A Primase-Induced Conformational Switch Controls the Stability of the Bacterial Replisome.

Recent studies of bacterial DNA replication have led to a picture of the replisome as an entity that freely exchanges DNA polymerases and displays intermittent coupling between the helicase and polymerase(s). Challenging the textbook model of the polymerase holoenzyme acting as a stable complex coordinating the replisome, these observations suggest a role of the helicase as the central organizing hub. We show here that the molecular origin of this newly found plasticity lies in the 500-fold increase in strength of the interaction between the polymerase holoenzyme and the replicative helicase upon association of the primase with the replisome. By combining in vitro ensemble-averaged and single-molecule assays, we demonstrate that this conformational switch operates during replication and promotes recruitment of multiple holoenzymes at the fork. Our observations provide a molecular mechanism for polymerase exchange and offer a revised model for the replication reaction that emphasizes its stochasticity.

Design of DNA rolling-circle templates with controlled fork topology to study mechanisms of DNA replication.

Rolling-circle DNA amplification is a powerful tool employed in biotechnology to produce large from small amounts of DNA. This mode of DNA replication proceeds via a DNA topology that resembles a replication fork, thus also providing experimental access to the molecular mechanisms of DNA replication. However, conventional templates do not allow controlled access to multiple fork topologies, which is an important factor in mechanistic studies. Here we present the design and production of a rolling-circle substrate with a tunable length of both the gap and the overhang, and we show its application to the bacterial DNA-replication reaction.

Structure-activity relationships of pyrazole-4-carbodithioates as antibacterials against methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus (MRSA) is a major cause of serious hospital-acquired infections and is responsible for significant morbidity and mortality in residential care facilities. New agents against MRSA are needed to combat rising resistance to current antibiotics. We recently reported 5-hydroxy-3-methyl-1-phenyl-1H-pyrazole-4-carbodithioate (HMPC) as a new bacteriostatic agent against MRSA that appears to act via a novel mechanism. Here, twenty nine analogs of HMPC were synthesized, their anti-MRSA structure-activity relationships evaluated and selectivity versus human HKC-8 cells determined. Minimum inhibitory concentrations (MIC) ranged from 0.5 to 64 µg/mL and up to 16-fold selectivity was achieved. The 4-carbodithioate function was found to be essential for activity but non-specific reactivity was ruled out as a contributor to antibacterial action. The study supports further work aimed at elucidating the molecular targets of this interesting new class of anti-MRSA agents.

The more the merrier: high-throughput single-molecule techniques.

The single-molecule approach seeks to understand molecular mechanisms by observing biomolecular processes at the level of individual molecules. These methods have led to a developing understanding that for many processes, a diversity of behaviours will be observed, representing a multitude of pathways. This realisation necessitates that an adequate number of observations are recorded to fully characterise this diversity. The requirement for large numbers of observations to adequately sample distributions, subpopulations, and rare events presents a significant challenge for single-molecule techniques, which by their nature do not typically provide very high throughput. This review will discuss many developing techniques which address this issue by combining nanolithographic approaches, such as zero-mode waveguides and DNA curtains, with single-molecule fluorescence microscopy, and by drastically increasing throughput of force-based approaches such as magnetic tweezers and laminar-flow techniques. These methods not only allow the collection of large volumes of single-molecule data in single experiments, but have also made improvements to ease-of-use, accessibility, and automation of data analysis.

Tunability of the CO adsorption energy on a Ni/Cu surface: Site change and coverage effects.

The adsorption energy of carbon monoxide on Ni ad-islands and ultra-thin films grown on the Cu(110) surface can be finely tuned via a complex interplay among diffusion, site change mechanisms, and coverage effects. The observed features of CO desorption can be explained in terms of migration of CO molecules from Cu to Ni islands, competition between bridge and on-top adsorption sites, and repulsive lateral adsorbate-adsorbate interactions. While the CO adsorption energy on clean Cu(110) is of the order of 0.5 eV, Ni-alloying allows for its controlled, continuous tunability in the 0.98-1.15 eV range with Ni coverage. Since CO is a fundamental reactant and intermediate in many heterogeneous catalytic (electro)-conversion reactions, insight into these aspects with atomic level detail provides useful information to potentially drive applicative developments. The tunability range of the CO adsorption energy that we measure is compatible with the already observed tuning of conversion rates by Ni doping of Cu single crystal catalysts for methanol synthesis from a CO2, CO, and H2 stream under ambient pressure conditions.

Reverse Water-Gas Shift or Sabatier Methanation on Ni(110)? Stable Surface Species at Near-Ambient Pressure.

The interaction of CO, CO2, CO + H2, CO2 + H2, and CO + CO2 + H2 with the nickel (110) single crystal termination has been investigated at 10(-1) mbar in situ as a function of the surface temperature in the 300-525 K range by means of infrared-visible sum frequency generation (IR-vis SFG) vibrational spectroscopy and by near-ambient pressure X-ray photoelectron spectroscopy (NAP-XPS). Several stable surface species have been observed and identified. Besides atomic carbon and precursors for graphenic C phases, five nonequivalent CO species have been distinguished, evidencing the role of coadsorption effects with H and C atoms, of H-induced activation of CO, and of surface reconstruction. At low temperature, carbonate species produced by the interaction of CO2 with atomic oxygen, which stems from the dissociation of CO2 into CO + O, are found on the surface. A metastable activated CO2(-) species is also detected, being at the same time a precursor state toward dissociation into CO and O in the reverse water-gas shift mechanism and a reactive species that undergoes direct conversion in the Sabatier methanation process. Finally, the stability of ethylidyne is deduced on the basis of our spectroscopic observations.

Watching cellular machinery in action, one molecule at a time.

Single-molecule manipulation and imaging techniques have become important elements of the biologist's toolkit to gain mechanistic insights into cellular processes. By removing ensemble averaging, single-molecule methods provide unique access to the dynamic behavior of biomolecules. Recently, the use of these approaches has expanded to the study of complex multiprotein systems and has enabled detailed characterization of the behavior of individual molecules inside living cells. In this review, we provide an overview of the various force- and fluorescence-based single-molecule methods with applications both in vitro and in vivo, highlighting these advances by describing their applications in studies on cytoskeletal motors and DNA replication. We also discuss how single-molecule approaches have increased our understanding of the dynamic behavior of complex multiprotein systems. These methods have shown that the behavior of multicomponent protein complexes is highly stochastic and less linear and deterministic than previously thought. Further development of single-molecule tools will help to elucidate the molecular dynamics of these complex systems both inside the cell and in solutions with purified components.

Single-molecule visualization of fast polymerase turnover in the bacterial replisome.

The Escherichia coli DNA replication machinery has been used as a road map to uncover design rules that enable DNA duplication with high efficiency and fidelity. Although the enzymatic activities of the replicative DNA Pol III are well understood, its dynamics within the replisome are not. Here, we test the accepted view that the Pol III holoenzyme remains stably associated within the replisome. We use in vitro single-molecule assays with fluorescently labeled polymerases to demonstrate that the Pol III* complex (holoenzyme lacking the ß2 sliding clamp), is rapidly exchanged during processive DNA replication. Nevertheless, the replisome is highly resistant to dilution in the absence of Pol III* in solution. We further show similar exchange in live cells containing labeled clamp loader and polymerase. These observations suggest a concentration-dependent exchange mechanism providing a balance between stability and plasticity, facilitating replacement of replisomal components dependent on their availability in the environment.

Bisecting Microfluidic Channels with Metallic Nanowires Fabricated by Nanoskiving.

This paper describes the fabrication of millimeter-long gold nanowires that bisect the center of microfluidic channels. We fabricated the nanowires by nanoskiving and then suspended them over a trench in a glass structure. The channel was sealed by bonding it to a complementary poly(dimethylsiloxane) structure. The resulting structures place the nanowires in the region of highest flow, as opposed to the walls, where it approaches zero, and expose their entire surface area to fluid. We demonstrate active functionality, by constructing a hot-wire anemometer to measure flow through determining the change in resistance of the nanowire as a function of heat dissipation at low voltage (<5 V). Further, passive functionality is demonstrated by visualizing individual, fluorescently labeled DNA molecules attached to the wires. We measure rates of flow and show that, compared to surface-bound DNA strands, elongation saturates at lower rates of flow and background fluorescence from nonspecific binding is reduced.

Reactivity of Carbon Dioxide on Nickel: Role of CO in the Competing Interplay between Oxygen and Graphene.

The catalytic conversion of carbon dioxide to synthetic fuels and other valuable chemicals is an issue of global environmental and economic impact. In this report we show by means of X-ray photoelectron spectroscopy in the millibar range that, on a Ni surface, the reduction of carbon dioxide is indirectly governed by the CO chemistry. While the growth of graphene and the carbide-graphene conversion can be controlled by selecting the reaction temperature, oxygen is mainly removed by CO, since oxygen reduction by hydrogen is a slow process on Ni. Even though there is still a consistent pressure gap with respect to industrial reaction conditions, the observed phenomena provide a plausible interpretation of the behavior of Ni/Cu based catalysts for CO2 conversion and account for a possible role of CO in the methanol synthesis process.