Analysis of the Essential Functions of the C-terminal Protein/Protein Interaction Domain of Saccharomyces cerevisiae pol epsilon and Its Unexpected Ability to Support Growth in the Absence of the DNA Polymerase Domain

As first observed by Wittenberg (Kesti, T., Flick, K., Keranen, S., Syvaoja, J. E., and Wittenburg, C. (1999) Mol. Cell 3, 679-685), we find that deletion mutants lacking the entire N-terminal DNA polymerase domain of yeast pol epsilon are viable. However, we now show that point mutations in DNA polymerase catalytic residues of pol epsilon are lethal. Taken together, the phenotypes of the deletion and the point mutants suggest that the polymerase of pol epsilon may normally participate in DNA replication but that another polymerase can substitute in its complete absence. Substitution is inefficient because the deletion mutants have serious defects in DNA replication. This observation raises the question of what is the essential function of the C-terminal half of pol epsilon . We show that the ability of the C-terminal half of the polymerase to support growth is disrupted by mutations in the cysteine-rich region, which disrupts both dimerization of the POL2 gene product and interaction with the essential DPB2 subunit, suggesting that this region plays an important architectural role at the replication fork even in the absence of the polymerase function. Finally, the S phase checkpoint, with respect to both induction of RNR3 transcription and cell cycle arrest, is intact in cells where replication is supported only by the C-terminal half of pol epsilon , but it is disrupted in mutants affecting the cysteine-rich region, suggesting that this domain directly affects the checkpoint rather than acting through the N-terminal polymerase active site

Role of the Putative Zinc Finger Domain of Saccharomyces cerevisiae DNA Polymerase epsilon in DNA Replication and the S/M Checkpoint Pathway

It has been proposed that C-terminal motifs of the catalytic subunit of budding yeast polymerase (pol) epsilon (POL2) couple DNA replication to the S/M checkpoint (Navas, T. A., Zheng, Z., and Elledge, S. J. (1995) Cell 80, 29-39). Scanning deletion analysis of the C terminus reveals that 20 amino acid residues between two putative C-terminal zinc fingers are essential for DNA replication and for an intact S/M cell cycle checkpoint. All mutations affecting the inter-zinc finger amino acids or the zinc fingers themselves are sensitive to methylmethane sulfonate and have reduced ability to induce RNR3, showing that the mutants are defective in the transcriptional response to DNA damage as well as the cell cycle response. The mutations affect the assembly of the pol epsilon holoenzyme. Two-hybrid assays show that the POL2 subunit interacts with itself, and that the replication and checkpoint mutants are specifically defective in the interaction, suggesting (but not proving) that direct or indirect dimerization may be important for the normal functions of pol epsilon . The POL2 C terminus is sufficient for interaction with DPB2, the essential and phylogenetically conserved subunit of pol epsilon , but not for interaction with DPB3. Neither Dpb3p nor Dpb2p homodimerizes in the two-hybrid assay

Subunit interactions within the Saccharomyces cerevisiae DNA polymerase ε (pol ε) complex - Demonstration of a dimeric pol ε

Saccharomyces cerevisiae DNA polymerase epsilon (pol ε) is essential for chromosomal replication. A major form of pol ε purified from yeast consists of at least four subunits: Pol2p, Dpb2p, Dpb3p, and Dpb4p. We have investigated the protein/protein interactions between these polypeptides by using expression of individual subunits in baculovirus-infected Sf9 insect cells and by using the yeast two-hybrid assay. The essential subunits, Pol2p and Dpb2p, interact directly in the absence of the other two subunits, and the C-terminal half of POL2, the only essential portion of Pol2p, is sufficient for interaction with Dpb2p. Dpb3p and Dpb4p, non-essential subunits, also interact directly with each other in the absence of the other two subunits. We propose that Pol2pzDpb2p and Dpb3pzDpb4p complexes interact with each other and document several interactions between individual members of the two respective complexes. We present biochemical evidence to support the proposal that pol ε may be dimeric in vivo. Gel filtration of the Pol2pzDpb2p complexes reveals a novel heterotetrameric form, consisting of two heterodimers of Pol2pzDpb2p. Dpb2p, but not Pol2p, exists as a homodimer, and thus the Pol2p dimerization may be mediated by Dpb2p. The pol2-E and pol2-F mutations that cause replication defects in vivo weaken the interaction between Pol2p and Dpb2p and also reduce dimerization of Pol2p. This suggests, but does not prove, that dimerization may also occur in vivo and be essential for DNA replication

Lubiprostone: a novel treatment for chronic constipation

Chronic constipation is highly prevalent, reduces patients’ quality of life, and imposes a significant health care burden on society. Lifestyle modifications and over-the-counter agents improve symptoms of constipation in some patients, however many patients have persistent symptoms and require the use of prescription medications. Three prescription medications are currently Food and Drug Administration (FDA) approved and available for the treatment of chronic constipation in adults. This review will focus on lubiprostone, the newest medication available for the treatment of chronic constipation. Lubiprostone is a bicyclic fatty acid metabolite analogue of prostaglandin E1. It activates specific chloride channels in the gastrointestinal tract to stimulate intestinal fluid secretion, increase gastrointestinal transit, and improve symptoms of constipation. This article will provide a brief overview on chloride channel function in the gastrointestinal tract, describe the structure, function, and pharmacokinetics of lubiprostone, and discuss the safety and efficacy of this new medication

Computer memories: the history of computer form

This paper looks at the computer as a truly global form. The similar beige boxes found in offices across the world are analysed from the perspective of design history rather than that of the history of science and technology. Through the exploration of an archive of computer manufacturer's catalogues and concurrent design texts, this paper examines the changes that have occurred in the production and consumption of the computer in the context of the workplace, from its inception as a room-sized mainframe operated through a console of flashing lights, to the personal computer as a 'universal' form, reproduced by many manufacturers. It shows how the computer in the past has been as diverse as any other product, and asks how and why it now appears as a standardised, sanitised object. In doing so our relationship with the office computer, past and present is explored, revealing a complex history of vicissitude.</p

Exome sequencing followed by large-scale genotyping suggests a limited role for moderately rare risk factors of strong effect in schizophrenia.

Schizophrenia is a severe psychiatric disorder with strong heritability and marked heterogeneity in symptoms, course, and treatment response. There is strong interest in identifying genetic risk factors that can help to elucidate the pathophysiology and that might result in the development of improved treatments. Linkage and genome-wide association studies (GWASs) suggest that the genetic basis of schizophrenia is heterogeneous. However, it remains unclear whether the underlying genetic variants are mostly moderately rare and can be identified by the genotyping of variants observed in sequenced cases in large follow-up cohorts or whether they will typically be much rarer and therefore more effectively identified by gene-based methods that seek to combine candidate variants. Here, we consider 166 persons who have schizophrenia or schizoaffective disorder and who have had either their genomes or their exomes sequenced to high coverage. From these data, we selected 5,155 variants that were further evaluated in an independent cohort of 2,617 cases and 1,800 controls. No single variant showed a study-wide significant association in the initial or follow-up cohorts. However, we identified a number of case-specific variants, some of which might be real risk factors for schizophrenia, and these can be readily interrogated in other data sets. Our results indicate that schizophrenia risk is unlikely to be predominantly influenced by variants just outside the range detectable by GWASs. Rather, multiple rarer genetic variants must contribute substantially to the predisposition to schizophrenia, suggesting that both very large sample sizes and gene-based association tests will be required for securely identifying genetic risk factors. © 2012 The American Society of Human Genetics

In vivo reconstitution of Saccharomyces cerevisiae DNA polymerase epsilon in insect cells - Purification and characterization

DNA polymerase epsilon (pol epsilon) is a multiple subunit complex consisting of at least four proteins, including catalytic Po12p, Dpb2p, Dpb3p, and Dpb4p. Pol epsilon has been shown to play essential roles in chromosomal DNA replication. Here, we report reconstitution of the yeast pol epsilon complex, which was expressed and purified from baculovirus-infected insect cells. During the purification, we were able to resolve the pol epsilon complex and truncated Po12p (140 kDa), as was observed initially with the pol epsilon purified from yeast. Biochemical characterization of subunit stoichiometry, salt sensitivity, processivity, and stimulation by proliferating cell nuclear antigen indicates that the reconstituted pol epsilon is functionally identical to native pol epsilon purified from yeast and is therefore useful for biochemical characterization of the interactions of pol epsilon with other replication, recombination, and repair proteins. Identification and characterization of a proliferating cell nuclear antigen consensus interaction domain on Po12p indicates that the motif is dispensable for DNA replication but is important for methyl methanesulfonate damage-induced DNA repair. Analysis of the putative zinc finger domain of Po12p for zinc binding capacity demonstrates that it binds zinc. Mutations of the conserved cysteines in the putative zinc finger domain reduced zinc binding, indicating that cysteine ligands are directly involved in binding zinc

Magnetoresistance, Micromagnetism, and Domain Wall Scattering in Epitaxial hcp Co Films

Large negative magnetoresistance (MR) observed in transport measurements of hcp Co films with stripe domains were recently reported and interpreted in terms of a novel domain wall (DW) scattering mechanism. Here detailed MR measurements, magnetic force microscopy, and micromagnetic calculations are combined to elucidate the origin of MR in this material. The large negative room temperature MR reported previously is shown to be due to ferromagnetic resistivity anisotropy. Measurements of the resistivity for currents parallel (CIW) and perpendicular to DWs (CPW) have been conducted as a function of temperature. Low temperature results show that any intrinsic effect of DWs scattering on MR of this material is very small compared to the anisotropic MR.Comment: 5 pages, 5 Figures, submitted to PR

A microfluidic 2×2 optical switch

A 2×2 microfluidic-based optical switch is proposed and demonstrated. The switch is made of an optically clear silicon elastomer, Polydimethylsiloxane (PDMS), using soft lithography. It has insertion loss smaller than 1 dB and extinction ratio on the order of 20 dB. The device is switching between transmission (bypass) and reflection (exchange) modes within less than 20 ms. © 2004 American Institute of Physics