ATPase cycle and DNA unwinding kinetics of RecG helicase

The superfamily 2 bacterial helicase, RecG, is a monomeric enzyme with a role in DNA repair by reversing stalled replication forks. The helicase must act specifically and rapidly to prevent replication fork collapse. We have shown that RecG binds tightly and rapidly to four-strand oligonucleotide junctions, which mimic a stalled replication fork. The helicase unwinds such DNA junctions with a step-size of approximately four bases per ATP hydrolyzed. To gain an insight into this mechanism, we used fluorescent stopped-flow and quenched-flow to measure individual steps within the ATPase cycle of RecG, when bound to a DNA junction. The fluorescent ATP analogue, mantATP, was used throughout to determine the rate limiting steps, effects due to DNA and the main states in the cycle. Measurements, when possible, were also performed with unlabeled ATP to confirm the mechanism. The data show that the chemical step of hydrolysis is the rate limiting step in the cycle and that this step is greatly accelerated by bound DNA. The ADP release rate is similar to the cleavage rate, so that bound ATP and ADP would be the main states during the ATP cycle. Evidence is provided that the main structural rearrangements, which bring about DNA unwinding, are linked to these states

The ATPase cycle of PcrA helicase and its coupling to translocation on DNA.

The superfamily 1 bacterial helicase PcrA has a role in the replication of certain plasmids, acting with the initiator protein (RepD) that binds to and nicks the double-stranded origin of replication. PcrA also translocates single-stranded DNA with discrete steps of one base per ATP hydrolyzed. Individual rate constants have been determined for the DNA helicase PcrA ATPase cycle when bound to either single-stranded DNA or a double-stranded DNA junction that also has RepD bound. The fluorescent ATP analogue 2'(3')-O-(N-methylanthraniloyl)ATP was used throughout all experiments to provide a complete ATPase cycle for a single nucleotide species. Fluorescence intensity and anisotropy stopped-flow measurements were used to determine rate constants for binding and release. Quenched-flow measurements provided the kinetics of the hydrolytic cleavage step. The fluorescent phosphate sensor MDCC-PBP was used to measure phosphate release kinetics. The chemical cleavage step is the rate-limiting step in the cycle and is essentially irreversible and would result in the bound ATP complex being a major component at steady state. This cleavage step is greatly accelerated by bound DNA, producing the high activation of this protein compared to the protein alone. The data suggest the possibility that ADP is released in two steps, which would result in bound ADP also being a major intermediate, with bound ADP.P(i) being a very small component. It therefore seems likely that the major transition in structure occurs during the cleavage step, rather than P(i) release. ATP rebinding could then cause reversal of this structural transition. The kinetic mechanism of the PcrA ATPase cycle is very little changed by potential binding to RepD, supporting the idea that RepD increases the processivity of PcrA by increasing affinity to DNA rather than affecting the enzymatic properties per se

Language and Reading Progress of Young Deaf and Hard-of-Hearing Children.

We examined the language and reading progress of 336 young DHH children in kindergarten, first and second grades. Trained assessors tested children's language, reading, and spoken and fingerspelled phonological awareness in the fall and spring of the school year. Children were divided into groups based on their auditory access and classroom communication: a spoken-only group (n = 101), a sign-only group (n = 131), and a bimodal group (n = 104). Overall, children showed delays in language and reading compared to norms established for hearing children. For language, vocabulary standard scores were higher than for English syntax. Although delayed in language, children made expected gains based on hearing norms from kindergarten to second grade. Reading scores declined from kindergarten to second grade. Spoken-only and bimodal children had similar word reading and reading comprehension abilities and higher scores than sign-only children. Spoken-only children had better spoken phonological awareness and nonword reading skills than the other two groups. The sign-only and bimodal groups made similar and significant gains in ASL syntax and fingerspelling phonological awareness

ATPase mechanism of the 5'-3' DNA helicase, RecD2: evidence for a pre-hydrolysis conformation change

The superfamily 1 helicase, RecD2, is a monomeric, bacterial enzyme with a role in DNA repair, but with 5'-3' activity unlike most enzymes from this superfamily. Rate constants were determined for steps within the ATPase cycle of RecD2 in the presence of ssDNA. The fluorescent ATP analog, mantATP (2'(3')-O-(N-methylanthraniloyl)ATP), was used throughout to provide a complete set of rate constants and determine the mechanism of the cycle for a single nucleotide species. Fluorescence stopped-flow measurements were used to determine rate constants for adenosine nucleotide binding and release, quenched-flow measurements were used for the hydrolytic cleavage step, and the fluorescent phosphate biosensor was used for phosphate release kinetics. Some rate constants could also be measured using the natural substrate, ATP, and these suggested a similar mechanism to that obtained with mantATP. The data show that a rearrangement linked to Mg(2+) coordination, which occurs before the hydrolysis step, is rate-limiting in the cycle and that this step is greatly accelerated by bound DNA. This is also shown here for the PcrA 3'-5' helicase and so may be a general mechanism governing superfamily 1 helicases. The mechanism accounts for the tight coupling between translocation and ATPase activity

HST/FOS Time-resolved spectral mapping of IP Pegasi at the end of an outburst

We report an eclipse mapping analysis of time-resolved ultraviolet spectroscopy covering three eclipses of the dwarf nova IP Pegasi on the late decline of the 1993 May outburst. The eclipse maps of the first run show evidence of one spiral arm, suggesting that spiral structures may still be present in the accretion disc 9 days after the onset of the outburst. In the spatially resolved spectra the most prominent lines appear in emission at any radius, being stronger in the inner disc regions. The spectrum of the gas stream is clearly distinct from the disc spectrum in the intermediate and outer disc regions, suggesting the occurrence of gas stream overflow. The full width half maximum of C IV is approximately constant with radius, in contrast to the expected $v\propto{R^{-1/2}}$ law for a gas in Keplerian orbits. This line probably originates in a vertically extended region (chromosphere + disc wind). The uneclipsed component contributes $\sim{4}$ % of the flux in C IV in the first run, and becomes negligible in the remaining runs. We fit stellar atmosphere models to the spatially resolved spectra. The radial run of the disc color temperature for the three runs is flatter than the expected $T\propto{R^{-3/4}}$ law for steady-state optically thick discs models, with $T\simeq{20000}$ K in the inner regions and $T\simeq{9000}$ K in the outer disc regions. The solid angles that result from the fits are smaller than expected from the parameters of the system. The radial run of the solid angle suggests that the disc is flared in outburst, and decreases in thickness toward the end of the outburst.Comment: 14 pages, 14 figures, in press in Astronomy & Astrophysic

Thermal analysis of stirling engine to power automotive alternator using heat from exhaust gases

AbstractThis paper investigates the development of small scale beta type Stirling engine to recover the exhaust heat from the main engine and to drive the alternator (decouple it from the main engine), thus providing the required electrical power for onboard devices. The ideal adiabatic model was used to predict the thermodynamic performance of the engine. CFD investigation was also carried out to optimise the heater and the cooler geometry of the Stirling engine. The results showed that it is possible to generate a power output of 1.5-2kWe at an ideal thermal efficiency of 40% and engine overall weight of 11-14kg

The AddAB helicase–nuclease catalyses rapid and processive DNA unwinding using a single Superfamily 1A motor domain

The oligomeric state of Superfamily I DNA helicases is the subject of considerable and ongoing debate. While models based on crystal structures imply that a single helicase core domain is sufficient for DNA unwinding activity, biochemical data from several related enzymes suggest that a higher order oligomeric species is required. In this work we characterize the helicase activity of the AddAB helicase–nuclease, which is involved in the repair of double-stranded DNA breaks in Bacillus subtilis. We show that the enzyme is functional as a heterodimer of the AddA and AddB subunits, that it is a rapid and processive DNA helicase, and that it catalyses DNA unwinding using one single-stranded DNA motor of 3′→5′ polarity located in the AddA subunit. The AddB subunit contains a second putative ATP-binding pocket, but this does not contribute to the observed helicase activity and may instead be involved in the recognition of recombination hotspot sequences

Discovery of seven T Tauri stars and a brown dwarf candidate in the nearby TW Hydrae Association

We report the discovery of five T Tauri star systems, two of which are resolved binaries, in the vicinity of the nearest known region of recent star formation, the TW Hydrae Association. The newly discovered systems display the same signatures of youth (namely high X-ray flux, large Li abundance and strong chromospheric activity) and the same proper motion as the original five members. These similarities firmly establish the group as a bona fide T Tauri association, unique in its proximity to Earth and its complete isolation from any known molecular clouds. At an age of ~10 Myr and a distance of ~50 pc, the association members are excellent candidates for future studies of circumstellar disk dissipation and the formation of brown dwarfs and planets. Indeed, as an example, our speckle imaging revealed a faint, very likely companion 2" north of CoD-33 7795 (TWA 5). Its color and brightness suggest a spectral type ~M8.5 which, at an age of ~10^7 years, implies a mass ~20 M(Jupiter).Comment: 6 pages, 4 figures and 1 table. AAS LaTeX aas2pp4.sty. To be published in Ap