End-bridging is required for pol μ to efficiently promote repair of noncomplementary ends by nonhomologous end joining

DNA polymerase μ is a member of the mammalian pol X family and reduces deletion during chromosome break repair by nonhomologous end joining (NHEJ). This biological role is linked to pol μ's ability to promote NHEJ of ends with noncomplementary 3′ overhangs, but questions remain regarding how it performs this role. We show here that synthesis by pol μ in this context is often rapid and, despite the absence of primer/template base-pairing, instructed by template. However, pol μ is both much less active and more prone to possible template independence in some contexts, including ends with overhangs longer than two nucleotides. Reduced activity on longer overhangs implies pol μ is less able to synthesize across longer gaps, arguing pol μ must bridge both sides of gaps between noncomplementary ends to be effective in NHEJ. Consistent with this argument, a pol μ mutant defective specifically on gapped substrates is also less active during NHEJ of noncomplementary ends both in vitro and in cells. Taken together, pol μ activity during NHEJ of noncomplementary ends can thus be primarily linked to pol μ's ability to work together with core NHEJ factors to bridge DNA ends and perform a template-dependent gap fill-in reaction

Expanding Cosmologies in Brane Geometries

Five dimensional gravity coupled, both in the bulk and on a brane, to a scalar Liouville field yields a geometry confined to a strip around the brane and with time dependent scale factors for the four geometry. In various limits known models can be recovered as well as a temporally expanding four geometry with a warp factor falling exponentially away from the brane. The effective theory on the brane has a time dependent Planck mass and ``cosmological constant''. Although the scale factor expands, the expansion is not an acceleration.Comment: 7 pages, LaTex/RevTex

The Genetic Drift Inventory: A Tool for Measuring What Advanced Undergraduates Have Mastered about Genetic Drift

Understanding genetic drift is crucial for a comprehensive understanding of biology, yet it is difficult to learn because it combines the conceptual challenges of both evolution and randomness. To help assess strategies for teaching genetic drift, we have developed and evaluated the Genetic Drift Inventory (GeDI), a concept inventory that measures upper-division students’ understanding of this concept. We used an iterative approach that included extensive interviews and field tests involving 1723 students across five different undergraduate campuses. The GeDI consists of 22 agree–disagree statements that assess four key concepts and six misconceptions. Student scores ranged from 4/22 to 22/22. Statements ranged in mean difficulty from 0.29 to 0.80 and in discrimination from 0.09 to 0.46. The internal consistency, as measured with Cronbach\u27s alpha, ranged from 0.58 to 0.88 across five iterations. Test–retest analysis resulted in a coefficient of stability of 0.82. The true–false format means that the GeDI can test how well students grasp key concepts central to understanding genetic drift, while simultaneously testing for the presence of misconceptions that indicate an incomplete understanding of genetic drift. The insights gained from this testing will, over time, allow us to improve instruction about this key component of evolution

The fidelity of DNA synthesis by yeast DNA polymerase zeta alone and with accessory proteins

DNA polymerase zeta (pol z) participates in several DNA transactions in eukaryotic cells that increase spontaneous and damage-induced mutagenesis. To better understand this central role in mutagenesis in vivo, here we report the fidelity of DNA synthesis in vitro by yeast pol z alone and with RFC, PCNA and RPA. Overall, the accessory proteins have little effect on the fidelity of pol z. Pol z is relatively accurate for single base insertion/deletion errors. However, the average base substitution fidelity of pol z is substantially lower than that of homologous B family pols a, d and «. Pol z is particularly error prone for substitutions in specific sequence con-texts and generates multiple single base errors clustered in short patches at a rate that is unprece-dented in comparison with other polymerases. The unique error specificity of pol z in vitro is consistent with Pol z-dependent mutagenic specificity reported in vivo. This fact, combined with the high rate of single base substitution errors and complex muta-tions observed here, indicates that pol z contributes to mutagenesis in vivo not only by extending mismatches made by other polymerases, but also by directly generating its own mismatches and then extending them

Does gender matter? A cross-national investigation of primary class-room discipline.

© 2018 Informa UK Limited, trading as Taylor & Francis GroupFewer than 15% of primary school teachers in both Germany and the UK are male. With the on-going international debate about educational performance highlighting the widening gender achievement gap between girl and boy pupils, the demand for more male teachers has become prevalent in educational discourse. Concerns have frequently been raised about the underachievement of boys, with claims that the lack of male ‘role models’ in schools has an adverse effect on boys’ academic motivation and engagement. Although previous research has examined ‘teaching’ as institutional talk, men’s linguistic behaviour in the classroom remains largely ignored, especially in regard to enacting discipline. Using empirical spoken data collected from four primary school classrooms in both the UK and in Germany, this paper examines the linguistic discipline strategies of eight male and eight female teachers using Interactional Sociolinguistics to address the question, does teacher gender matter?Peer reviewedFinal Accepted Versio

The Major Roles of DNA Polymerases Epsilon and Delta at the Eukaryotic Replication Fork Are Evolutionarily Conserved

Coordinated replication of eukaryotic genomes is intrinsically asymmetric, with continuous leading strand synthesis preceding discontinuous lagging strand synthesis. Here we provide two types of evidence indicating that, in fission yeast, these two biosynthetic tasks are performed by two different replicases. First, in Schizosaccharomyces pombe strains encoding a polδ-L591M mutator allele, base substitutions in reporter genes placed in opposite orientations relative to a well-characterized replication origin are strand-specific and distributed in patterns implying that Polδ is primarily involved in lagging strand replication. Second, in strains encoding a polε-M630F allele and lacking the ability to repair rNMPs in DNA due to a defect in RNase H2, rNMPs are selectively observed in nascent leading strand DNA. The latter observation demonstrates that abundant rNMP incorporation during replication can be tolerated and that they are normally removed in an RNase H2-dependent manner. This provides strong physical evidence that Polε is the primary leading strand replicase. Collectively, these data and earlier results in budding yeast indicate that the major roles of Polδ and Polε at the eukaryotic replication fork are evolutionarily conserved

A novel fluorescence-based assay for the rapid detection and quantification of cellular deoxyribonucleoside triphosphates

Current methods for measuring deoxyribonucleoside triphosphates (dNTPs) employ reagent and labor-intensive assays utilizing radioisotopes in DNA polymerase-based assays and/or chromatography-based approaches. We have developed a rapid and sensitive 96-well fluorescence-based assay to quantify cellular dNTPs utilizing a standard real-time PCR thermocycler. This assay relies on the principle that incorporation of a limiting dNTP is required for primer-extension and Taq polymerase-mediated 5–3′ exonuclease hydrolysis of a dual-quenched fluorophore-labeled probe resulting in fluorescence. The concentration of limiting dNTP is directly proportional to the fluorescence generated. The assay demonstrated excellent linearity (R2 > 0.99) and can be modified to detect between ∼0.5 and 100 pmol of dNTP. The limits of detection (LOD) and quantification (LOQ) for all dNTPs were defined as <0.77 and <1.3 pmol, respectively. The intra-assay and inter-assay variation coefficients were determined to be <4.6% and <10%, respectively with an accuracy of 100 ± 15% for all dNTPs. The assay quantified intracellular dNTPs with similar results obtained from a validated LC–MS/MS approach and successfully measured quantitative differences in dNTP pools in human cancer cells treated with inhibitors of thymidylate metabolism. This assay has important application in research that investigates the influence of pathological conditions or pharmacological agents on dNTP biosynthesis and regulation