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

Realistic Rashba and Dressehaus spin-orbit coupling for neutral atoms

We describe a new class of atom-laser coupling schemes which lead to spin-orbit coupled Hamiltonians for ultra-cold neutral atoms. By properly setting the optical phases, a pair of degenerate pseudospin states emerge as the lowest energy states in the spectrum, and are thus immune to collisionally induced decay. These schemes use $N$ cyclically coupled ground or metastable internal states. We specialize to two situations: a three level case giving fixed Rashba coupling, and a four-level case that adds a controllable Dresselhaus contribution. We describe an implementation of the four level scheme for \Rb87 and analyze the sensitivity of our approach to realistic experimental limitations and imperfections. Lastly, we argue that no laser coupling scheme can give pure Rashba or Dresselhaus coupling: akin to condensed matter systems, higher order terms spoil the symmetry of these couplings. However, for sufficiently intense laser fields the continuous rotational symmetry approximately holds, making the Rashba Hamiltonian applicable for cold atoms.Comment: 5 pages, 4 figures, accepted as a PRA Brief Repor

Usability and feasibility of consumer-facing technology to reduce unsafe medication use by older adults

Background Mobile health technology can improve medication safety for older adults, for instance, by educating patients about the risks associated with anticholinergic medication use. Objective This study's objective was to test the usability and feasibility of Brain Buddy, a consumer-facing mobile health technology designed to inform and empower older adults to consider the risks and benefits of anticholinergics. Methods Twenty-three primary care patients aged ≥60 years and using anticholinergic medications participated in summative, task-based usability testing of Brain Buddy. Self-report usability was assessed by the System Usability Scale and performance-based usability data were collected for each task through observation. A subset of 17 participants contributed data on feasibility, assessed by self-reported attitudes (feeling informed) and behaviors (speaking to a physician), with confirmation following a physician visit. Results Overall usability was acceptable or better, with 100% of participants completing each Brain Buddy task and a mean System Usability Scale score of 78.8, corresponding to “Good” to “Excellent” usability. Observed usability issues included higher rates of errors, hesitations, and need for assistance on three tasks, particularly those requiring data entry. Among participants contributing to feasibility data, 100% felt better informed after using Brain Buddy and 94% planned to speak to their physician about their anticholinergic related risk. On follow-up, 82% reported having spoken to their physician, a rate independently confirmed by physicians. Conclusion Consumer-facing technology can be a low-cost, scalable intervention to improve older adults’ medication safety, by informing and empowering patients. User-centered design and evaluation with demographically heterogeneous clinical samples uncovers correctable usability issues and confirms the value of interventions targeting consumers as agents in shared decision making and behavior change

Modular tunable coupler for superconducting qubits

The development of modular and versatile quantum interconnect hardware is a key next step in the scaling of quantum information platforms to larger size and greater functionality. For superconducting quantum systems, fast and well-controlled tunable circuit couplers will be paramount for achieving high fidelity and resource efficient connectivity, whether for performing two-qubit gate operations, encoding or decoding a quantum data bus, or interfacing across modalities. Here we propose a versatile and internally-tunable double-transmon coupler (DTC) architecture that implements tunable coupling via flux-controlled interference in a three-junction dcSQUID. Crucially, the DTC possesses an internally defined zero-coupling state that is independent of the coupled data qubits or circuit resonators. This makes it particular attractive as a modular and versatile design element for realizing fast and robust linear coupling in several applications such as high-fidelity two-qubit gate operations, qubit readout, and quantum bus interfacing

Addressing Urban High-Poverty School Teacher Attrition by Addressing Urban High Poverty School Teacher Retention: Why Effective Teachers Perservere

Recruiting and retaining quality teachers specific for high-poverty schools in urban areas is a national concern, especially in light of the No Child Left Behind federal legislation. The educational realities, detrimental effects of poverty, and human despair that often depress low-income communities can prove to be quite overpowering for many teachers new to the profession and significantly contribute to high levels of teacher absenteeism, attrition rates, and teacher shortages. Examining this issue through a new lens, that being through the eyes of effective urban high-poverty school educators, has the potential to spark spirited conversations and debates among policy makers and educators alike so that significant polices and efforts can be developed and implemented. Therefore, the intent of this study was to develop a profile of high-quality educators who remain in urban high-poverty schools within a large metropolitan school district, and identify the indicators that influence them to remain. The results indicated that teachers who are African American, older, and more experienced define the profile of teachers most likely to remain beyond the first three years in this demanding setting. Additionally, these teachers reported that they remain because they believe they are well suited for teaching in high-poverty schools. Unless more attention is given to teacher retention, and why some educators are successful and persevere in even the most hard-to-staff schools, teacher attrition will continue to be a national concern