Site-specific Reaction Research Articles

Reactions of boron cluster anions: implication of site-specific chemical neutralization pathways

Boron cluster anions were studied in a fast-flow tube reaction apparatus. The clusters were reacted with methyl iodide, and based on comparisons with aluminum clusters, it is suggested that we have observed the formation of neutral B n CH 3 clusters. While isotopic peak broadening and mass coincidences prohibit quantitative conclusions, analogies with previous experiments involving aluminum clusters allow us to speculate as to the nature of the neutralization process in these reactions. It is believed that the type of site-specific reactions implied here may correspond directly to the reactions observed with aluminum clusters.

Chronic vulvar purpura: persistent pigmented purpuric dermatitis (lichen aureus) of the vulva or plasma cell (Zoon's) vulvitis?

Lichen aureus is localized variant of persistent pigmented purpuric dermatitis that typically affects the legs and can be associated with delayed hypersensitivity reactions or vascular abnormalities. Plasma cell vulvitis (Zoon's vulvitis) is a rare condition that frequently contains hemosiderin deposits and is suspected to be a mucosal reaction pattern due to variety of insults, most often local irritation or trauma. A 50-year-old female with longstanding complaints of spotting, vulvar dryness, irritation, and dyspareunia presented with circumscribed, purpuric, erythematous vulvar patches. Past estrogen cream treatment evoked symptoms of discomfort. On biopsy, siderophages and extravasated red blood cells were found in conjunction with a lichenoid, lymphocyte and plasma cell infiltrate, and dilated dermal and intraepithelial vessels. Reported herein is an unusual vulvar dermatosis that is best classified as a localized variant of persistent pigmented dermatosis (lichen aureus) but overlaps clinically and histologically with Zoon's vulvitis. This constellation of findings may represent a site-specific mucosal reaction to an erosive process that could either be inflammatory (hypersensitivity reaction) and/or traumatic in nature.

Kinetics of Reactions of Cl Atoms with Ethane, Chloroethane, and 1,1-Dichloroethane

Reactions of Cl atoms with ethane (1), chloroethane (2), and 1,1-dichloroethane (3) were studied experimentally with the discharge flow/resonance fluorescence technique over wide ranges of temperatures and at pressures between 2.3 and 9.2 Torr. Results of earlier investigations of the reactions of Cl atoms with ethane and chloroethanes are analyzed and compared with the results of the current work. The rate constants of the reaction of Cl atoms with ethane obtained in the current study (k1 = (4.91 × 10-12)T0.47 exp(−82 K/T) cm3 molecule-1 s-1, 299−1002 K) agree with the results of earlier determinations. Combination of the temperature dependence of k1 obtained in the current work with the existing (mostly at lower temperatures) literature data results in the expression k1 = (7.23 × 10-13)T0.70 exp(+117 K/T) cm3 molecule-1 s-1 (203−1400 K). Rate constants of the reactions of Cl atoms with chloroethane and 1,1-dichloroethane demonstrate different temperature dependences in the low-temperature (room temperature to 378 K) and the high-temperature (484−810 K) regions. The differences are due to the regeneration of Cl atoms at higher temperatures as a consequence of the fast thermal decomposition of radical products with a chlorine atom in the β position. This information enables quantitative differentiation between the site-specific reaction channels. The results of the current study, combined with those of earlier relative-rate experiments, were used to derive expressions for the temperature dependences of the rates of the site-specific abstraction channels: k2a(T) = (2.76 ± 0.28) × 10-11 exp(−455 ± 44 K/T) (296−810 K), k2b(T) = (1.92 ± 0.47) × 10-11 exp(−789 ± 84 K/T) (296−378 K), k3a(T) = (1.46 ± 0.21) × 10-11 exp(−733 ± 58 K/T) (293−810 K), and k3b(T) = (2.98 ± 1.42) × 10-11 exp(−1686 ± 160 K/T) (293−378 K) cm3 molecule-1 s-1, where channels a and b signify abstraction of hydrogen atoms in the α and β positions, respectively.

Purely site-specific chemisorption and conformation of trimethylamine on Si(100)c(4 x 2).

One of the fundamental points of interest on the Si(100) surface is how the spatial localization of electron density on the buckled silicon dimer controls the site-specific reaction toward different Lewis acid and Lewis base molecules. We have investigated the adsorption of trimethylamine (TMA) on Si(100)c(4x2) using scanning tunneling microscopy (STM) at 80 K. The adsorbed TMA appears as a triangle-shaped bright protrusion in the occupied-state STM image. The triangle-shaped protrusion is ascribed to three methyl groups in the adsorbed TMA. The center of the protrusion is located on the down atom site, which indicates that the adsorption of TMA occurs only on the down dimer atom. Thus, TMA adsorption on Si(100)c(4x2) is found to be purely site-specific on the down dimer atom and can be categorized in Lewis acid-base reaction.

Crystallization and preliminary X-ray crystallographic analysis of the excisionase-DNA complex from bacteriophage lambda.

Bacteriophage lambda uses an elegantly regulated and highly directional site-specific DNA-recombination reaction to integrate and excise its genome. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which dramatically stimulates excision by orchestrating the assembly of a higher order nucleoprotein structure that excises the prophage. The Xis protein stabilizes this recombination intermediate by substantially altering the trajectory of viral DNA and by cooperatively interacting with the lambda integrase (Int) protein. In an attempt to understand how Xis controls the directionality of bacteriophage lambda recombination, co-crystals of the DNA-binding domain of Xis in complex with its binding site within the P-arm of the phage have been obtained using the hanging-drop vapor-diffusion method. Using sodium acetate as a precipitating reagent, the Xis-DNA complex crystallizes in space group C2, with unit-cell parameters a = 80.2, b = 72.7, c = 38.8 A, beta = 104.1 degrees. These crystals diffract beyond 1.5 A resolution and are well suited for structural analysis using X-ray crystallography.

Identification of the lambda integrase surface that interacts with Xis reveals a residue that is also critical for Int dimer formation.

Lambda integrase (Int) is a heterobivalent DNA-binding protein that together with the accessory DNA-bending proteins IHF, Fis, and Xis, forms the higher-order protein-DNA complexes that execute integrative and excisive recombination at specific loci on the chromosomes of phage lambda and its Escherichia coli host. The large carboxyl-terminal domain of Int is responsible for binding to core-type DNA sites and catalysis of DNA cleavage and ligation reactions. The small amino-terminal domain (residues 1-70), which specifies binding to arm-type DNA sites distant from the regions of strand exchange, consists of a three-stranded beta-sheet, proposed to recognize the cognate DNA site, and an alpha-helix. We report here that a site on this alpha-helix is critical for both homomeric interactions between Int protomers and heteromeric interactions with Xis. The mutant E47A, which was identified by alanine-scanning mutagenesis, abolishes interactions between Int and Xis bound at adjacent binding sites and reduces interactions between Int protomers bound at adjacent arm-type sites. Concomitantly, this residue is essential for excisive recombination and contributes to the efficiency of the integrative reaction. NMR titration data with a peptide corresponding to Xis residues 57-69 strongly suggest that the carboxyl-terminal tail of Xis and the alpha-helix of the aminoterminal domain of Int comprise the primary interaction surface for these two proteins. The use of a common site on lambda Int for both homotypic and heterotypic interactions fits well with the complex regulatory patterns associated with this site-specific recombination reaction.

Ionization of a critical adenosine residue in the neurospora Varkud Satellite ribozyme active site.

The Varkud Satellite (VS) ribozyme catalyzes a site-specific self-cleavage reaction that generates 5'-OH and 2',3'-cyclic phosphate products. Other ribozymes that perform an equivalent reaction appear to employ ionization of an active site residue, either to neutralize the negatively charged transition state or to act as a general acid-base catalyst. To test for important base ionization events in the VS ribozyme ligation reaction, we performed nucleotide analogue interference mapping (NAIM) with a series of ionization-sensitive adenosine and cytidine analogues. A756, a catalytically critical residue located within the VS active site, was the only nucleotide throughout the VS ribozyme that displayed the pH-dependent interference pattern characteristic of functional base ionization. We observed unique rescue of 8-azaadenosine (pK(a) 2.2) and purine riboside (pK(a) 2.1) interference at A756 at reduced reaction pH, suggestive of an ionization-specific effect. These results are consistent with protonation and/or deprotonation of A756 playing a direct role in the VS ribozyme reaction mechanism. In addition, NAIM experiments identified several functional groups within the RNA that play important roles in ribozyme folding and/or catalysis. These include residues in helix II, helix VI (730 loop), the II-III-VI and III-IV-V helix junctions, and loop V.

Mechanism of Inhibition of Site-specific Recombination by the Holliday Junction-trapping Peptide WKHYNY: Insights into Phage λ Integrase-mediated Strand Exchange

Holliday junctions are central intermediates in site-specific recombination reactions mediated by tyrosine recombinases. Because these intermediates are extremely transient, only artificially assembled Holliday junctions have been available for study. We have recently identified hexapeptides that cause the accumulation of natural Holliday junctions of bacteriophage λ Integrase (Int)-mediated reactions. We now show that one of these peptides acts after the first DNA cleavage event to stabilize protein-bound junctions and to prevent their resolution. The peptide acts before the step affected by site affinity ( saf) mutations in the core region, in agreement with a model that the peptide stabilizes the products of strand exchange (i.e. Holliday junctions) while saf mutations reduce ligation of exchanged strands. Strand exchange events leading to Holliday junctions in phage λ integration and excision are asymmetric, presumably because interactions between Int and some of its core-binding sites determine the order of strand cleavage. We have compared the structure of Holliday junctions in one unidirectional and in two bidirectional Int-mediated pathways and show that the strand cleavage steps are much more symmetric in the bidirectional pathways. Thus Int–DNA interactions which determine the order of top and bottom strand cleavage and exchange are unique in each recombination pathway.

Molecular typing of dengue virus type 2 in Brazil.

Strain typing is a critical tool for molecular epidemiological analysis and can provide important information about the spread of dengue viruses. Here, we performed a molecular characterization of DEN-2 viruses isolated in Brazil during 1990-2000 from geographically and temporally distinct areas in order to investigate the genetic distribution of this serotype circulating in the country. Restriction site-specific polymerase chain reaction (RSS)-PCR presented the same pattern for all 52 Brazilian samples, showing the circulation of just one DEN-2 variant. Phylogenetic analysis using progressive pairwise alignments from 240-nucleotide sequences of the E/NS1 junction in 15 isolates showed that they belong to genotype III (Jamaica genotype).

Regulation of Directionality in Bacteriophage λ Site-specific Recombination: Structure of the Xis Protein

Upon induction of a bacteriophage λ lysogen, a site-specific recombination reaction excises the phage genome from the chromosome of its bacterial host. A critical regulator of this process is the phage-encoded excisionase (Xis) protein, which functions both as a DNA architectural factor and by cooperatively recruiting integrase to an adjacent binding site specifically required for excision. Here we present the three-dimensional structure of Xis and the results of a structure-based mutagenesis study to define the molecular basis of its function. Xis adopts an unusual “winged”-helix motif that is modeled to interact with the major- and minor-grooves of its binding site through a single α-helix and loop structure (“wing”), respectively. The C-terminal tail of Xis, which is required for cooperative binding with integrase, is unstructured in the absence of DNA. We propose that asymmetric bending of DNA by Xis positions its unstructured C-terminal tail for direct contacts with the N-terminal DNA-binding domain of integrase and that an ensuing disordered to ordered transition of the tail may act to stabilize the formation of the tripartite integrase–Xis–DNA complex required for phage excision.

MULTI-SUGAR ANALYSIS SYSTEM USING A NOVEL GLUCOSE-3-DEHYDROGENASE ELECTRODE

A flavoenzyme, glucose-3-dehydrogenase (G3DH) from Halomonas sp. α-15 strain, possessed broad substrate specificity and site-specific reaction against various sugars. We constructed an amperometric enzyme electrode employing G3DH and also developed a multi-sugar analysis system combined with the G3DH sensor and sugar analysis chromatography. This system can discriminate the 4 components of sugar, i.e., glucose, 1,5-anhydro-D-glucitol, trehalose, and N-carbamoyl-β-D-glucopyranosylamine mixture and it can determine each concentration from 2 mM to 10 mM.

Site-specific labeling of supercoiled DNA at the A+T rich sequences.

Progress in structural biology studies of supercoiled DNA and its complexes with regulatory proteins depends on the availability of reliable and routine procedures for site-specific labeling of circular molecules. For this, we made use of oligonucleotide uptake by plasmid DNA under negative superhelical tension. Subsequent circularization of the oligonucleotide label facilitated by an oligonucleotide scaffold results in its threading between the two strands of duplex DNA. Several lines of evidence, including direct AFM mapping of the label, show that the circular oligonucleotide is stably localized at its target, an A+T rich region. The specific binding mode when the oligonucleotide threads the double helix results in a DNA kink that tends to occupy an apical position in a plectonemically wound supercoiled DNA, similar to the positioning of an A-tract bend. Site-specific labels may allow visualization techniques, such as electron and atomic force microscopies, to reliably map protein binding sites, identify local alternative structures in supercoiled DNA, and monitor structural dynamics of DNA molecules in real time. Site-specific oligonucleotide reactions with DNA may also have application in biotechnology and gene therapy.

Signatures of site-specific reaction of H2 on Cu(100)

Six-dimensional quantum dynamical calculations are presented for the reaction of (v,j) H2 on Cu(100), at normal incidence, for v=0–1 and j=0–5. The dynamical calculations employed a potential energy surface computed with density functional theory, using the generalized gradient approximation and a slab representation for the adsorbate-substrate system. The aim of the calculations was to establish signatures from which experiments could determine the dominant reaction site of H2 on the surface and the dependence of the reaction site on the initial rovibrational state of H2. Two types of signatures were found. First, we predict that, at energies near threshold, the reaction of (v=1) H2 is rotationally enhanced, because it takes place at the top site, which has an especially late barrier and a reaction path with a high curvature. On the other hand, we predict the reaction to be almost independent of j for (v=0) H2, which reacts at the bridge site. Second, we predict that, at collision energies slightly above threshold for which the reaction probabilities of the (v=0) and (v=1) states are comparable, the rotational quadrupole alignment of (v=1) reacting molecules should be larger than that of (v=0) reacting molecules, for j=1, 4, and 5. For (j=2) H2, the opposite should be true, and for (j=3) H2, the rotational quadrupole alignment should be approximately equal for (v=1) and (v=0) H2. These differences can all be explained by the difference in the predicted reaction site for (v=1) and (v=0) H2 (top and bridge) and by the differences in the anisotropy of the potential at the reaction barrier geometries associated with these sites. Our predictions can be tested in associative desorption experiments, using currently available experimental techniques.

Site-specific DNA cleavage by Chlorella virus topoisomerase II.

The DNA cleavage reaction of topoisomerase II is central to the catalytic activity of the enzyme and is the target for a number of important anticancer drugs. Unfortunately, efforts to characterize this fundamental reaction have been limited by the low levels of DNA breaks normally generated by the enzyme. Recently, however, a type II topoisomerase with an extraordinarily high intrinsic DNA cleavage activity was isolated from Chlorella virus PBCV-1. To further our understanding of this enzyme, the present study characterized the site-specific DNA cleavage reaction of PBCV-1 topoisomerase II. Results indicate that the viral enzyme cleaves DNA at a limited number of sites. The DNA cleavage site utilization of PBCV-1 topoisomerase II is remarkably similar to that of human topoisomerase IIalpha, but the viral enzyme cleaves these sites to a far greater extent. Finally, PBCV-1 topoisomerase II displays a modest sensitivity to anticancer drugs and DNA damage in a site-specific manner. These findings suggest that PBCV-1 topoisomerase II represents a unique model with which to dissect the DNA cleavage reaction of eukaryotic type II topoisomerases.

Measuring the Site-Specific Reactivity of Impurities: The Pronounced Effect of Dissolved Oxygen on Silicon Etching

The reactivity of dissolved O2, a ubiquitous impurity in aqueous solutions, with H-terminated Si(111) surfaces was quantified using a new technique that exploits kinetic competition between an impurity and an etchant to produce impurity-concentration-dependent changes in the steady-state etch morphology. This method is applicable to any impurity that alters the etch morphology. The impurity-induced changes are quantified using scanning tunneling microscopy measurements and atomistic, concentration-dependent kinetic Monte Carlo simulations. The site-specific reactivity of O2(aq) is surprisingly anisotropic. Oxidation of the highly strained dihydride step site is 4 times faster than oxidation of the relatively unstrained monohydride step site. Both steps are 104 times more reactive than terrace sites. The observed site-specific reaction rates are highly correlated to local strain. FTIR measurements of the Si−H stretch vibration showed that dissolved O2 inserts O atoms into surface Si−Si back-bonds without removing the H-termination. Dissolved O2 does not attack Si−H bonds, because neither Si−H consumption nor silanol production is observed.

Characterization of hard metal dusts from sintering and detonation coating processes and comparative hydroxyl radical production.

Dust samples from sintering and detonation coating hard-metal processes were characterized, compared, and contrasted for morphology, composition, and generation of hydroxyl radicals. Inhalation of respirable hard-metal (sintered carbide) dusts from hard-metal processes is known to cause fibrotic and asthmatic lung disease. Scanning electron microscopy/energy-dispersive X-ray analysis was used for morphology, composition, and elemental distribution. An electron spin resonance (ESR) spin trapping technique was used to detect hydroxyl radical generation. Samples were incubated with air-saturated buffer solutions containing a spin trap and analyzed by ESR for the presence of *OH in solution. Postdetonation coating samples often had surface contamination of Co on the WC particles, as shown by elemental mapping of individual particles; this was not evident in predetonation samples or unsintered materials in this study. ESR measurements show that both detonation-gun materials were capable of generating *OH , while the WC, cobalt, and presintered mixture did not produce detectable amounts of *OH radicals. The DMPO/*OH adduct formation was apparently facilitated by Fe-mediated reactions for predetonation dusts, and by Fe-mediated site-specific reactions for postdetonation dusts. The overspray materials from the detonation-gun process produced 9-fold more *OH radicals than the predetonation coating mixture. Overall, this study indicates there are substantial differences between postdetonation materials and both predetonation and unsintered hard-metal process materials with respect to morphology, elemental distribution, and *OH radical generation reactions and that these differences may be important in the toxic potential of those materials.

Site-specific Photo-cross-linking between λ Integrase and Its DNA Recombination Target

The site-specific recombinase (Int) of bacteriophage lambda is a heterobivalent DNA-binding protein and is composed of three domains as follows: an amino-terminal domain that binds with high affinity to "arm-type" sequences within the recombination target DNA (att sites), a carboxyl-terminal domain that contains all of the catalytic functions, and a central domain that contributes significantly to DNA binding at the "core-type" sequences where DNA cleavage and ligation are executed. We constructed a family of core-type DNA oligonucleotides, each of which contained the photoreactive analog 4-thiodeoxythymidine (4-thioT) at a different position. When tested for their respective abilities to promote covalent cross-links with Int after irradiation with UV light at 366 nm, one oligonucleotide stood out dramatically. The 4-thioT substitution on the DNA strand opposite the site of Int cleavage led to photo-induced cross-linking efficiencies of approximately 20%. The efficiency and specificity of Int binding and cleavage at this 4-thioT-substituted core site was shown to be largely uncompromised, and its ability to participate in a full site-specific recombination reaction was reduced only slightly. Identification of the photo-cross-linked residue as Lys-141 in the central domain provides, along with other results, several insights about the nature of core-type DNA recognition by the bivalent recombinases of the lambda Int family.

Osmoregulation of dimer resolution at the plasmid pJHCMW1 mwr locus by Escherichia coli XerCD recombination.

Xer-mediated dimer resolution at the mwr site of plasmid pJHCMW1 is osmoregulated in Escherichia coli. Whereas under low-salt conditions, the site-specific recombination reaction is efficient, under high-salt conditions, it proceeds inefficiently. Regulation of dimer resolution is independent of H-NS and is mediated by changes in osmolarity rather than ionic effects. The low level of recombination at high salt concentrations can be overcome by high levels of PepA or by mutating the ARG box to a sequence closer to the E. coli ARG box consensus. The central region of the mwr core recombination site plays a role in regulation of site-specific recombination by the osmotic pressure of the medium.

The Drosophila Pipsqueak protein defines a new family of helix-turn-helix DNA-binding proteins.

Many prokaryotic and eukaryotic DNA-binding proteins use a helix-turn-helix (HTH) structure for DNA recognition. Here we describe a new family of eukaryotic HTH proteins, the Pipsqueak (Psq) family, which includes proteins from fungi, sea urchins, nematodes, insects, and vertebrates. Three subgroups of the Psq family can be distinguished. Like the HTH proteins of the prokaryotic resolvase family, members of the CENP-B/transposase subgroup catalyze site-specific recombination reactions. This functional conservation, together with a primary sequence similarity between the resolvase and Psq DNA-binding domains, suggests that the resolvase and Psq families are evolutionarily linked. More than half of the newly identified Drosophila Psq proteins contain a BTB protein-protein interaction domain. All proteins of this BTB subgroup belong to the conserved Tramtrack group of BTB-domain proteins. About half of the members of the Tramtrack group contain a Psq domain, while the other half is made up of proteins that contain a zinc finger domain. Thus, nearly all members of this group appear to be DNA-binding proteins. Among other developmental regulators, the Drosophila cell death protein E93 was found to contain a Psq motif and to define a third subgroup of Psq domain proteins. The high sequence conservation of the E93 Psq motif allowed the identification of E93 orthologs in humans and lower metazoans.

Genetic manipulation of mouse embryonic stem cells by mutant lambda integrase.

Mutant lambda integrases catalyze site-specific recombination reactions inside mammalian cells. Here we demonstrate that the integrase system can be used to eliminate resistance marker genes from the genome of mouse embryonic stem cells. So-called integrative and excisive recombination pathways led to the precise deletion of the neomycin gene, which was inserted together with a flanking pair of directly repeated recombination sites into the ROSA26 locus by standard targeting techniques. The excision of the resistance gene led to the expression of enhanced green fluorescence protein, which served as a means to sort out cells that had undergone site-specific recombination. Southern analysis and DNA sequencing confirmed that strand exchange reactions had occurred in the genome as expected. Hence, the integrase system may be used in conjunction with other site-specific recombinases as a tool in genome manipulation protocols.