent, and (ii) a succession of chi sites prevents degradation of HMW by RecBCD enzyme. These results demonstrate chi activity in plasmids. The rolling circle mechanism produces a sigma structure during plasmid replication; we propose that the double-stranded DNA tail of this sigma form allows RecBCD entry; the tail is degraded unless it is protected by a chi site. By analogy, a principal role of chi in the survival of lambda red-gam- mutants in wild-type strains may be to protect rolling circle concatemers (in late replication) from degradation by RecBCD.

Gene conversion in the Escherichia coli RecF pathway: a successive half crossing-over model.

Gene conversion--apparently non-reciprocal transfer of sequence information between homologous DNA sequences--has been reported in various organisms. Frequent association of gene conversion with reciprocal exchange (crossing-over) of the flanking sequences in meiosis has formed the basis of the current view that gene conversion reflects events at the site of interaction during homologous recombination. In order to analyze mechanisms of gene conversion and homologous recombination in an Escherichia coli strain with an active RecF pathway (recBC sbcBC), we first established in cells of this strain a plasmid carrying two mutant neo genes, each deleted for a different gene segment, in inverted orientation. We then selected kanamycin-resistant plasmids that had reconstituted an intact neo+ gene by homologous recombination. We found that all the neo+ plasmids from these clones belonged to the gene-conversion type in the sense that they carried one neo+ gene and retained one of the mutant neo genes. This apparent gene conversion was, however, only very rarely accompanied by apparent crossing-over of the flanking sequences. This is in contrast to the case in a rec+ strain or in a strain with an active RecE pathway (recBC sbcA). Our further analyses, especially comparisons with apparent gene conversion in the rec+ strain, led us to propose a mechanism for this biased gene conversion. This "successive half crossing-over model" proposes that the elementary recombinational process is half crossing-over in the sense that it generates only one recombinant DNA duplex molecule, and leaves one or two free end(s), out of two parental DNA duplexes. The resulting free end is, the model assumes, recombinogenic and frequently engages in a second round of half crossing-over with the recombinant duplex. The products resulting from such interaction involving two molecules of the plasmid would be classified as belonging to the gene-conversion type without crossing-over. We constructed a dimeric molecule that mimics the intermediate form hypothesized in this model and introduced it into cells. Biased gene conversion products were obtained in this reconstruction experiment. The half crossing-over mechanism can also explain formation of huge linear multimers of bacterial plasmids, the nature of transcribable recombination products in bacterial conjugation, chromosomal gene conversion not accompanied by flanking exchange (like that in yeast mating-type switching), and antigenic variation in microorganisms.

Nonconservative recombination in Escherichia coli.

Homologous recombination between two duplex DNA molecules might result in two duplex DNA molecules (conservative) or, alternatively, it might result in only one recombinant duplex DNA molecule (nonconservative). Here we present evidence that the mode of homologous recombination is nonconservative in an Escherichia coli strain with an active RecF pathway (a recBC sbcBC mutant). We employed plasmid substrates that enable us to recover both recombination products. These plasmids carry two mutant alleles of neo gene in direct orientation, two drug-resistance marker genes, and two compatible replication origins. After their transfer to the cells followed by immediate selection for the recombination to neo+, we could recover only one recombination product. A double-strand break at the region of homology increased this nonconservative recombination. If a nonconservative exchange should leave an end, this end may stimulate another exchange. Such "successive half crossing-over events" can explain several recombination-related phenomena in E. coli, including the origin of plasmid linear multimers and of transcribable, nonreplicated recombination products, and also in yeast and mammalian cells.

Hydrogen Peroxide-induced DNA Damages &lt;italic&gt;In Vitro&lt;/italic&gt; Revealed by Electron Microscopy

A plasmid pSV2neo (5.6 kb) which is a recombinant SV40-based vector was subjected to hydrogen peroxide treatment in presence of iron in vitro (Fenton reaction). Transmission electron microscopy revealed that H2O2 through the Fenton reaction introduced different types of damages in DNA. Besides the expected nicked circular and linear molecules, single-stranded loops and plasmid multimers could be characterized. These observations on DNA damages might be helpful for better understanding of the effect of hydrogen peroxide on living cells in vivo.