Srs2 Disassembles Rad51 Filaments by a Protein-Protein Interaction Triggering ATP Turnover and Dissociation of Rad51 from DNA

Rad51 is a DNA recombinase functioning in the repair of DNA double-strand breaks and the generation of genetic diversity by homologous recombination (HR). In the presence of ATP, Rad51 self-assembles into an extended polymer on single-stranded DNA to catalyze strand exchange. Inappropriate HR causes genomic instability, and it is normally prevented by remodeling enzymes that antagonize the activities of Rad51 nucleoprotein filaments. In yeast, the Srs2 helicase/translocase suppresses HR by clearing Rad51 polymers from single-stranded DNA. We have examined the mechanism of disassembly of Rad51 nucleoprotein filaments by Srs2 and find that a physical interaction between Rad51 and the C-terminal region of Srs2 triggers ATP hydrolysis within the Rad51 filament, causing Rad51 to dissociate from DNA. This allosteric mechanism explains the biological specialization of Srs2 as a DNA motor protein that antagonizes HR

Single stranded DNA translocation of E. coli UvrD monomer is tightly coupled to ATP hydrolysis

E. coli UvrD is an SF1A helicase/translocase that functions in several DNA repair pathways. A UvrD monomer is a rapid and processive single-stranded (ss) DNA translocase, but is unable to unwind DNA processively in vitro. Based on data at saturating ATP (500 μM) we proposed a non-uniform stepping mechanism in which a UvrD monomer translocates with biased (3′ to 5′) directionality while hydrolyzing 1 ATP per DNA base translocated, but with a kinetic step-size of 4–5 nucleotides/step, suggesting a pause occurs every 4–5 nucleotides translocated. To further test this mechanism we examined UvrD translocation over a range of lower ATP concentrations (10–500 μM ATP), using transient kinetic approaches. We find a constant ATP coupling stoichiometry of ~1 ATP/DNA base translocated even at the lowest ATP concentration examined (10 μM) indicating that ATP hydrolysis is tightly coupled to forward translocation of a UvrD monomer along ssDNA with little slippage or futile ATP hydrolysis during translocation. The translocation kinetic step size remains constant at 4–5 nucleotides/step down to 50 μM ATP, but increases to ~7 nucleotides/step at 10 μM ATP. These results suggest that UvrD pauses more frequently during translocation at low ATP, but with little futile ATP hydrolysis

Ensemble Methods for Monitoring Enzyme Translocation along Single Stranded Nucleic Acids

We review transient kinetic methods developed to study the mechanism of translocation of nucleic acid motor proteins. One useful stopped-flow fluorescence method monitors arrival of the translocase at the end of a fluorescently labeled nucleic acid. When conducted under single-round conditions the time courses can be analyzed quantitatively using n-step sequential models to determine the kinetic parameters for translocation (rate, kinetic step size and processivity). The assay and analysis discussed here can be used to study enzyme translocation along a linear lattice such as ssDNA or ssRNA. We outline the methods for experimental design and two approaches, along with their limitations, that can be used to analyze the time courses. Analysis of the full time courses using n-step sequential models always yields an accurate estimate of the translocation rate. An alternative semi-quantitative “time to peak” analysis yields accurate estimates of translocation rates only if the enzyme initiates translocation from a unique site on the nucleic acid. However, if initiation occurs at random sites along the nucleic acid, then the “time to peak” analysis can yield inaccurate estimates of even the rates of translocation depending on the values of other kinetic parameters, especially the rate of dissociation of the translocase. Thus, in those cases analysis of the full time course is needed to obtain accurate estimates of translocation rates

Putting it all together: intrinsic and extrinsic mechanisms governing proteasome biogenesis

Background The 26S proteasome is at the heart of the ubiquitin-proteasome system, which is the key cellular pathway for the regulated degradation of proteins and enforcement of protein quality control. The 26S proteasome is an unusually large and complicated protease comprising a 28-subunit core particle (CP) capped by one or two 19-subunit regulatory particles (RP). Multiple activities within the RP process incoming ubiquitinated substrates for eventual degradation by the barrel-shaped CP. The large size and elaborate architecture of the proteasome have made it an exceptional model for understanding mechanistic themes in macromolecular assembly. Objective In the present work, we highlight the most recent mechanistic insights into proteasome assembly, with particular emphasis on intrinsic and extrinsic factors regulating proteasome biogenesis. We also describe new and exciting questions arising about how proteasome assembly is regulated and deregulated in normal and diseased cells. Methods A comprehensive literature search using the PubMed search engine was performed, and key findings yielding mechanistic insight into proteasome assembly were included in this review. Results Key recent studies have revealed that proteasome biogenesis is dependent upon intrinsic features of the subunits themselves as well as extrinsic factors, many of which function as dedicated chaperones. Conclusion Cells rely on a diverse set of mechanistic strategies to ensure the rapid, efficient, and faithful assembly of proteasomes from their cognate subunits. Importantly, physiological as well as pathological changes to proteasome assembly are emerging as exciting paradigms to alter protein degradation in vivo

The Influence of Modest Weight Gain on Taste and Smell Acuity in College Freshmen

Poster from the 2016 Food & Nutrition Conference & Expo. Poster Session: Food/Nutrition Science; Education; Management; Food Services/Culinary; Research

Vestibular afferent responses to linear accelerations in the alert squirrel monkey

The spontaneous activity of 40 otolith afferents and 44 canal afferents was recorded in 4 alert, intact squirrel monkeys. Polarization vectors and response properties of otolith afferents were determined during static re-orientations relative to gravity and during Earth-horizontal, sinusoidal, linear oscillations. Canal afferents were tested for sensitivity to linear accelerations. For regular otolith afferents, a significant correlation between upright discharge rate and sensitivity to dynamic acceleration in the horizontal plane was observed. This correlation was not present in irregular units. The sensitivity of otolith afferents to both static tilts and dynamic linear acceleration was much greater in irregularly discharging units than in regularly discharging units. The spontaneous activity and static and dynamic response properties of regularly discharging otolith afferents were similar to those reported in barbiturate-anesthetized squirrel monkeys. Irregular afferents also had similar dynamic response properties when compared to anesthetized monkeys. However, this sample of irregular afferents in alert animals had higher resting discharge rates and greater sensitivity to static tilts. The majority of otolith polarization vectors were oriented near the horizontal in the plane of the utricular maculae; however, directions of maximum sensitivity were different during dynamic and static testing. Canal afferents were not sensitive to static tilts or linear oscillations of the head

Translocation of Saccharomyces cerevisiae Pif1 helicase monomers on single-stranded DNA

In Saccharomyces cerevisiae Pif1 participates in a wide variety of DNA metabolic pathways both in the nucleus and in mitochondria. The ability of Pif1 to hydrolyse ATP and catalyse unwinding of duplex nucleic acid is proposed to be at the core of its func-tions. We recently showed that upon binding to DNA Pif1 dimerizes and we proposed that a dimer of Pif1 might be the species poised to catalysed DNA un-winding. In this work we show that monomers of Pif1 are able to translocate on single-stranded DNA with 50 to 30 directionality. We provide evidence that the translocation activity of Pif1 could be used in activities other than unwinding, possibly to displace proteins from ssDNA. Moreover, we show that monomers of Pif1 retain some unwinding activity although a dimer is clearly a better helicase, suggesting that regulation of the oligomeric state of Pif1 could play a role in its functioning as a helicase or a translocase. Finally, although we show that Pif1 can translocate on ssDNA, the translocation profiles suggest the presence on ssDNA of two populations of Pif1, both able to translocate with 50 to 30 directionality