SNAP-8 materials report for January - June 1964. Volume I - Electrical insulation development

SNAP-8 electrical insulation developmen

Monomeric PcrA helicase processively unwinds plasmid lengths of DNA in the presence of the initiator protein RepD

The helicase PcrA unwinds DNA during asymmetric replication of plasmids, acting with an initiator protein, in our case RepD. Detailed kinetics of PcrA activity were measured using bulk solution and a single-molecule imaging technique to investigate the oligomeric state of the active helicase complex, its processivity and the mechanism of unwinding. By tethering either DNA or PcrA to a microscope coverslip surface, unwinding of both linear and natural circular plasmid DNA by PcrA/RepD was followed in real-time using total internal reflection fluorescence microscopy. Visualization was achieved using a fluorescent single-stranded DNA-binding protein. The single-molecule data show that PcrA, in combination with RepD, can unwind plasmid lengths of DNA in a single run, and that PcrA is active as a monomer. Although the average rate of unwinding was similar in single-molecule and bulk solution assays, the single-molecule experiments revealed a wide distribution of unwinding speeds by different molecules. The average rate of unwinding was several-fold slower than the PcrA translocation rate on single-stranded DNA, suggesting that DNA unwinding may proceed via a partially passive mechanism. However, the fastest dsDNA unwinding rates measured in the single-molecule unwinding assays approached the PcrA translocation speed measured on ssDNA

Monitoring the Health of Computer Networks with Visualization - VAST 2012 Mini Challenge 1 Award: "Efficient Use of Visualization"

The complex computer networks of large organisations contain many machines of many types, used in many geographic locations. Although system administrators should monitor the health of each machine, they need to do so within the context of the whole computer network. Our visualization presents the health of a fictitious financial institution's computer network at a snapshot in time and over a time range, and preserves the important aspects of each facility's administrative and geographic context. Using the "Bank of Money" VAST Challenge dataset, our visualization allowed us to correctly identify several areas of concern, as well as hypothesise about their causes

Opening of DNA double strands by helicases. Active versus passive opening

Helicase opening of double-stranded nucleic acids may be "active" (the helicase directly destabilizes the dsNA to promote opening) or "passive" (the helicase binds ssNA available due to a thermal fluctuation which opens part of the dsNA). We describe helicase opening of dsNA, based on helicases which bind single NA strands and move towards the double-stranded region, using a discrete ``hopping'' model. The interaction between the helicase and the junction where the double strand opens is characterized by an interaction potential. The form of the potential determines whether the opening is active or passive. We calculate the rate of passive opening for the helicase PcrA, and show that the rate increases when the opening is active. Finally, we examine how to choose the interaction potential to optimize the rate of strand separation. One important result is our finding that active opening can increase the unwinding rate by 7 fold compared to passive opening.Comment: 13 pages, 3 figure

The AddAB helicase–nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A motor domain

The oligomeric state of Superfamily I DNA helicases is the subject of considerable and ongoing debate. While models based on crystal structures imply that a single helicase core domain is sufficient for DNA unwinding activity, biochemical data from several related enzymes suggest that a higher order oligomeric species is required. In this work we characterize the helicase activity of the AddAB helicase–nuclease, which is involved in the repair of double-stranded DNA breaks in Bacillus subtilis. We show that the enzyme is functional as a heterodimer of the AddA and AddB subunits, that it is a rapid and processive DNA helicase, and that it catalyses DNA unwinding using one single-stranded DNA motor of 3′→5′ polarity located in the AddA subunit. The AddB subunit contains a second putative ATP-binding pocket, but this does not contribute to the observed helicase activity and may instead be involved in the recognition of recombination hotspot sequences

Atmospheric pressure plasma hydrophilic modification of a silicone surface

Presented in part at the 1st International Conference on Structural Adhesive Bonding (AB2011), Porto, Portugal, 7-8 July 2011.The aim of this study was the creation of a silicone hydrophilic surface prior to bonding. Modifications in wettability and adhesion properties of silicone were performed with an atmospheric plasma torch (APPT). Surface energy variations of the substrate, both pristine and APPT-treated, were evaluated through contact angle measurements, studying the hydrophobic recovery of the samples up to 24 hours of aging. Compositional and topographical changes induced by APPT and aging were studied by attenuated total multiple reflection mode infrared spectroscopy (ATR-FTIR), X-ray photoelectron spectroscopy (XPS), mechanical profilometry, scanning electron microscopy (SEM), and atomic force microscopy (AFM), respectively. Adhesion pull-off tests were performed on silicone-aluminium stud joints using three commercial adhesives, which were Sikaflex®-252, polyurethane-based, Loctite®-330, urethane methacrylate ester-based acrylic, and Terostat®-922, modified silicone. Although experimental data of all the bonding specimens led to an undesired adhesive failure, it was found that APPT-treated samples gave higher adhesive strength than the pristine ones, which was explained by the higher surface energy, thus more wettable material, after APPT. This effect remained stable for just 1 h, when the substrate began its hydrophobic recovery, reaching the original surface energy values after 24 h of aging.Financial support from the Universidad Carlos III de Madrid Foundation and Chemistry and Materials Technological Institute ‘‘Álvaro Alonso Barba’’ are acknowledged, as well as from the Universidad Pontificia Comillas (ICAI) (Spain)

X-Ray photoelectron spectroscopy and mass spectrometry studies of X-ray-processed solid CO2

Solid CO2 films have been grown on a stainless steel substrate and processed by X-ray bombardment for up to 6 hr.. The reactions induced were monitored using X-ray photoelectron spectroscopy (XPS) and mass spectrometry. The XPS results are twofold: direct X-ray photolysis of the CO2 ice produced CO and an unidentified O product, possibly atomic O; secondary effects resulting from surface reactions between CO, O, and residual H from the vacuum environment produced H2CO, CH3OH, and a water ice cap on the CO2 film. The rate of production of CO from direct X-ray photolysis of CO2 is measured to be 5.4 × 102 molecule photon-1, corresponding to a formation cross section of 4.7 × 10-20 cm2. The growth rate for the water cap is calculated to be 2.6 × 10-4 monolayers s-1 for a partial pressure of H equal to 2 × 10-10 Torr. The appearance of gas-phase products from the film showed a time lag which indicates that the diffusion of the product species in the bulk CO2 is affected by some time-dependent process, possibly the creation of defects in the film. A model for the observed time dependence of the dissociation products in the gas phase yields diffusion coefficients in the CO2 of 5 × 10-12 and 1 × 10-12 cm2 s-1, for O and CO, respectively