Dynamic assembly of Hda and the sliding clamp in the regulation of replication licensing

Regulatory inactivation of DnaA (RIDA) is one of the major regulatory mechanisms of prokaryotic replication licensing. In RIDA, the Hda-sliding clamp complex loaded onto DNA directly interacts with adenosine triphosphate (ATP)-bound DnaA and stimulates the hydrolysis of ATP to inactivate DnaA. A prediction is that the activity of Hda is tightly controlled to ensure that replication initiation occurs only once per cell cycle. Here, we determined the crystal structure of the Hda-�� clamp complex. This complex contains two pairs of Hda dimers sandwiched between two �� clamp rings to form an octamer that is stabilized by three discrete interfaces. Two separate surfaces of Hda make contact with the �� clamp, which is essential for Hda function in RIDA. The third interface between Hda monomers occludes the active site arginine finger, blocking its access to DnaA. Taken together, our structural and mutational analyses of the Hda-�� clamp complex indicate that the interaction of the �� clamp with Hda controls the ability of Hda to interact with DnaA. In the octameric Hda-�� clamp complex, the inability of Hda to interact with DnaA is a novel mechanism that may regulate Hda function. ? The Author(s) 2017.113Ysciescopu

The CNC Workbook : An introduction to computer numerical control

viii.310 : 28 c

Pondera: comparador de alternativas de alojamiento

Hace una o dos décadas, planificar unas vacaciones era un asunto complejo. Para la mayoría de nosotros, suponía una visita a una agencia de viajes que nos presentaba un conjunto de opciones limitadas: un puñado de destinos, fechas de salida y regreso restringidas y pocas posibilidades de adaptar el viaje a nuestros propios gustos. Hoy en día, gracias a Internet, tenemos literalmente el mundo al alcance de la mano. Pero, aunque la tecnología ha democratizado el acto de buscar y reservar vacaciones, también ha creado lo que los psicólogos llaman la paradoja de la elección. Cuantas más opciones tenemos, más ansiosos e indecisos nos sentimos. Y eso es exactamente lo que se ve en los datos sobre viajes. Por ejemplo, según un análisis de los datos de clics entre dispositivos, utilizando más de 300.000 términos de búsqueda relacionados con los viajes: el tiempo medio de la reserva de un alojamiento dura la increíble cifra de 36 días e implica 45 puntos de contacto a través de varios dispositivos y tipos de sitios web. Cuando un viajero está abrumado de opciones, el instinto es investigar un poco más y, al tener a disposición tanta cantidad de información que no es capaz de digerir, termina fatigado y decidiendo irracionalmente. Este documento tiene por objetivo el análisis de la manera en la que un sistema podría dar soporte a la toma de decisión para la reserva de alojamiento, así como la implementación del propio sistema. Desarrollamos el documento de manera iterativa, para lograr que sea lo más representativo respecto a cómo fue el proceso de desarrollo del sistema. Es por eso que se parte de una investigación profunda de la problemática y una descripción de las soluciones que cubren esa problemática en el mercado actual. Con estos datos se define una propuesta de solución a alto nivel. Partiendo de la propuesta de solución, se itera sobre cada aspecto de ésta...Fil: Cobresi Serravalle, Ticiana María. Universidad Católica de Córdoba. Facultad de Ingeniería; ArgentinaFil: Nanfara, Nara Abril. Universidad Católica de Córdoba. Facultad de Ingeniería; Argentin

Identification of β Clamp-DNA Interaction Regions That Impair the Ability of <i>E</i>. <i>coli</i> to Tolerate Specific Classes of DNA Damage

<div><p>The <i>E</i>. <i>coli dnaN</i>-encoded β sliding clamp protein plays a pivotal role in managing the actions on DNA of the 5 bacterial DNA polymerases, proteins involved in mismatch repair, as well as several additional proteins involved in DNA replication. Results of <i>in vitro</i> experiments indicate that the loading of β clamp onto DNA relies on both the DnaX clamp loader complex as well as several discrete sliding clamp-DNA interactions. However, the importance of these DNA interactions to <i>E</i>. <i>coli</i> viability, as well as the ability of the β clamp to support the actions of its numerous partner proteins, have not yet been examined. To determine the contribution of β clamp-DNA interactions to the ability of <i>E</i>. <i>coli</i> to cope with different classes of DNA damage, we used alanine scanning to mutate 22 separate residues mapping to 3 distinct β clamp surfaces known or nearby those known to contact the DNA template, including residues P20-L27 (referred to here as loop I), H148-Y154 (loop II) and 7 different residues lining the central pore of the β clamp through which the DNA template threads. Twenty of these 22 <i>dnaN</i> mutants supported bacterial growth. While none of these 20 conferred sensitivity to hydrogen peroxide or ultra violet light, 12 were sensitized to NFZ, 5 were sensitized to MMS, 8 displayed modestly altered frequencies of DNA damage-induced mutagenesis, and 2 may be impaired for supporting <i>hda</i> function. Taken together, these results demonstrate that discrete β clamp-DNA interaction regions contribute to the ability of <i>E</i>. <i>coli</i> to tolerate specific classes of DNA damage.</p></div

Abilities of mutant <i>dnaN</i> alleles to support <i>E</i>. <i>coli</i> viability.

<p>Abilities of mutant <i>dnaN</i> alleles to support <i>E</i>. <i>coli</i> viability.</p

Ability of mutant <i>dnaN</i> alleles to support <i>E</i>. <i>coli</i> growth.

<p>Growth rates of <i>dnaN</i> strains bearing mutations in <b>(A)</b> loop I, <b>(B)</b> loop II or <b>(C)</b> the central pore of the β clamp were measured as described in <i>Materials and Methods</i>. Results represent an average of 4 determinations ± one standard deviation.</p

<i>oriC</i>/<i>terC</i> ratios in the different <i>dnaN</i> mutant strains.

<p>The <i>oriC</i>/<i>terC</i> ratio in the indicated mutant <i>dnaN</i> strains was measured as described in <i>Materials and Methods</i>. Results represent the average of 3 determinations ± one standard deviation. Symbols: *, <i>p</i> < 0.05; **, <i>p</i> < 0.001.</p

Epistasis of <i>dnaN-G22A</i> and <i>dnaN-R24A</i> with Δ<i>dinB</i>.

<p>Double mutants bearing Δ<i>dinB</i> and the indicated <i>dnaN</i> allele were examined for <b>(A)</b> MMS sensitivity and <b>(B)</b> MMS-induced mutagenesis (1.0 mM) as described in <i>Materials and Methods</i>. Results presented in panel B represent the average of 3 determinations from two independent clones for each strain ± one standard deviation. Symbols: *, <i>p</i> < 0.05.</p

Steady-state expression levels of mutant β clamp proteins.

<p>Steady-state expression levels of mutant β clamp proteins.</p