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

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

ATPase cycle and DNA unwinding kinetics of RecG helicase

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

Metatranscriptomes from diverse microbial communities: assessment of data reduction techniques for rigorous annotation

Background Metatranscriptome sequence data can contain highly redundant sequences from diverse populations of microbes and so data reduction techniques are often applied before taxonomic and functional annotation. For metagenomic data, it has been observed that the variable coverage and presence of closely related organisms can lead to fragmented assemblies containing chimeric contigs that may reduce the accuracy of downstream analyses and some advocate the use of alternate data reduction techniques. However, it is unclear how such data reduction techniques impact the annotation of metatranscriptome data and thus affect the interpretation of the results. Results To investigate the effect of such techniques on the annotation of metatranscriptome data we assess two commonly employed methods: clustering and de-novo assembly. To do this, we also developed an approach to simulate 454 and Illumina metatranscriptome data sets with varying degrees of taxonomic diversity. For the Illumina simulations, we found that a two-step approach of assembly followed by clustering of contigs and unassembled sequences produced the most accurate reflection of the real protein domain content of the sample. For the 454 simulations, the combined annotation of contigs and unassembled reads produced the most accurate protein domain annotations. Conclusions Based on these data we recommend that assembly be attempted, and that unassembled reads be included in the final annotation for metatranscriptome data, even from highly diverse environments as the resulting annotations should lead to a more accurate reflection of the transcriptional behaviour of the microbial population under investigation

Benchmarking pKa prediction

Background: pKa values are a measure of the protonation of ionizable groups in proteins. Ionizable groups are involved in intra-protein, protein-solvent and protein-ligand interactions as well as solubility, protein folding and catalytic activity. The pKa shift of a group from its intrinsic value is determined by the perturbation of the residue by the environment and can be calculated from three-dimensional structural data. Results: Here we use a large dataset of experimentally-determined pKas to analyse the performance of different prediction techniques. Our work provides a benchmark of available software implementations: MCCE, MEAD, PROPKA and UHBD. Combinatorial and regression analysis is also used in an attempt to find a consensus approach towards pKa prediction. The tendency of individual programs to over- or underpredict the pKa value is related to the underlying methodology of the individual programs. Conclusion: Overall, PROPKA is more accurate than the other three programs. Key to developing accurate predictive software will be a complete sampling of conformations accessible to protein structures

Computational analysis of eukaryotic metatranscriptomes from diverse marine environments

Phytoplankton are photosynthetic microbes that form the basis of the marine food web and are estimated to produce over half of all oxygen in the Earth's atmosphere. Recent advances in high-throughput DNA sequencing technologies have allowed scientists to sample the set of genes actively transcribed from communities of microbes in-situ. This set of transcripts (the metatranscriptome) provides a snapshot of actively transcribed genes at the time of sampling, and can provide insights into microbial metabolism and their relationship with their environment. In this thesis we present the computational analysis of eukaryotic phytoplankton metatranscriptome data sampled from representative marine environments; the simulation of metatranscriptome data for benchmarking computational tools; and analysis carried out on a newly sequenced eukaryotic phytoplankton genome. Transcripts a�liated with ribosomal proteins and associated with translation dominated in all but the Equatorial Paci�c metatranscriptome sample. Hierarchical clustering of the metatranscriptome samples by taxa produced two groups: the diatom dominated and the alveolate dominated. However, clustering by Gene Ontology terms clustered the samples by environment type (tropical, temperate and polar), producing a gradient of translation-associated transcripts which increased as the in-situ temperature of the samples decreased. A strong i correlation (R = 0:9) was detected between the relative proportion of transcripts associated with temperature and the in-situ temperature. Laboratory experiments on model diatom species under control conditions con�rmed that as the in-situ temperature decreases, these model diatoms produce more transcripts and consequently more ribosomal proteins. A translational e�ciency experiment demonstrated that the rate of translation decreased under low temperatures for a model diatom species. This suggested that the increased production of ribosomes acts as a compensatory mechanism under low temperatures. As more ribosomes require more phosphate-rich rRNAs we hypothesised that this could have an impact on biogeochemical cycles (E.g. the Red�eld ratio of Nitrate (N) to Phosphate (P)). This was modelled by our collaborators from the University of Exeter, who produced a global phytoplankton cell model of resource allocation. They showed how the N:P ratio di�ers across latitudinal temperature zones and predicted the impact of increasing temperature on global N:P

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

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

Truth, “Conspiracy Theorists”, and Theories: An Ethnographic Study of “Truth-Seeking” in Contemporary Britain

This thesis is an ethnographic study of a culture comprised of real-life “conspiracy theorists” living in contemporary Britain, based on fieldwork undertaken in 2014-2016. Within much popular and academic discourse, “conspiracy theorist” is a pejorative label that invokes a delusional person who subscribes to distortive, dangerous, and disempowering ideas; these assumptions are justified by viewing such ideas as unwarranted knowledge-claims. This thesis challenges these assumptions by turning instead to a cultural context in which such ideas are fully warranted, using a multi-sited method of participant observation and interviewing to provide a qualitative study of the so-called “Truth Movement”. While this “movement” is shown to lack formal status or structure, I argue that the (un-)likeminded affiliates of this uneasy collective are united by a shared orientation of “truth-seeking”. Across three separate sites, “truth-seekers” wrestle with common ideas, discovering empowering truths amidst a wider world they commonly perceive as conspired by a hidden, malign elite. Interviews reveal what this world looks like from the insider perspective, including the “waking up” narratives of conversion into this subjectively-plausible alternative outlook. In the chapter focussing on alternative health, I argue that “conspiracy theories”, and potential solutions, are embodied in everyday experiences and practices. I investigate the significance of “false-flag” theories about the 9/11 attacks for modern truth-seekers. The internal conflicts of the truth movement are explored in the more contentious fields of the “flat earth” theories, and “freeman” theories about the legal system, where I argue that these topics reveal the essential attraction of contemporary “conspiracy theory”: the recurring affirmation of the sacred character of humankind

PIP-DB:the protein isoelectric point database

A protein's isoelectric point or pI corresponds to the solution pH at which its net surface charge is zero. Since the early days of solution biochemistry, the pI has been recorded and reported, and thus literature reports of pI abound. The Protein Isoelectric Point database (PIP-DB) has collected and collated these data to provide an increasingly comprehensive database for comparison and benchmarking purposes. A web application has been developed to warehouse this database and provide public access to this unique resource. PIP-DB is a web-enabled SQL database with an HTML GUI front-end. PIP-DB is fully searchable across a range of properties

A survey of new PIs in the UK

The challenges facing a new independent group leader, principal investigator (PI) or university lecturer are formidable: secure funding, recruit staff and students, establish a research programme, give lectures, and carry out various administrative duties. Here we report the results of a survey of individuals appointed as new group leaders, PIs or university lecturers in the UK between 2012 and 2018. The concerns expressed include difficulties in recruiting PhD students, maintaining a good work-life balance and securing permanent positions. Gender differences were also found in relation to starting salary and success with research funding. We make recommendations to employers and funders to address some of these concerns, and offer advice to those applying for PI positions

Three Essays on the Economics of Fisheries Management

Problems arising from the common-pool nature of fisheries are well known (Gordon 1954). Recent research estimates the lost economic benefits from suboptimal fisheries production to be on the order of $50 billion annually (Arnason et al. 2009). Among the most promising management tools for improving fisheries performance are closed areas (marine reserves) and rights-based management (catch shares) (Worm et al. 2009). This collection of essays examines the emergence and performance of these two policy tools from an economics perspective.The first chapter examines the determinants of the transition from command and control regulation to rights-based management in common-pool resources. I develop a conceptual framework describing a regulator's decision to adopt a rights-based management regime in a common-pool resource and empirically test the hypotheses advanced in the framework with a duration analysis of rights-based management program adoption in a group of federally managed Alaska fisheries. Consistent with the conceptual framework, I find that rent dissipation along input cost and product value dimensions increases likelihood of program adoption, and high transaction costs decrease likelihood of program adoption. I find mixed evidence that resource depletion increases probability of program adoption.The second chapter examines the capacity of marine reserves to function as a cooperation mechanism in transboundary fisheries, stocks shared by two or more jurisdictions. Using a game theoretic model, we find that marine reserve implementation can achieve first-best equilibrium economic outcomes. We illustrate our theoretical predictions with a case study of the Chilean loco fishery. The third chapter estimates the economic and biological outcomes of the implementation of a marine reserve network at the California Channel Islands in 2003 on the Santa Barbara Area red sea urchin fishery. The paper develops an integrated bioeconomic model of the fishery consisting of an age structured population model and a discrete choice model of fishermen behavior linked via the fishing mortality rate. We calibrate the model using a 15-year panel dataset of fishery data and use the calibrated model to simulate outcomes under a counterfactual no-reserve scenario. We find that eight years after reserve implementation, urchin biomass is 53.7% higher, fishery yield is 11.3% lower, and fishery revenue is 10.9% lower compared to simulated outcomes under a counterfactual no-reserve policy