A plasmid-based lacZα gene assay for DNA polymerase fidelity measurement

A significantly improved DNA polymerase fidelity assay, based on a gapped plasmid containing the lacZα reporter gene in a single-stranded region, is described. Nicking at two sites flanking lacZα, and removing the excised strand by thermocycling in the presence of complementary competitor DNA, is used to generate the gap. Simple methods are presented for preparing the single-stranded competitor. The gapped plasmid can be purified, in high amounts and in a very pure state, using benzoylated-naphthoylated DEAE-cellulose, resulting in a low background mutation frequency (~1 × 10(-4)). Two key parameters, the number of detectable sites and the expression frequency, necessary for measuring polymerase error rates have been determined. DNA polymerase fidelity is measured by gap filling in vitro, followed by transformation into Escherichia coli and scoring of blue/white colonies and converting the ratio to error rate. Several DNA polymerases have been used to fully validate this straightforward and highly sensitive system

PrimPol is required for replicative tolerance of G quadruplexes in vertebrate cells

G quadruplexes (G4s) can present potent blocks to DNA replication. Accurate and timely replication of G4s in vertebrates requires multiple specialized DNA helicases and polymerases to prevent genetic and epigenetic instability. Here we report that PrimPol, a recently described primase-polymerase (PrimPol), plays a crucial role in the bypass of leading strand G4 structures. While PrimPol is unable to directly replicate G4s, it can bind and reprime downstream of these structures. Disruption of either the catalytic activity or zinc-finger of PrimPol results in extreme G4-dependent epigenetic instability at the BU-1 locus in avian DT40 cells, indicative of extensive uncoupling of the replicative helicase and polymerase. Together, these observations implicate PrimPol in promoting restart of DNA synthesis downstream of, but closely coupled to, G4 replication impediments

Uncontrollable Modes in Double Wall Panels

Double-glazed windows have a poor transmission loss at low frequency. Since the passive means are more or less exhausted one could think of using an active controller to increase the transmission loss. In the work presented here two speakers in the cavity between the panes are used as actuators. A modal model is derived and validated with data from a laser scanner and measured transfer functions on the structure. From an analysis of this model it is shown that for certain configurations of the double wall panel some modes of the coupled system are uncontrollable and unobservable by speakers and microphones in the cavity, thus limiting the achievable controller performance. This theoretical result is verified by feedforward control experiments on two types of double-glazed windows. For the fully controllable window the transmission loss achieved by the active controller is about twice as large as for the window with uncontrollable modes

Human PrimPol mutation associated with high myopia has a DNA replication defect

PrimPol is a primase-polymerase found in humans, and other eukaryotes, involved in bypassing lesions encountered during DNA replication. PrimPol employs both translesion synthesis and repriming mechanisms to facilitate lesion bypass by the replisome. PrimPol has been reported to be a potential susceptibility gene associated with the development of myopia. Mutation of tyrosine 89 to aspartic acid (PrimPolY89D) has been identified in a number of cases of high myopia, implicating it in the aetiology of this disorder. Here, we examined whether this mutation resulted in any changes in the molecular and cellular activities associated with human PrimPol. We show that PrimPolY89D has a striking decrease in primase and polymerase activities. The hydrophobic ring of tyrosine is important for retaining wild-type extension activity. We also demonstrate that the decreased activity of PrimPolY89D is associated with reduced affinities for DNA and nucleotides, resulting in diminished catalytic efficiency. Although the structure and stability of PrimPolY89D is altered, its fidelity remains unchanged. This mutation also reduces cell viability after DNA damage and significantly slows replication fork rates in vivo. Together, these findings establish that the major DNA replication defect associated with this PrimPol mutant is likely to contribute to the onset of high myopia

Ammonia decomposition enhancement by Cs-Promoted Fe/Al2O3 catalysts

A range of Cs-doped Fe/Al2O3 catalysts were prepared for the ammonia decomposition reaction. Through time on-line studies it was shown that at all loadings of Cs investigated the activity of the Fe/Al2O3 catalysts was enhanced, with the optimum Cs:Fe being ca. 1. Initially, the rate of NH3 decomposition was low, typically < 10% equilibrium conversion (99.7%@500°C) recorded after 1 h. All catalysts exhibited an induction period (typically ca. 10 h) with the conversion reaching a high of 67% equilibrium conversion for Cs:Fe = 0.5 and 1. The highest rate of decomposition observed was attributed to the balance between increasing the concentration of Cs without blocking the active site. Analysis of H2-TPR and XPS measurements indicated that Cs acts as an electronic promoter. Previously, Cs has been shown to act as a promoter for Ru, where Cs alters the electron density of the active site, thereby facilitating the recombination of N2 which is considered the rate determining step. In addition, XRD and N2 adsorption measurements suggest that with higher Cs loadings deactivation of the catalytic activity is due to a layer of CsOH that forms on the surface and blocks active sites

Dual RNA-seq of pathogen and host

Abstract | A comprehensive understanding of host-pathogen interactions requires a knowledge of the associated gene expression changes in both the pathogen and the host. Traditional, probe-dependent approaches using microarrays or reverse transcription PCR typically require the pathogen and host cells to be physically separated before gene expression analysis. However, the development of the probe-independent RNA sequencing (RNA-seq) approach has begun to revolutionize transcriptomics. Here, we assess the feasibility of taking transcriptomics one step further by performing &apos;dual RNA-seq&apos;, in which gene expression changes in both the pathogen and the host are analysed simultaneously. R E V I E W S 618 | SEPTEMBER 2012 | VOLUME 10 www.nature.com/reviews/micro R E V I E W S © 2012 Macmillan Publishers Limited. All rights reserved Nature Reviews | Microbiology Compare with target genome (or genomes), and enumerate sequence

MLP: a MATLAB toolbox for rapid and reliable auditory threshold estimation

In this paper, we present MLP, a MATLAB toolbox enabling auditory thresholds estimation via the adaptive Maximum Likelihood procedure proposed by David Green (1990, 1993). This adaptive procedure is particularly appealing for those psychologists that need to estimate thresholds with a good degree of accuracy and in a short time. Together with a description of the toolbox, the current text provides an introduction to the threshold estimation theory and a theoretical explanation of the maximum likelihood adaptive procedure. MLP comes with a graphical interface and it is provided with several built-in, classic psychoacoustics experiments ready to use at a mouse click

Human PrimPol is a highly error-prone polymerase regulated by single-stranded DNA binding proteins

PrimPol is a recently identified polymerase involved in eukaryotic DNA damage tolerance, employed in both re-priming and translesion synthesis mechanisms to bypass nuclear and mitochondrial DNA lesions. In this report, we investigate how the enzymatic activities of human PrimPol are regulated. We show that, unlike other TLS polymerases, PrimPol is not stimulated by PCNA and does not interact with it in vivo. We identify that PrimPol interacts with both of the major single-strand binding proteins, RPA and mtSSB in vivo. Using NMR spectroscopy, we characterize the domains responsible for the PrimPol-RPA interaction, revealing that PrimPol binds directly to the N-terminal domain of RPA70. In contrast to the established role of SSBs in stimulating replicative polymerases, we find that SSBs significantly limit the primase and polymerase activities of PrimPol. To identify the requirement for this regulation, we employed two forward mutation assays to characterize PrimPol's replication fidelity. We find that PrimPol is a mutagenic polymerase, with a unique error specificity that is highly biased towards insertion-deletion errors. Given the error-prone disposition of PrimPol, we propose a mechanism whereby SSBs greatly restrict the contribution of this enzyme to DNA replication at stalled forks, thus reducing the mutagenic potential of PrimPol during genome replication

Mechanism of opiate mediated systemic and coronary vasoconstriction

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/24142/1/0000399.pd

Molecular dissection of the domain architecture and catalytic activities of human PrimPol

PrimPol is a primase–polymerase involved in nuclear and mitochondrial DNA replication in eukaryotic cells. Although PrimPol is predicted to possess an archaeo-eukaryotic primase and a UL52-like zinc finger domain, the role of these domains has not been established. Here, we report that the proposed zinc finger domain of human PrimPol binds zinc ions and is essential for maintaining primase activity. Although apparently dispensable for its polymerase activity, the zinc finger also regulates the processivity and fidelity of PrimPol's extension activities. When the zinc finger is disrupted, PrimPol becomes more promutagenic, has an altered translesion synthesis spectrum and is capable of faithfully bypassing cyclobutane pyrimidine dimer photolesions. PrimPol's polymerase domain binds to both single- and double-stranded DNA, whilst the zinc finger domain binds only to single-stranded DNA. We additionally report that although PrimPol's primase activity is required to restore wild-type replication fork rates in irradiated PrimPol−/− cells, polymerase activity is sufficient to maintain regular replisome progression in unperturbed cells. Together, these findings provide the first analysis of the molecular architecture of PrimPol, describing the activities associated with, and interplay between, its functional domains and defining the requirement for its primase and polymerase activities during nuclear DNA replication