11,343 research outputs found

    ATPase cycle and DNA unwinding kinetics of RecG helicase

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    The superfamily 2 bacterial helicase, RecG, is a monomeric enzyme with a role in DNA repair by reversing stalled replication forks. The helicase must act specifically and rapidly to prevent replication fork collapse. We have shown that RecG binds tightly and rapidly to four-strand oligonucleotide junctions, which mimic a stalled replication fork. The helicase unwinds such DNA junctions with a step-size of approximately four bases per ATP hydrolyzed. To gain an insight into this mechanism, we used fluorescent stopped-flow and quenched-flow to measure individual steps within the ATPase cycle of RecG, when bound to a DNA junction. The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle. Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism. The data show that the chemical step of hydrolysis is the rate limiting step in the cycle and that this step is greatly accelerated by bound DNA. The ADP release rate is similar to the cleavage rate, so that bound ATP and ADP would be the main states during the ATP cycle. Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states

    Seeking Signs of Transparency: Audit, Materiality and Monuments to Active Citizenship in New Delhi

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    In New Delhi the Chief Information Commissioner has ordered that municipal councillors should “proactively disclose” details of the amounts spent on public works via Hindi language notice boards displayed in every city ward. As “appropriate” technology designed to reach out to the common people the noticeboards are part of an ongoing technomoral project to develop and democratise citizen engagement with urban governance. An audit of the noticeboards carried out by Information Commission officials and Right to Information activists reveals that many are badly positioned or assembled from inappropriate materials. As such they are judged to be unreliable actants in the project to prefigure a more open and transparent city administration. A focus on the materiality and temporal orientation of the noticeboards however reveals them to be productive in other ways. We come to understand the noticeboards as monuments to earlier projects of active citizenship and as ongoing sites of contest or collaboration between actors concerned with their audit and remediation

    Fallen World Ethics and Third World Politics

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    Phrasing Bimanual Interaction for Visual Design

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    Architects and other visual thinkers create external representations of their ideas to support early-stage design. They compose visual imagery with sketching to form abstract diagrams as representations. When working with digital media, they apply various visual operations to transform representations, often engaging in complex sequences. This research investigates how to build interactive capabilities to support designers in putting together, that is phrasing, sequences of operations using both hands. In particular, we examine how phrasing interactions with pen and multi-touch input can support modal switching among different visual operations that in many commercial design tools require using menus and tool palettes—techniques originally designed for the mouse, not pen and touch. We develop an interactive bimanual pen+touch diagramming environment and study its use in landscape architecture design studio education. We observe interesting forms of interaction that emerge, and how our bimanual interaction techniques support visual design processes. Based on the needs of architects, we develop LayerFish, a new bimanual technique for layering overlapping content. We conduct a controlled experiment to evaluate its efficacy. We explore the use of wearables to identify which user, and distinguish what hand, is touching to support phrasing together direct-touch interactions on large displays. From design and development of the environment and both field and controlled studies, we derive a set methods, based upon human bimanual specialization theory, for phrasing modal operations through bimanual interactions without menus or tool palettes

    The ATPase cycle of PcrA helicase and its coupling to translocation on DNA.

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    The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP.P(i) being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P(i) release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se

    ATPase mechanism of the 5'-3' DNA helicase, RecD2: evidence for a pre-hydrolysis conformation change

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    The superfamily 1 helicase, RecD2, is a monomeric, bacterial enzyme with a role in DNA repair, but with 5'-3' activity unlike most enzymes from this superfamily. Rate constants were determined for steps within the ATPase cycle of RecD2 in the presence of ssDNA. The fluorescent ATP analog, mantATP (2'(3')-O-(N-methylanthraniloyl)ATP), was used throughout to provide a complete set of rate constants and determine the mechanism of the cycle for a single nucleotide species. Fluorescence stopped-flow measurements were used to determine rate constants for adenosine nucleotide binding and release, quenched-flow measurements were used for the hydrolytic cleavage step, and the fluorescent phosphate biosensor was used for phosphate release kinetics. Some rate constants could also be measured using the natural substrate, ATP, and these suggested a similar mechanism to that obtained with mantATP. The data show that a rearrangement linked to Mg(2+) coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA. This is also shown here for the PcrA 3'-5' helicase and so may be a general mechanism governing superfamily 1 helicases. The mechanism accounts for the tight coupling between translocation and ATPase activity

    How do meters mediate? Energy meters, boundary objects and household transitions in Australia and the United Kingdom

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    This paper investigates the changing role of an integral but often overlooked technology within our energy systems: the meter. Empirical cases from the United Kingdom and Australia demonstrate the repurposing of the energy meter. No longer just an instrument of metrology, the meter is increasingly seen by utilities and governments as a key enabling technology for a raft of objectives, from tariff reform to peak load reduction. We draw on the Science and Technology Studies concept of a boundary object to explore these changes. A boundary object is conceptualised as positioned between different social worlds – such as those of householders, government, and utilities – and as having sufficient interpretive flexibility to mediate between their distinct interests. Here we use the boundary object concept to explain the ways in which the meter is being reconfigured, and in particular to analyse the role of householders in the transition to digital meters

    Population Characteristics within the Portland-Vancouver MSA

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    In this article, we will explore population characteristics in different parts of the Portland MSA using Census Public Use Microsample (PUMS) data from 2005-2007. For the spatial component, we will use a Census-defined small area geography called Public Use Microsample Area (PUMA), designed to follow existing county boundaries and contain around 100,000 people. (The Census reports the one-year and three-year ACS data only to the detail of this geography in order to maintain the privacy of survey respondents and to improve the precision of the estimates.) We will sometimes focus on a couple of example PUMAs to show the interesting differences within the region. (The information shown is available for all PUMAs in this case, but for the sake of space, we will Population Characteristics within the PortlandVancouver MSA Webb Sprague, Emily Picha, Sheila Martin Institute of Metropolitan Studies, PSU May 2010 just focus on a few.
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