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Replication forks, chromatin loops and dormant replication origins
J Julian Blow, Xin Ge
Genome Biology , 2008, DOI: 10.1186/gb-2008-9-12-244
Abstract: It is critical that chromosomal DNA is precisely duplicated during S phase of the eukaryotic cell cycle, with no sections of DNA left unreplicated or replicated more than once. There is a considerable plasticity in this process because cells license many potential replication origins, of which only a small percentage are used in any one cell cycle, with the others remaining 'dormant'. This means that the usage of replication origins can change under different circumstances. For example, dormant replication origins can be activated when replication forks are inhibited to allow timely completion of the replication programme. A recent paper published in Nature by Courbet et al. [1] illustrates this plasticity of replication origin usage and shows that it is associated with longer-term changes to the organization of chromatin loops. The changes to chromatin organization can then directly affect the way that replication origins are used in subsequent cell cycles.The precise duplication of large eukaryotic chromosomes is a dauntingly complex task. For the DNA to be completely replicated, replication forks need to be initiated at thousands of replication origins scattered throughout the genome. This is made more difficult by the fact that replication forks can frequently stall, for example if they encounter damaged bases. It is also crucial that each replication origin does not fire more than once in a single S phase, as this would lead to local amplification of the DNA.During late mitosis and early G1, the cell licenses replication origins for use in the upcoming S phase by loading protein complexes composed of Mcm proteins (Mcm2-7 complexes) onto the origin DNA [2,3]. During S phase, Mcm2-7 at licensed origins can initiate replication forks. The Mcm2-7 complex moves with the replication forks, providing the essential DNA helicase activity that unwinds the DNA. This means that when an origin initiates a pair of forks, it is converted into the unlicensed state and cannot f
Reconstitution of licensed replication origins on Xenopus sperm nuclei using purified proteins
Peter J Gillespie, Anatoliy Li, J Julian Blow
BMC Biochemistry , 2001, DOI: 10.1186/1471-2091-2-15
Abstract: Here we show that a combination of purified nucleoplasmin, the origin recognition complex (ORC), Cdc6, RLF-B/Cdt1 and Mcm2-7 can promote functional origin licensing and the assembly of Mcm2-7 onto Xenopus sperm nuclei. The reconstituted reaction is inhibited by geminin, a specific RLF-B/Cdt1 inhibitor. Interestingly, the purified ORC used in the reconstitution had apparently lost the Orc6 subunit, suggesting that Orc6 is not essential for replication licensing. We use the reconstituted system to make a preliminary analysis of the different events occuring during origin assembly, and examine their nucleotide requirements. We show that the loading of Xenopus ORC onto chromatin is strongly stimulated by both ADP, ATP and ATP-γ-S whilst the loading of Cdc6 and Cdt1 is stimulated only by ATP or ATP-γ-S.Nucleoplasmin, ORC, Cdc6, RLF-B/Cdt1 and Mcm2-7 are the only proteins required for functional licensing and the loading of Mcm2-7 onto chromatin. The requirement for nucleoplasmin probably only reflects a requirement to decondense sperm chromatin before ORC can bind to it. Use of this reconstituted system should allow a full biochemical analysis of origin licensing and Mcm2-7 loading.During S phase of the eukaryotic cell division cycle the entire genome must be faithfully duplicated. The many thousands of replication forks involved in this process must be co-ordinated to ensure that, despite the very large quantities of DNA involved, no section of DNA is left unreplicated and no section of DNA is replicated more than once. Cells achieve this by dividing the process of replication origin activation into two distinct phases [1-3]. During late mitosis and early G1, proteins are assembled onto replication origins which culminates in the origin becoming 'licensed' for a single round of DNA replication by loading complexes of Mcm2-7 proteins [4-8]. In yeast, a 'pre-replicative complex' (pre-RC) forms a footprint over replication origins during G1 [9] which may well correspond to
Chromosome replication: from ORC to fork
Conrad A Nieduszynski, Anne D Donaldson, J Julian Blow
Genome Biology , 2001, DOI: 10.1186/gb-2001-2-12-reports4030
Abstract: The Cold Spring Harbor Laboratory holds a biennial meeting on eukaryotic DNA replication that provides a broad view of what is happening in this field. The scope of the latest meeting ranged from the mechanistics of replication fork proteins, through tying the cell cycle to replication, to viral replication strategies. Here we pick out some major themes of this exciting meeting, focusing mainly on work that has yet to be published.Previous work has revealed that chromosomal DNA replication is controlled by the sequential assembly of 'pre-replicative complex' (pre-RC) proteins onto specialized DNA sequences, the replication origins, early in the cell cycle. One crucial step in this process is the loading of the minichromosome maintenance (Mcm) proteins Mcm2-Mcm7 onto DNA, which results in the origin becoming 'licensed' for only a single round of DNA replication. Loading of the proteins of the origin recognition complex (ORC) onto replication origins is the earliest known step in pre-RC assembly. Detailed characterization of ORC, which consists of six subunits, Orc1-Orc6, was reported by a number of groups.Sanjay Vashee (Johns Hopkins University School of Medicine, Baltimore, USA) described the interactions between recombinant human ORC subunits, and proposed that Orc2, Orc3 and Orc4 form a core upon which Orc5 and then Orc1 can be assembled. The reported results suggested that only a minority of Orc6 molecules is associated with this Orc1-Orc5 complex. Consistent with this, Supriya Prasanth (Cold Spring Harbor Laboratory, New York, USA) reported distinct subcellular localization patterns for human Orc6 and Orc2 and Igor Chesnokov (University of California, Berkeley, USA) reported that less than 50% of Drosophila Orc6 associates with the other ORC subunits. In both studies, Orc6 was seen to localize to the mid-body, the cytoplasmic bridge between two daughter cells, by telophase. Drosophila Orc6 is nevertheless required for ORC to bind specifically to replication orig
A Xenopus Dbf4 homolog is required for Cdc7 chromatin binding and DNA replication
Pedro Jares, M Gloria Luciani, J Julian Blow
BMC Molecular Biology , 2004, DOI: 10.1186/1471-2199-5-5
Abstract: We have cloned a Xenopus homologue of Dbf4 (XDbf4), the sequence of which confirms the results of Furukhori et al. We have analysed the role of XDbf4 in DNA replication using cell-free extracts of Xenopus eggs. Our results indicate that XDbf4 is the regulatory subunit of XCdc7 required for DNA replication. We show that XDbf4 binds to chromatin during interphase, but unlike XCdc7, its chromatin association is independent of pre-RC formation, occurring in the absence of licensing, XCdc6 and XORC. Moreover, we show that the binding of XCdc7 to chromatin is dependent on the presence of XDbf4, whilst under certain circumstances XDbf4 can bind to chromatin in the absence of XCdc7. We provide evidence that the chromatin binding of XDbf4 that occurs in the absence of licensing depends on checkpoint activation.We have identified XDbf4 as a functional activator of XCdc7, and show that it is required to recruit XCdc7 to chromatin. Our results also suggest that XCdc7 and XDbf4 are differentially regulated, potentially responding to different cell cycle signals.The eukaryote genome is organised into multiple chromosomes, whose replication must be strictly controlled in order to ensure that the DNA is replicated once and only once in each cell cycle. DNA replication initiates from multiple origins, whose activation can be divided into two stages. In the first stage, pre-RCs are assembled at replication origins by the sequential binding of the origin recognition complex (ORC), Cdc6, Cdt1 and Mcm2-7 (the MCM/P1 proteins) [1,2]. This assembly takes places during late mitosis and G1, and results in the origin becoming "licensed" for DNA replication. The second stage occurs during S phase and involves the activation of licensed origins by the action of two S phase-promoting kinases: S-phase promoting CDKs and Cdc7/Dbf4, leading to the loading of Cdc45 and the initiation of a pair of replication forks.Cdc7 is a serine/threonine kinase, conserved from yeast to humans that is required fo
Optimal Placement of Origins for DNA Replication
Jens Karschau,J. Julian Blow,Alessandro P. S. de Moura
Quantitative Biology , 2012, DOI: 10.1103/PhysRevLett.108.058101
Abstract: DNA replication is an essential process in biology and its timing must be robust so that cells can divide properly. Random fluctuations in the formation of replication starting points, called origins, and the subsequent activation of proteins lead to variations in the replication time. We analyse these stochastic properties of DNA and derive the positions of origins corresponding to the minimum replication time. We show that under some conditions the minimization of replication time leads to the grouping of origins, and relate this to experimental data in a number of species showing origin grouping.
Optimisation of the two-dimensional gel electrophoresis protocol using the Taguchi approach
Guennadi A Khoudoli, Iain M Porter, J Julian Blow, Jason R Swedlow
Proteome Science , 2004, DOI: 10.1186/1477-5956-2-6
Abstract: Our optimisation resulted in increased protein solubility, improved resolution and reproducibility of 2D gels, using a wide variety of samples. With the updated protocol we routinely detected approximately 4-fold more polypeptides on samples containing complex protein mixtures resolved on small format 2D gels. In addition the pI and size ranges over which proteins could be resolved was substantially improved. Moreover, with improved sample loading and resolution, analysis of individual spots by immunoblotting and mass spectrometry revealed previously uncharacterised posttranscriptional modifications in a variety of chromatin proteins.While the optimised RB (oRB) is specific to the gels and analysis approach we use, our use of the Taguchi method should be generally applicable to a broad range of electrophoresis and analysis systems.During cell cycle progression different functional protein complexes associate with and dissociate from chromosomal DNA [1,2]. We have taken a proteomic strategy to identify and then characterize proteins that are bound to chromatin at defined stages of the cell cycle in cell free extracts derived from Xenopus eggs. The combination of 2D gel electrophoresis (2DE) and Mass Spectroscopy (MS) are powerful tools for this analysis.2DE is capable of resolving thousands of proteins in a single separation procedure [3]. Development of immobilised pH gradients (IPG) coupled with pre-cast gradient polyacrylamide gels and introduction of new sensitive fluorescent stains have considerably simplified and greatly improved the capacity, sensitivity and reproducibility of 2D gels. These recent technological advances do not however eliminate a number of difficulties associated with the separation of proteins by 2DE. One major problem is the solubilisation of protein mixtures during isoelectric focusing (IEF), (reviewed in [4]). In addition, reduction and alkylation of protein samples for 2DE has not yet been fully optimised [5,6]. As a consequence, convent
Superhydrophobicity on hairy surfaces
M. L. Blow,J. M. Yeomans
Physics , 2010, DOI: 10.1021/la101847b
Abstract: We investigate the wetting properties of surfaces patterned with fine elastic hairs, with an emphasis on identifying superhydrophobic states on hydrophilic hairs. We formulate a two dimensional model of a large drop in contact with a row of equispaced elastic hairs and, by minimising the free energy of the model, identify the stable and metastable states. In particular we concentrate on "partially suspended" states, where the hairs bend to support the drop -- singlet states where all hairs bend in the same direction, and doublet states where neighbouring hairs bend in opposite directions -- and find the limits of stability of these configurations in terms of material contact angle, hair flexibility, and system geometry. The drop can remain suspended in a singlet state at hydrophilic contact angles, but doublets exist only when the hairs are hydrophobic. The system is more likely to evolve into a singlet state if the hairs are inclined at the root. We discuss how, under limited circumstances, the results can be modified to describe an array of hairs in three dimensions. We find that now both singlets and doublets can exhibit superhydrophobic behaviour on hydrophilic hairs. We discuss the limitations of our approach and the directions for future work.
In-Motes EYE: A Real Time Application for Automobiles in Wireless Sensor Networks  [PDF]
Dimitrios Georgoulas, Keith Blow
Wireless Sensor Network (WSN) , 2011, DOI: 10.4236/wsn.2011.35018
Abstract: Wireless sensor networks have been identified as one of the key technologies for the 21st century. In order to overcome their limitations such as fault tolerance and conservation of energy, we propose a middleware solution, In-Motes. In-Motes stands as a fault tolerant platform for deploying and monitoring applications in real time offers a number of possibilities for the end user giving him in parallel the freedom to experiment with various parameters, in an effort the deployed applications to run in an energy efficient manner inside the network. The proposed scheme is evaluated through the In-Motes EYE application, aiming to test its merits under real time conditions. In-Motes EYE application which is an agent based real time In-Motes application developed for sensing acceleration variations in an environment. The application was tested in a prototype area, road alike, for a period of four months.
Wireless Sensor Network Management and Functionality: An Overview  [PDF]
Dimitrios GEORGOULAS, Keith BLOW
Wireless Sensor Network (WSN) , 2009, DOI: 10.4236/wsn.2009.14032
Abstract: Sensor networks are dense wireless networks of small, low-cost sensors, which collect and disseminate en-vironmental data. Wireless sensor networks facilitate monitoring and controlling of physical environments from remote locations with better accuracy. They have applications in a variety of fields such as environ-mental monitoring; military purposes and gathering sensing information in inhospitable locations. Sensor nodes have various energy and computational constraints because of their inexpensive nature and adhoc method of deployment. Considerable research has been focused at overcoming these deficiencies through more energy efficient routing, localization algorithms and system design. Our survey presents the funda-mentals of wireless sensor network, thus providing the necessary background required for understanding the organization, functionality and limitations of those networks. The middleware solution is also investigated through a critical presentation and analysis of some of the most well established approaches.
The collapse transition on superhydrophobic surfaces
H. Kusumaatmaja,M. L. Blow,A. Dupuis,J. M. Yeomans
Physics , 2008, DOI: 10.1209/0295-5075/81/36003
Abstract: We investigate the transition between the Cassie-Baxter and Wenzel states of a slowly evaporating, micron-scale drop on a superhydrophobic surface. In two dimensions analytical results show that there are two collapse mechanisms. For long posts the drop collapses when it is able to overcome the free energy barrier presented by the hydrophobic posts. For short posts, as the drop loses volume, its curvature increases allowing it to touch the surface below the posts. We emphasise the importance of the contact line retreating across the surface as the drop becomes smaller: this often preempts the collapse. In a quasi-three dimensional simulation we find similar behaviour, with the additional feature that the drop can de-pin from all but the peripheral posts, so that its base resembles an inverted bowl.
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