DNA-strand exchange promoted by RecA protein in the absence of ATP: implications for the mechanism of energy transduction in protein-promoted nucleic acid transactions.

DNA-strand exchange promoted by Escherichia coli RecA protein normally requires the presence of ATP and is accompanied by ATP hydrolysis, thereby implying a need for ATP hydrolysis. Previously, ATP hydrolysis was shown not to be required; here we demonstrate furthermore that a nucleoside triphosphate cofactor is not required for DNA-strand exchange. A gratuitous allosteric effector consisting of the noncovalent complex of ADP and aluminum fluoride, ADP.AIF4-, can both induce the high-affinity DNA-binding state of RecA protein and support the homologous pairing and exchange of up to 800-900 bp of DNA. These results demonstrate that induction of the functionally active, high-affinity DNA-binding state of RecA protein is needed for RecA protein-promoted DNA-strand exchange and that there is no requirement for a high-energy nucleotide cofactor for the exchange of DNA strands. Consequently, the free energy needed to activate the DNA substrates for DNA-strand exchange is not derived from ATP hydrolysis. Instead, the needed free energy is derived from ligand binding and is transduced to the DNA via the associated ligand-induced structural transitions of the RecA protein-DNA complex; ATP hydrolysis simply destroys the effector ligand. This concept has general applicability to the mechanism of energy transduction by proteins.

Kinetics and mechanism of polyamide ("peptide") nucleic acid binding to duplex DNA.

To elucidate the mechanism of recognition of double-stranded DNA (dsDNA) by homopyrimidine polyamide ("peptide") nucleic acid (PNA) leading to the strand-displacement, the kinetics of the sequence-specific PNA/DNA binding have been studied. The binding was monitored with time by the gel retardation and nuclease S1 cleavage assays. The experimental kinetic curves obey pseudo-first-order kinetics and the dependence of the pseudo-first-order rate constant, kps, on PNA concentration, P, obeys a power law kps approximately P gamma with 2 < gamma < 3. The kps values for binding of decamer PNA to dsDNA target sites with one mismatch are hundreds of times slower than for the correct site. A detailed kinetic scheme for PNA/DNA binding is proposed that includes two major steps of the reaction of strand invasion: (i) a transient partial opening of the PNA binding site on dsDNA and incorporation of one PNA molecule with the formation of an intermediate PNA/DNA duplex and (ii) formation of a very stable PNA2/DNA triplex. A simple theoretical treatment of the proposed kinetic scheme is performed. The interpretation of our experimental data in the framework of the proposed kinetic scheme leads to the following conclusions. The sequence specificity of the recognition is essentially provided at the "search" step of the process, which consists in the highly reversible transient formation of duplex between one PNA molecule and the complementary strand of duplex DNA while the other DNA strand is displaced. This search step is followed by virtually irreversible "locking" step via PNA2/DNA triplex formation. The proposed mechanism explains how the binding of homopyrimidine PNA to dsDNA meets two apparently mutually contradictory features: high sequence specificity of binding and remarkable stability of both correct and mismatched PNA/DNA complexes.

The Structure of theCyprinid herpesvirus 3ORF112-Zα·Z-DNA Complex Reveals a Mechanism of Nucleic Acids Recognition Conserved with E3L, a Poxvirus Inhibitor of Interferon Response

In vertebrate species, the innate immune system down-regulates protein translation in response to viral infection through the action of the double-stranded RNA (dsRNA)-activated protein kinase (PKR). In some teleost species another protein kinase, Z-DNA-dependent protein kinase (PKZ), plays a similar role but instead of dsRNA binding domains, PKZ has Zα domains. These domains recognize the left-handed conformer of dsDNA and dsRNA known as Z-DNA/Z-RNA. Cyprinid herpesvirus 3 infects common and koi carp, which have PKZ, and encodes the ORF112 protein that itself bears a Zα domain, a putative competitive inhibitor of PKZ. Here we present the crystal structure of ORF112-Zα in complex with an 18-bp CpG DNA repeat, at 1.5 Å. We demonstrate that the bound DNA is in the left-handed conformation and identify key interactions for the specificity of ORF112. Localization of ORF112 protein in stress granules induced in Cyprinid herpesvirus 3-infected fish cells suggests a functional behavior similar to that of Zα domains of the interferon-regulated, nucleic acid surveillance proteins ADAR1 and DAI.

The isotope composition of selenium in chondrites constrains the depletion mechanism of volatile elements in solar system materials

Solar nebula processes led to a depletion of volatile elements in different chondrite groups when compared to the bulk chemical composition of the solar system deduced from the Sun's photosphere. For moderately-volatile elements, this depletion primarily correlates with the element condensation temperature and is possibly caused by incomplete condensation from a hot solar nebula, evaporative loss from the precursor dust, and/or inherited from the interstellar medium. Element concentrations and interelement ratios of volatile elements do not provide a clear picture about responsible mechanisms. Here, the abundance and stable isotope composition of the moderately- to highly-volatile element Se are investigated in carbonaceous, ordinary, and enstatite chondrites to constrain the mechanism responsible for the depletion of volatile elements in planetary bodies of the inner solar system and to define a δ(82/78)Se value for the bulk solar system. The δ(82/78)Se of the studied chondrite falls are identical within their measurement uncertainties with a mean of −0.20±0.26‰ (2 s.d., n=14n=14, relative to NIST SRM 3149) despite Se abundance depletions of up to a factor of 2.5 with respect to the CI group. The absence of resolvable Se isotope fractionation rules out a kinetic Rayleigh-type incomplete condensation of Se from the hot solar nebula or partial kinetic evaporative loss on the precursor material and/or the parent bodies. The Se depletion, if acquired during partial condensation or evaporative loss, therefore must have occurred under near equilibrium conditions to prevent measurable isotope fractionation. Alternatively, the depletion and cooling of the nebula could have occurred simultaneously due to the continuous removal of gas and fine particles by the solar wind accompanied by the quantitative condensation of elements from the pre-depleted gas. In this scenario the condensation of elements does not require equilibrium conditions to avoid isotope fractionation. The results further suggest that the processes causing the high variability of Se concentrations and depletions in ordinary and enstatite chondrites did not involve any measurable isotope fractionation. Different degrees of element depletions and isotope fractionations of the moderately-volatile elements Zn, S, and Se in ordinary and enstatite chondrites indicate that their volatility is controlled by the thermal stabilities of their host phases and not by the condensation temperature under canonical nebular conditions.

Dynamic Precision Phenotyping Reveals Mechanism of Crop Tolerance to Root Herbivory

The western corn rootworm (WCR) is a major pest of maize that is well adapted to most crop management strategies. Breeding for tolerance is a promising alternative to combat WCR, but is currently constrained by a lack of physiological understanding and phenotyping tools. We developed dynamic precision phenotyping approaches using carbon-11 with positron emission tomography, root autoradiography and radiometabolite flux analysis to understand maize tolerance to WCR. Our results reveal that WCR attack induces specific patterns of lateral root growth which are associated with a shift in auxin biosynthesis from indole-3-pyruvic acid to indole-3-acetonitrile. WCR attack also increases transport of newly synthesized amino acids to the roots, including the accumulation of glutamine. Finally, the regrowth zones of WCR attacked roots show an increase in glutamine turnover which strongly correlates with the induction of indole-3-acetonitrile-dependent auxin biosynthesis. In summary, our findings identify local changes in the auxin flux network as a promising marker for induced WCR tolerance.

Report of workshop on isolation, characterization, and mechanism of action of proteases and antiproteases /

Mode of access: Internet.

The mechanism of filtration /

Photostatic reproduction.

Mechanism of dimensional instability /

"Classification cancelled or changed to UNCLASSIFIED by authority of TIS Change Notice No. 1849 by ASB TISOR, date 8/14/55."

The mechanism of adjustment, Canadian balance of payments, 1946-1957.

Includes bibliographical references.

Elements of electro-biology, or the voltaic mechanism of man of electro-pathology, especially of the nervous system and of electro-therapeutics /

Mode of access: Internet.

A study of the mechanism of radiation-induced gelation in monomer-polymer mixtures : report for the period May 1, 1961 - October 31, 1962 /

"October 31, 1962."