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Copy of R6K plasmid

papers based of R6K Replication model

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Transcript of Copy of R6K plasmid

Antecedentes Modelo de Replicación Plásmido R6K Sistema de chaperonas DnaK (DnaK,DnaJ,GrpE) ¿Como se monomeriza la proteina pi? (acción de la chaperona DnaK-DnaJ-GrpE ) El plásmido R6K necesita de la proteina pi (monómero) para poder iniciar la replicación en el origen gamma; y de monómeros y dímeros para realizar la replicación de origenes distantes (alfa y beta).
> [ ] de dímeros que monómeros en la célula.
Debe existir un mecanismo que mantanga una proporción dímero:monómero.
¿Que proteinas se necesitan para monomerizar los dímeros de pi? Chaperonas Familia de proteinas Clp Y (ClpA y ClpX) 1. El sistema DnaK convierte a los dímeros Pi Wt a monómeros The chaperone effect was not due to global refolding of a denatured form of WT π, rather it specifically converted the protein that bound to iterons as a monomer. 2. ¿Cual es la concentración optima necesaria de DnaK, DnaJ, and GrpE para la interaccion con los Iterones? The DnaK but not the individual proteins were needed for the activation of WT π. Despite the fact that DnaJ is known to be a chaperone in its own right in other systems, it did not activate π without the presence of DnaK and GrpE.
3. ¿ las proteinas ClpX and ClpB pueden activar a pi? Although there was apparent cooperation between ClpX and the DnaK system in the enhancement of DNA binding, further experiments showed that the enhancement was not manifested in the activation of DNA replication in vitro as presented in Table II.
ClpB by itself had insignificant ability to activate DNA binding of WT π. Once again, as presented in Table II, no physiologically significant cooperation was observed in terms of enhancement of in vitro replication catalyzed by WT π.
Systematic omission experiments showed that the enhancement in DNA binding of WT π to the iteron DNA was both chaperone and ATP-dependent. 4. Activacion de la proteina Pi por sistema DnaK (in vitro) The DnaK chaperone system, under these conditions,
promoted vigorous replication yielding up to -250 pmol
of reaction products
5. [ ] componentes proteicos (chaperonas) necesarios para regular la activación de monómeros de pi Although the addition of either DnaJ and DnaK or GrpE and DnaK resulted
in 30% replication in comparison with the complete system, the presence
of all three chaperone components, namely DnaK, DnaJ, and GrpE, was
needed for optimal replication.
6. ¿cual es el sitio de inicio de la replicación al cual se une proteinas pi WT activadas? Activated WT π was initiating replication at or
close to the in vivo location of ori γ and therefore
the replication was deemed to be origin-specific.
6. ¿ hay una interacción física entre pi WT y DnaJ? π interacts with DnaA, DnaB, and DnaG and also with DnaJ.
It should be kept in mind that the possibility of conjoint
interaction of WT with DnaJ and DnaK could not be ruled
out by the results.
La proteína π que codifica R6K, posee una especificidad aparente con proteínas chaperonas, al menos bajo las condiciones en las que se realizo los experimentos.

La chaperona que permite la activación de π no cambia la especificidad del origen de la proteína. Además, el sitio de unión de la proteína es detectado por la incorporación de ddNTP.

Existe una interacción física entre DnaJ y π. ¿Que papel cumple la proteina Pi en la replicación del plasmido R6K? Los dímeros π WT son inertes y son activados
por el sistema de chaperonas DnaK,
monómerizandolos al entrar en contacto con
el DNA.

Estructura ¿Como es la estrutura de pi?
(Cristalografia ) 1. ¿ Como es la estructura de la proteína Pi? The protein comprises two domains, the N-terminal domain (NTD) and the C-terminal domains (CTD), and these are topologically related. The NTD of π starts with β1 that forms a two-stranded sheet with its counterpart across the molecular dyad, b1’. After α1 and α 2, the chain leads to β2, the first β-strand of the three-stranded sheet, followed by α3 and then α 4, which is the major DNA-recognition element of the NTD. The NTD in finishes with the remaining two strands of the three-stranded β-sheet (β3 and 4), with the ‘‘wing’’ comprising the connection between these, followed by the helix α6.
2. ¿Cual es la estructura que toma el DNA en la union iteron-pi? Although most of the base pairs conform closely to Watson
and Crick geometry, some are separated and thus appear to
be sites where the DNA is being melted. Compared with the
iteron of DNA in complex with RepE, the DNA bound to is
markedly more bent ( 30° compared with 20° for RepE).
3. ¿Existe interacción entre pi y DnaB? The majority of the mutations that lower binding of to
DnaB map to the top of the molecule. Mutations at these
sites do not interfere with DNA binding, so the defect in
DNA replication of these mutants likely arises by lowered
binding to DnaB. Curiously, these sites map very closely
to the same region containing the high-copynumber
mutations that promotes formation of monomers and yet,
in these assays, DNA is not present, and so DnaB must be
binding to a dimer of π. La estructura del complejo π (monómero) con un iteron ha dilucidado hallazgos sobre el inicio de la replicación (ensamblaje de DnaB, curvamiento del DNA), y a su vez, revela sitios de interacción π-DnaB potenciales . El Plásmido R6K
(Generalidades) Sm: Streptomycin
Amp: Ampicillin Modificado de: http.//w.bioscience.org/1999/v4/d/actis/fulltext.htm Astrid Nausa-Diana Mayorga
Biología molecular de plásmidos
I-2011 ¿Como regula pi la replicación de Ori- ?
(Monómeros-Dímeros de pi ) En ensayos de unión in vitro a menudo descuidan la influencia de factores como la arquitectura de ADN, proteínas chaperonas, y otras proteínas generadas por el hospedero.

Una unión cooperativa de monómeros π en dos iterones puede dilucidar un mecanismo de regulación del número de copias del plásmido. En primer lugar, ofrece una explicación de cómo Ori γ está saturado de monómeros π cuando dímeros abundan. Por ejemplo, las interacciones cooperativas pueden prevenir o detener monómeros de disociarse de los iterones conformar nuevamente dímeros, aumentando la posibilidad de reclutar a más monómeros de π para iterones adyacentes. En un iteron simple, el reconocimiento de la DNA primasa (DnaG) es esencial para la replicacion de Ori-α. Estos resultados son consistentes contribuyen a la interpretación de que el complejo π-DnaA activa Ori-α a distancia (formación de looping) ¿ Que elementos interactuan en la replicación por Ori-Gamma? ¿Como actuan monómeros-dímeros de pi en la regulación de la replicación
(Handcuffing) π is primarily dimeric in solution and strong
evidence suggests that dimers inhibit replication,
while monomers bind the seven iterons of γ ori to
activate replication (π monomers contact a
larger segment of DNA than π dimers)

γ 1. ¿Ocurre interacciones cooperativas por parte de la proteína pi in vivo? Helical orientation of DNA is more important than distance for cooperative interactions.
2. Unión de pi ·wt, pi ·P106L^F107S and pi ·M36A^M38A a un fragmento de 2 iterones Only a minimal fraction π·wt of binds as two monomers to a two-iteron DNA fragment in vitro (chaperons action). In the absence of chaperones, an increase in π·wt concentration results in an increase in dimer binding.
In π variant (π·P106L^F107S), a steep binding curve was observed with the fraction of one monomer shifting to two monomers over a relatively small change in protein concentration, an indication of cooperative binding. 3. Cuantificación de monómeros pi unidos a dos iterones termodinámicamente idénticos Pi monomers bind to ori-gamma with positive cooperativity, and the distance and orientation between adjacent binding
sites influence these cooperative interactions. 4. Cuantificación de monómeros pi unidos a dos iterones termodinámicamente pi binds with greater cooperativity to the two-iteron* probe than the two-iteron*+10 probe and binds with little cooperativity to the two-iteron*+5 probe. the estimated cooperativity involved in pi binding to two heterogeneous iterons is greater than the estimated cooperativity involved in binding two wt iterons
Determine which of these
elements were necessary for Ori-α function
1. ¿Cual es la secuencia mínima necesaria de Ori-alfa para el mantenimiento del plásmido? the iteron, the G site (see below), and the sequence between the two were essential elements for replication. he βHR and the sequence following the G site were needed for optimal plasmid propagation in the absence of IHF. (starting immediately downstream of the 3’-end of the iteron and extending up to the G site). 2. ¿El “G site” es un componente esencial de Ori-alfa in vitro and in vivo data, taken together, support the conclusion that the primase-loading G site was an essential component of ori-γ
4. ¿hay un efecto neutralizante en Ori-alfa cuando se emplea regiones mutadas de Ori-? 3. Sitio de inicio de Ori-alfa (Gel 2D-plásmidos derivados) Both the complete γ-α and the mini γ-α replicons
generated bubble arcs in addition to more intense
Y arcs and some double Y arcs (initiation bubble
within approximately the same restriction fragment)
1. ¿PI aumenta los fragmentos ligados que contienen a Ori-Gamma? Handcuffing, mediated by the Rep protein π, occurs efficiently between two DNA fragments containing iterons. π dimers bind an individual iteron by using a single subunit, thus leaving the second subunit free to engage a second iteron

2. El numero de copias es π deficiente bajo concentraciones de DNA limite (Handcuffing) 3. Dímeros de pi (variantes) aumentan la ligación bajo concentraciones de DNA limite
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