Understanding how the crowded interior of cells stabilizes DNA/DNA and DNA/RNA hybrids–in silico predictions and in vitro evidence

Amplification of DNA in vivo occurs in intracellular environments characterized by macromolecular crowding (MMC). In vitro Polymerase-chain-reaction (PCR), however, is non-crowded, requires thermal cycling for melting of DNA strands, primer-template hybridization and enzymatic primer-extension. The temperature-optima for primer-annealing and extension are strikingly disparate which predicts primers to dissociate from template during extension thereby compromising PCR efficiency. We hypothesized that MMC is not only important for the extension phase in vivo but also during PCR by stabilizing nucleotide hybrids. Novel atomistic Molecular Dynamics simulations elucidated that MMC stabilizes hydrogen-bonding between complementary nucleotides. Real-time PCR under MMC confirmed that melting-temperatures of complementary DNA–DNA and DNA–RNA hybrids increased by up to 8°C with high specificity and high duplex-preservation after extension (71% versus 37% non-crowded). MMC enhanced DNA hybrid-helicity, and drove specificity of duplex formation preferring matching versus mismatched sequences, including hair-pin-forming DNA- single-strands

Solution structure of an arsenate reductase-related protein, YffB, from Brucella melitensis, the etiological agent responsible for brucellosis

B. melitensis is a NIAID Category B microorganism that is responsible for brucellosis and is a potential agent for biological warfare. Here, the solution structure of the 116-residue arsenate reductase-related protein Bm-YffB (BR0369) from this organism is reported

Structure of a Nudix hydrolase (MutT) in the Mg2+-­bound state from Bartonella henselae, the bacterium responsible for cat scratch fever

B. henselae is the etiological agent responsible for cat scratch fever (bartonellosis). The crystal structure of the smaller of the two Nudix hydrolases encoded in the genome of B. henselae, Bh-MutT, was determined to 2.1 Å resolution

eParticipation in the EU: Re-focusing on social media and young citizens for reinforcing European identity

Purpose: The purpose of this paper is to research the key role of eParticipation and social media in the construction and diffusion of a European identity for European citizens, as a valuable means of acculturalisation, through the creation of a common sense of belonging and self-identifying with the European ideals. Design/methodology/approach: The paper argues that the limited success of current EU institutions' communication strategy and eParticipation initiatives could be partly attributed to a communication gap between the means currently used on the one hand, and the preferences of targeted audiences on the other. Findings: This communication gap is demonstrated by combining empirical data on EU eParticipation initiatives addressing young people, young citizens' involvement in EU affairs, the penetration of social media on young citizen groups and the social media presence of EU political entities. Research limitations/implications: These empirical data could be enriched with more detailed statistics, and monitored across time to identity advancements and changing trends. Practical implications: The paper proposes, therefore, that the focus for eParticipation instruments be redirected to social media due to their comparative advantages as regards their great visibility, their notable level of penetration into current social groups and their potential of targeting specific audiences and becoming an integral part of these audiences' everyday life. Originality/value: The paper believes this approach can contribute to improving eParticipation ventures in terms of their actual appeal to young citizens and contribution to the construction and diffusion of a European identity. © Emerald Group Publishing Limited

Comparative thermal denaturation of Thermus aquaticus and Escherichia coli type 1 DNA polymerases.

Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5' nuclease domain. Removal of the 5' nuclease domains produces the 'large fragment' domains of Pol 1 and Taq, termed Klenow and Klentaq respectively. Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature T(m) of 37 and 100 degrees C respectively at pH 9.5. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5' nuclease domains of each melting separately. The 5' nuclease domain of Taq denatures as a separate peak, 10 degrees C before the Klentaq domain. Melting of the 5' nuclease domain of Pol 1 overlaps with the Klenow fragment. Presence of the 5' nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq. Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins. Melting monitored by CD yields slightly lower T(m) values, but almost identical van't Hoff enthalpy Delta H values, consistent with two-state unfolding followed by an irreversible kinetic step. Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow

Structure Guided Understanding of NAD⁺ Recognition in Bacterial DNA Ligases

NAD⁺-dependent DNA ligases (LigA) are essential bacterial enzymes that catalyze phosphodiester bond formation during DNA replication and repair processes. Phosphodiester bond formation proceeds through a 3-step reaction mechanism. In the first step, the LigA adenylation domain interacts with NAD⁺ to form a covalent enzyme-AMP complex. Although it is well established that the specificity for binding of NAD⁺ resides within the adenylation domain, the precise recognition elements for the initial binding event remain unclear. We report here the structure of the adenylation domain from <i>Haemophilus influenzae</i> LigA. This structure is a first snapshot of a LigA-AMP intermediate with NAD⁺ bound to domain 1a in its open conformation. The binding affinities of NAD⁺ for adenylated and nonadenylated forms of the <i>H. influenzae</i> LigA adenylation domain were similar. The combined crystallographic and NAD⁺-binding data suggest that the initial recognition of NAD⁺ is via the NMN binding region in domain 1a of LigA

The stability of Taq

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases has been performed by obtaining full Gibbs-Helmholtz stability curves of the free energy of folding (ΔG) versus temperature. This analysis provides the temperature dependencies of the folding enthalpy and entropy (ΔH and ΔS), and the heat capacity (ΔCp) of folding. If increased or enhanced non-covalent bonding in the native state is responsible for enhanced thermal stabilization of a protein, as is often proposed, then an enhanced favourable folding enthalpy should, in general, be observed for thermophilic proteins. However, for the Klenow-Klentaq homologous pair, the folding enthalpy (ΔHfold) of Klentaq is considerably less favorable than that of Klenow at all temperatures. In contrast, it is found that Klentaq\u27s extreme free energy of folding (ΔGfold) originates from a significantly reduced entropic penalty of folding (ΔSfold). Furthermore, the heat capacity changes upon folding are similar for Klenow and Klentaq. Along with this new data, comparable extended analysis of available thermodynamic data for 17 other mesophilic-thermophilic protein pairs (where enough applicable thermodynamic data exists) shows a similar pattern in seven of the 18 total systems. When analyzed with this approach, the more familiar reduced ΔCp mechanism for protein thermal stabilization (observed in a different six of the 18 systems) frequently manifests as a temperature dependent shift from enthalpy driven stabilization to a reduced-entropic-penalty model. © 2013 Wiley Periodicals, Inc