Primase-polymerases are a functionally diverse superfamily of replication and repair enzymes

Until relatively recently, DNA primases were viewed simply as a class of proteins that synthesize short RNA primers requisite for the initiation of DNA replication. However, recent studies have shown that this perception of the limited activities associated with these diverse enzymes can no longer be justified. Numerous examples can now be cited demonstrating how the term 'DNA primase' only describes a very narrow subset of these nucleotidyltransferases, with the vast majority fulfilling multifunctional roles from DNA replication to damage tolerance and repair. This article focuses on the archaeo-eukaryotic primase (AEP) superfamily, drawing on recently characterized examples from all domains of life to highlight the functionally diverse pathways in which these enzymes are employed. The broad origins, functionalities and enzymatic capabilities of AEPs emphasizes their previous functional misannotation and supports the necessity for a reclassification of these enzymes under a category called primase-polymerases within the wider functional grouping of polymerases. Importantly, the repositioning of AEPs in this way better recognizes their broader roles in DNA metabolism and encourages the discovery of additional functions for these enzymes, aside from those highlighted here

A First Comparison of the responses of a He4-based fast-neutron detector and a NE-213 liquid-scintillator reference detector

A first comparison has been made between the pulse-shape discrimination characteristics of a novel $^{4}$He-based pressurized scintillation detector and a NE-213 liquid-scintillator reference detector using an Am/Be mixed-field neutron and gamma-ray source and a high-resolution scintillation-pulse digitizer. In particular, the capabilities of the two fast neutron detectors to discriminate between neutrons and gamma-rays were investigated. The NE-213 liquid-scintillator reference cell produced a wide range of scintillation-light yields in response to the gamma-ray field of the source. In stark contrast, due to the size and pressure of the $^{4}$He gas volume, the $^{4}$He-based detector registered a maximum scintillation-light yield of 750~keV$_{ee}$ to the same gamma-ray field. Pulse-shape discrimination for particles with scintillation-light yields of more than 750~keV$_{ee}$ was excellent in the case of the $^{4}$He-based detector. Above 750~keV$_{ee}$ its signal was unambiguously neutron, enabling particle identification based entirely upon the amount of scintillation light produced.Comment: 23 pages, 7 figures, Nuclear Instruments and Methods in Physics Research Section A review addresse

Overcoming High Energy Backgrounds at Pulsed Spallation Sources

Instrument backgrounds at neutron scattering facilities directly affect the quality and the efficiency of the scientific measurements that users perform. Part of the background at pulsed spallation neutron sources is caused by, and time-correlated with, the emission of high energy particles when the proton beam strikes the spallation target. This prompt pulse ultimately produces a signal, which can be highly problematic for a subset of instruments and measurements due to the time-correlated properties, and different to that from reactor sources. Measurements of this background have been made at both SNS (ORNL, Oak Ridge, TN, USA) and SINQ (PSI, Villigen, Switzerland). The background levels were generally found to be low compared to natural background. However, very low intensities of high-energy particles have been found to be detrimental to instrument performance in some conditions. Given that instrument performance is typically characterised by S/N, improvements in backgrounds can both improve instrument performance whilst at the same time delivering significant cost savings. A systematic holistic approach is suggested in this contribution to increase the effectiveness of this. Instrument performance should subsequently benefit.Comment: 12 pages, 8 figures. Proceedings of ICANS XXI (International Collaboration on Advanced Neutron Sources), Mito, Japan. 201

Tagging fast neutrons from an 241Am/9Be source

We report on an investigation of the fast-neutron spectrum emitted by 241Am/9Be. Well-understood shielding, coincidence, and time-of-flight measurement techniques are employed to produce a continuous, polychromatic, energy-tagged neutron beam.Comment: 17 pages, 7 figures, submitted to Journal of Applied Radiation and Isotope

Identical oligomeric and fibrillar structures captured from the brains of R6/2 and knock-in mouse models of Huntington's disease

Huntington's disease (HD) is a late-onset neurodegenerative disorder that is characterized neuropathologically by the presence of neuropil aggregates and nuclear inclusions. However, the profile of aggregate structures that are present in the brains of HD patients or of HD mouse models and the relative contribution of specific aggregate structures to disease pathogenesis is unknown. We have used the Seprion ligand to develop a highly sensitive enzyme-linked immunosorbent assay (ELISA)-based method for quantifying aggregated polyglutamine in tissues from HD mouse models. We used a combination of electron microscopy, atomic force microscopy (AFM) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) to investigate the aggregate structures isolated by the ligand. We found that the oligomeric, proto-fibrillar and fibrillar aggregates extracted from the brains of R6/2 and HdhQ150 knock-in mice were remarkably similar. Using AFM, we determined that the nanometre globular oligomers isolated from the brains of both mouse models have dimensions identical to those generated from recombinant huntingtin exon 1 proteins. Finally, antibodies that detect exon 1 Htt epitopes differentially recognize the ligand-captured material on SDS–PAGE gels. The Seprion-ligand ELISA provides an assay with good statistical power for use in preclinical pharmacodynamic therapeutic trials or to assess the effects of the genetic manipulation of potential therapeutic targets on aggregate load. This, together with the ability to identify a spectrum of aggregate species in HD mouse tissues, will contribute to our understanding of how these structures relate to the pathogenesis of HD and whether their formation can be manipulated for therapeutic benefit

Response of a Li-glass/multi-anode photomultiplier detector to collimated thermal-neutron beams

The response of a position-sensitive Li-glass scintillator detector being developed for thermal-neutron detection with 6 mm position resolution has been investigated using collimated beams of thermal neutrons. The detector was moved perpendicularly through the neutron beams in 0.5 to 1.0 mm horizontal and vertical steps. Scintillation was detected in an 8 X 8 pixel multi-anode photomultiplier tube on an event-by-event basis. In general, several pixels registered large signals at each neutron-beam location. The number of pixels registering signal above a set threshold was investigated, with the maximization of the single-hit efficiency over the largest possible area of the detector as the primary goal. At a threshold of ~50% of the mean of the full-deposition peak, ~80% of the events were registered in a single pixel, resulting in an effective position resolution of ~5 mm in X and Y. Lower thresholds generally resulted in events demonstrating higher pixel multiplicities, but these events could also be localized with ~5 mm position resolution.Comment: 23 pages, 8 figure

The Interplay between PolyQ and Protein Context Delays Aggregation by Forming a Reservoir of Protofibrils

Polyglutamine (polyQ) diseases are inherited neurodegenerative disorders caused by the expansion of CAG codon repeats, which code for polyQ in the corresponding gene products. These diseases are associated with the presence of amyloid-like protein aggregates, induced by polyQ expansion. It has been suggested that the soluble aggregates rather than the mature fibrillar aggregates are the toxic species, and that the aggregation properties of polyQ can be strongly modulated by the surrounding protein context. To assess the importance of the protein carrier in polyQ aggregation, we have studied the misfolding pathway and the kinetics of aggregation of polyQ of lengths above (Q41) and below (Q22) the pathological threshold fused to the well-characterized protein carrier glutathione S-transferase (GST). This protein, chosen as a model system, is per se able to misfold and aggregate irreversibly, thus mimicking the behaviour of domains of naturally occurring polyQ proteins. We prove that, while it is generally accepted that the aggregation kinetics of polyQ depend on its length and are faster for longer polyQ tracts, the presence of GST alters the polyQ aggregation pathway and reverses this trend. Aggregation occurs through formation of a reservoir of soluble intermediates whose populations and kinetic stabilities increase with polyQ length. Our results provide a new model that explains the toxicity of expanded polyQ proteins, in which the interplay between polyQ regions and other aggregation-prone domains plays a key role in determining the aggregation pathway