The solution structure of the amino-terminal domain of human DNA polymerase ε subunit B is homologous to C-domains of AAA+ proteins

DNA polymerases α, δ and ε are large multisubunit complexes that replicate the bulk of the DNA in the eukaryotic cell. In addition to the homologous catalytic subunits, these enzymes possess structurally related B subunits, characterized by a carboxyterminal calcineurin-like and an aminoproximal oligonucleotide/oligosaccharide binding-fold domain. The B subunits also share homology with the exonuclease subunit of archaeal DNA polymerases D. Here, we describe a novel domain specific to the N-terminus of the B subunit of eukaryotic DNA polymerases ε. The N-terminal domain of human DNA polymerases ε (Dpoe2NT) expressed in Escherichia coli was characterized. Circular dichroism studies demonstrated that Dpoe2NT forms a stable, predominantly α-helical structure. The solution structure of Dpoe2NT revealed a domain that consists of a left-handed superhelical bundle. Four helices are arranged in two hairpins and the connecting loops contain short β-strand segments that form a short parallel sheet. DALI searches demonstrated a striking structural similarity of the Dpoe2NT with the α-helical subdomains of ATPase associated with various cellular activity (AAA+) proteins (the C-domain). Like C-domains, Dpoe2NT is rich in charged amino acids. The biased distribution of the charged residues is reflected by a polarization and a considerable dipole moment across the Dpoe2NT. Dpoe2NT represents the first C-domain fold not associated with an AAA+ protein

The high fidelity and unique error signature of human DNA polymerase ε

Bulk replicative DNA synthesis in eukaryotes is highly accurate and efficient, primarily because of two DNA polymerases (Pols): Pols δ and ε. The high fidelity of these enzymes is due to their intrinsic base selectivity and proofreading exonuclease activity which, when coupled with post-replication mismatch repair, helps to maintain human mutation rates at less than one mutation per genome duplication. Conditions that reduce polymerase fidelity result in increased mutagenesis and can lead to cancer in mice. Whereas yeast Pol ε has been well characterized, human Pol ε remains poorly understood. Here, we present the first report on the fidelity of human Pol ε. We find that human Pol ε carries out DNA synthesis with high fidelity, even in the absence of its 3′→5′ exonucleolytic proofreading and is significantly more accurate than yeast Pol ε. Though its spectrum of errors is similar to that of yeast Pol ε, there are several notable exceptions. These include a preference of the human enzyme for T→A over A→T transversions. As compared with other replicative DNA polymerases, human Pol ε is particularly accurate when copying homonucleotide runs of 4–5 bases. The base pair substitution specificity and high fidelity for frameshift errors observed for human Pol ε are distinct from the errors made by human Pol δ

Stand Out in Class: restructuring theclassroom environment to reducesedentary behaviour in 9–10-year-olds—study protocol for a pilot clusterrandomised controlled trial

Background: Sedentary behaviour (sitting) is a highly prevalent negative health behaviour, with individuals of allages exposed to environments that promote prolonged sitting. Excessive sedentary behaviour adversely affects health inchildren and adults. As sedentary behaviour tracks from childhood into adulthood, the reduction of sedentary time inyoung people is key for the prevention of chronic diseases that result from excessive sitting in later life. The sedentaryschool classroom represents an ideal setting for environmentalchange, through the provision of sit-stand desks. Whilstthe use of sit-stand desks in classrooms demonstrates positiveeffects in some key outcomes, evidence is currently limitedby small samples and/or short intervention durations, withfewstudiesadoptingrandomisedcontrolledtrial(RCT)designs. This paper describes the protocol of a pilot cluster RCT of a sit-stand desk interventioninprimaryschoolclassrooms.Methods/Design:A two-arm pilot cluster RCT will be conducted in eight primary schools (four intervention, four control)with at least 120 year 5 children (aged 9–10 years). Sit-stand desks will replace six standard desks in the interventionclassrooms. Teachers will be encouraged to ensure all pupils are exposed to the sit-stand desks for at least 1 h/dayon average using a rotation system. Schools assigned to the control arm will continue with their usual practice, noenvironmental changes will be made to their classrooms. Measurements will be taken at baseline, beforerandomisation, and at the end of the schools’academic year. In this study, the primary outcomes of interest will beschool and participant recruitment and attrition, acceptability of the intervention, and acceptability and complianceto the proposed outcome measures (including activPAL-measured school-time and school-day sitting, accelerometer-measured physical activity, adiposity, blood pressure, cognitive function, academic progress, engagement, andbehaviour) for inclusion in a definitive trial. A full process evaluation and an exploratory economic evaluation willalso be conducted to further inform a definitive tria

Defective interaction between Pol2p and Dpb2p, subunits of DNA polymerase epsilon, contributes to a mutator phenotype in Saccharomyces cerevisiae.

Most of the prokaryotic and eukaryotic replicative polymerases are multi-subunit complexes. There are several examples indicating that noncatalytic subunits of DNA polymerases may function as fidelity factors during replication process. In this work, we have further investigated the role of Dpb2p, a noncatalytic subunit of DNA polymerase epsilon holoenzyme from Saccharomyces cerevisiae in controlling the level of spontaneous mutagenesis. The data presented indicate that impaired interaction between catalytic Pol2p subunit and Dpb2p is responsible for the observed mutator phenotype in S. cerevisiae strains carrying different mutated alleles of the DPB2 gene. We observed a significant correlation between the decreased level of interaction between different mutated forms of Dpb2p towards a wild-type form of Pol2p and the strength of mutator phenotype that they confer. We propose that structural integrity of the Pol epsilon holoenzyme is essential for genetic stability in S. cerevisiae cells