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John F. X. Diffley - One of the best experts on this subject based on the ideXlab platform.
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cryo em structure of a licensed DNA Replication Origin
Nature Communications, 2017Co-Authors: Max E Douglas, Julia Locke, Andrea Nans, John F. X. Diffley, Alessandro CostaAbstract:Eukaryotic Origins of Replication are licensed upon loading of the MCM helicase motor onto DNA. ATP hydrolysis by MCM is required for loading and the post-catalytic MCM is an inactive double hexamer that encircles duplex DNA. Origin firing depends on MCM engagement of Cdc45 and GINS to form the CMG holo-helicase. CMG assembly requires several steps including MCM phosphorylation by DDK. To understand Origin activation, here we have determined the cryo-EM structures of DNA-bound MCM, either unmodified or phosphorylated, and visualize a phospho-dependent MCM element likely important for Cdc45 recruitment. MCM pore loops touch both the Watson and Crick strands, constraining duplex DNA in a bent configuration. By comparing our new MCM–DNA structure with the structure of CMG–DNA, we suggest how the conformational transition from the loaded, post-catalytic MCM to CMG might promote DNA untwisting and melting at the onset of Replication. Origins of Replication are licensed by loading of MCM onto DNA, and Origin firing depends on interaction with Cdc45 and GINS to form two CMG holo-helicases. Here, authors determine the cryo-EM structures of DNA-bound MCM and visualise a phospho-dependent MCM element important for Cdc45 recruitment.
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cryo em structure of a licensed DNA Replication Origin
Nature Communications, 2017Co-Authors: Max E Douglas, Julia Locke, Andrea Nans, John F. X. Diffley, Alessandro CostaAbstract:Eukaryotic Origins of Replication are licensed upon loading of the MCM helicase motor onto DNA. ATP hydrolysis by MCM is required for loading and the post-catalytic MCM is an inactive double hexamer that encircles duplex DNA. Origin firing depends on MCM engagement of Cdc45 and GINS to form the CMG holo-helicase. CMG assembly requires several steps including MCM phosphorylation by DDK. To understand Origin activation, here we have determined the cryo-EM structures of DNA-bound MCM, either unmodified or phosphorylated, and visualize a phospho-dependent MCM element likely important for Cdc45 recruitment. MCM pore loops touch both the Watson and Crick strands, constraining duplex DNA in a bent configuration. By comparing our new MCM–DNA structure with the structure of CMG–DNA, we suggest how the conformational transition from the loaded, post-catalytic MCM to CMG might promote DNA untwisting and melting at the onset of Replication.
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chromatin controls DNA Replication Origin selection lagging strand synthesis and Replication fork rates
Molecular Cell, 2017Co-Authors: Christoph F Kurat, Joseph T P Yeeles, Harshil Patel, Anne Early, John F. X. DiffleyAbstract:The integrity of eukaryotic genomes requires rapid and regulated chromatin Replication. How this is accomplished is still poorly understood. Using purified yeast Replication proteins and fully chromatinized templates, we have reconstituted this process in vitro. We show that chromatin enforces DNA Replication Origin specificity by preventing non-specific MCM helicase loading. Helicase activation occurs efficiently in the context of chromatin, but subsequent replisome progression requires the histone chaperone FACT (facilitates chromatin transcription). The FACT-associated Nhp6 protein, the nucleosome remodelers INO80 or ISW1A, and the lysine acetyltransferases Gcn5 and Esa1 each contribute separately to maximum DNA synthesis rates. Chromatin promotes the regular priming of lagging-strand DNA synthesis by facilitating DNA polymerase α function at Replication forks. Finally, nucleosomes disrupted during Replication are efficiently re-assembled into regular arrays on nascent DNA. Our work defines the minimum requirements for chromatin Replication in vitro and shows how multiple chromatin factors might modulate Replication fork rates in vivo.
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regulated eukaryotic DNA Replication Origin firing with purified proteins
Nature, 2015Co-Authors: Joseph T P Yeeles, Anne Early, Tom D Deegan, Agnieszka Janska, John F. X. DiffleyAbstract:Eukaryotic cells initiate DNA Replication from multiple Origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at Replication Origins during G1 phase, and then converted to the active CMG (Cdc45–MCM–GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA Replication initiation with 16 purified Replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the Origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes Replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA Replication. It has long been a goal to reconstitute eukaryotic DNA Replication; here a purified in vitro system from budding yeast containing 16 factors, themselves composed of 42 polypeptides, fulfils the staged process of Origin-dependent initiation, including its regulation by kinases. It has been a long-desired goal to be able to reconstitute a eukaryotic system of DNA Replication from its earliest stages of Origin firing using purified proteins. However, the greater complexity of eukaryotes compared to bacterial and phage systems has hampered this development. But now John Diffley and colleagues have successfully reconstituted the initial events of budding yeast DNA Replication in vitro. The purified system contains 42 proteins, comprising 16 complexes, and fulfills the staged process of Origin-dependent initiation, including its regulation by kinases.
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regulated eukaryotic DNA Replication Origin firing with purified proteins
Nature, 2015Co-Authors: Joseph T P Yeeles, Anne Early, Tom D Deegan, Agnieszka Janska, John F. X. DiffleyAbstract:Eukaryotic cells initiate DNA Replication from multiple Origins, which must be tightly regulated to promote precise genome duplication in every cell cycle. To accomplish this, initiation is partitioned into two temporally discrete steps: a double hexameric minichromosome maintenance (MCM) complex is first loaded at Replication Origins during G1 phase, and then converted to the active CMG (Cdc45-MCM-GINS) helicase during S phase. Here we describe the reconstitution of budding yeast DNA Replication initiation with 16 purified Replication factors, made from 42 polypeptides. Origin-dependent initiation recapitulates regulation seen in vivo. Cyclin-dependent kinase (CDK) inhibits MCM loading by phosphorylating the Origin recognition complex (ORC) and promotes CMG formation by phosphorylating Sld2 and Sld3. Dbf4-dependent kinase (DDK) promotes Replication by phosphorylating MCM, and can act either before or after CDK. These experiments define the minimum complement of proteins, protein kinase substrates and co-factors required for regulated eukaryotic DNA Replication.
Marcel Méchali - One of the best experts on this subject based on the ideXlab platform.
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DNA Replication Origins, Development, and Cancer
Hormonal Control of Cell Cycle, 2020Co-Authors: Marcel MéchaliAbstract:DNA Replication is at the heart of living organisms. Themain purpose of any organism, even the simplest, is the duplication of the genetic information and its transmission to the offspring. In simple organisms, such as the bacteria Echerichia coli, a single DNA Replication Origin is used, fromwhich Replication forksmigrate in opposite directions to allowthe duplication of the entire genome. In complex eukaryotes, such as multicellular organisms,multiple DNAReplication Origins are used to replicate chromosomes,which arehighly regulatedduring Sphase.Here, Iwill review how the positions of these Origins are regulated and how DNA Replication Origins might be regulatory elements that also control development and cell identity. I will also consider how DNA Replication Origins are regulated within each cell cycle in order to avoid re-Replication, an event thatmight lead to genetic damage and neoplasic evolution.
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11 DNA Replication during Animal Development and Its Relevance to Gene Expression
Cold Spring Harbor Monograph Archive, 2020Co-Authors: Damien Grégoire, Marcel MéchaliAbstract:Animal development has to coordinate cell proliferation and differentiation in order to produce, from a single egg cell, an organism containing billions of cells and tens of different cell types. For this to happen, the DNA Replication program must be adapted to different cell fates and to specific transcriptional programs. Intrinsic properties of Replication can be modulated, such as fork speed, temporal order of Replication, and Origin usage, and the changes can be linked to developmental programs in diverse ways. The changes in Replication timing that occur during different stages of development or in conjunction with differentiation have been deeply investigated and are reviewed in Chapter 10. Here, we review the changes in DNA Replication that occur during animal development, with a special focus on the regulation of Origin usage in relation to development and transcription. Indeed, the replicon hypothesis was proposed in 1962 as a way to explain how DNA Replication might be linked to cell growth (Jacob et al. 1964). In multicellular organisms, DNA Replication must be linked not only to cell growth but also to cell differentiation. Accordingly, the evolution of metazoans might have involved a transition from the strict use of sequence-specific Replication Origins to a recognition of DNA Replication Origin more adapted to the adoption of different cell fates in the different tissues of an organism (Mechali 2001). FIRST ROUND OF Replication The first Replication event during animal development occurs a few hours after fertilization, as a prerequisite for the first division. This event has...
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Metazoan DNA Replication Origins.
Current Opinion in Cell Biology, 2019Co-Authors: Olivier Ganier, Paulina Prorok, Ildem Akerman, Marcel MéchaliAbstract:DNA Replication starts with the opening of DNA at sites called DNA Replication Origins. From the single sequence-specific DNA Replication Origin of the small Escherichia coli genome, up to thousands of Origins that are necessary to replicate the large human genome, strict sequence specificity has been lost. Nevertheless, genome-wide analyses performed in the recent years, using different mapping methods, demonstrated that there are precise locations along the metazoan genome from which Replication initiates. These sites contain relaxed sequence consensus and epigenetic features. There is flexibility in the choice of Origins to be used during a given cell cycle, probably imposed by evolution and developmental constraints. Here, we will briefly describe their main features.
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The ORC ubiquitin ligase OBI1 promotes DNA Replication Origin firing
Nature Communications, 2019Co-Authors: Philippe Coulombe, Joelle Nassar, Isabelle Peiffer, Axel Delamarre, Stéphane Bocquet, Slavica Stanojcic, Yvon Sterkers, Marcel MéchaliAbstract:DNA Replication is initiated at defined genomic sites called Origins of Replication following ORC pre-replicative complex assembly. Here the authors identify a protein ubiquitylating ORC that is involved in Origin activation and may act as a selector of Origins to be fired.AbstractDNA Replication initiation is a two-step process. During the G1-phase of the cell cycle, the ORC complex, CDC6, CDT1, and MCM2–7 assemble at Replication Origins, forming pre-replicative complexes (pre-RCs). In S-phase, kinase activities allow fork establishment through (CDC45/MCM2–7/GINS) CMG-complex formation. However, only a subset of all potential Origins becomes activated, through a poorly understood selection mechanism. Here we analyse the pre-RC proteomic interactome in human cells and find C13ORF7/RNF219 (hereafter called OBI1, for ORC-ubiquitin-ligase-1) associated with the ORC complex. OBI1 silencing result in defective Origin firing, as shown by reduced CMG formation, without affecting pre-RC establishment. OBI1 catalyses the multi-mono-ubiquitylation of a subset of chromatin-bound ORC3 and ORC5 during S-phase. Importantly, expression of non-ubiquitylable ORC3/5 mutants impairs Origin firing, demonstrating their relevance as OBI1 substrates for Origin firing. Our results identify a ubiquitin signalling pathway involved in Origin activation and provide a candidate protein for selecting the Origins to be fired.
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the chromatin environment shapes DNA Replication Origin organization and defines Origin classes
Genome Research, 2015Co-Authors: Christelle Cayrou, Isabelle Peiffer, Benoit Ballester, Romain Fenouil, Jean-christophe Andrau, Jacques Van Helden, Philippe Coulombe, Marcel MéchaliAbstract:To unveil the still-elusive nature of metazoan Replication Origins, we identified them genome-wide and at unprecedented high-resolution in mouse ES cells. This allowed initiation sites (IS) and initiation zones (IZ) to be differentiated. We then characterized their genetic signatures and organization and integrated these data with 43 chromatin marks and factors. Our results reveal that Replication Origins can be grouped into three main classes with distinct organization, chromatin environment, and sequence motifs. Class 1 contains relatively isolated, low-efficiency Origins that are poor in epigenetic marks and are enriched in an asymmetric AC repeat at the initiation site. Late Origins are mainly found in this class. Class 2 Origins are particularly rich in enhancer elements. Class 3 Origins are the most efficient and are associated with open chromatin and polycomb protein-enriched regions. The presence of Origin G-rich Repeated elements (OGRE) potentially forming G-quadruplexes (G4) was confirmed at most Origins. These coincide with nucleosome-depleted regions located upstream of the initiation sites, which are associated with a labile nucleosome containing H3K64ac. These data demonstrate that specific chromatin landscapes and combinations of specific signatures regulate Origin localization. They explain the frequently observed links between DNA Replication and transcription. They also emphasize the plasticity of metazoan Replication Origins and suggest that in multicellular eukaryotes, the combination of distinct genetic features and chromatin configurations act in synergy to define and adapt the Origin profile.
Bruce Stillman - One of the best experts on this subject based on the ideXlab platform.
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evolution of DNA Replication Origin specification and gene silencing mechanisms
bioRxiv, 2020Co-Authors: Y Hu, Christian Speck, Ammar Tareen, Yj Sheu, William T Ireland, Huilin Li, Leemor Joshuator, Justin B Kinney, Bruce StillmanAbstract:DNA Replication in eukaryotic cells initiates from chromosomal locations, called Replication Origins, that bind the Origin Recognition Complex (ORC) prior to S phase. Origin establishment is guided by well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific Origins. At present, the mechanistic and evolutionary reasons for this difference are unclear. A 3.9 A structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to Origin DNA revealed, among other things, that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove1. We show that this Orc4 α-helix mediates the sequence-specificity of Origins in S. cerevisiae. Specifically, mutations were identified within this &alpha-helix that alter the sequence-dependent activity of individual Origins as well as change global genomic Origin firing patterns. This was accomplished using a massively parallel Origin selection assay analyzed using a custom mutual-information-based modeling approach and a separate analysis of whole-genome Replication profiling and statistics. Interestingly, the sequence specificity of DNA Replication initiation, as mediated by the Orc4 α-helix, has evolved in close conjunction with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.
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the architecture of the DNA Replication Origin recognition complex in saccharomyces cerevisiae
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Zhiqiang Chen, Christian Speck, Patricia Wendel, Chunyan Tang, Bruce Stillman, H LiAbstract:The Origin recognition complex (ORC) is conserved in all eukaryotes. The six proteins of the Saccharomyces cerevisiae ORC that form a stable complex bind to Origins of DNA Replication and recruit prereplicative complex (pre-RC) proteins, one of which is Cdc6. To further understand the function of ORC we recently determined by single-particle reconstruction of electron micrographs a low-resolution, 3D structure of S. cerevisiae ORC and the ORC–Cdc6 complex. In this article, the spatial arrangement of the ORC subunits within the ORC structure is described. In one approach, a maltose binding protein (MBP) was systematically fused to the N or the C termini of the five largest ORC subunits, one subunit at a time, generating 10 MBP-fused ORCs, and the MBP density was localized in the averaged, 2D EM images of the MBP-fused ORC particles. Determining the Orc1–5 structure and comparing it with the native ORC structure localized the Orc6 subunit near Orc2 and Orc3. Finally, subunit–subunit interactions were determined by immunoprecipitation of ORC subunits synthesized in vitro. Based on the derived ORC architecture and existing structures of archaeal Orc1–DNA structures, we propose a model for ORC and suggest how ORC interacts with Origin DNA and Cdc6. The studies provide a basis for understanding the overall structure of the pre-RC.
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sequential initiation of lagging and leading strand synthesis by two different polymerase complexes at the sv40 DNA Replication Origin
Nature, 1990Co-Authors: Toshiki Tsurimoto, Thomas Melendy, Bruce StillmanAbstract:Enzymatic synthesis of DNA from the simian virus 40 Origin of DNA Replication has been reconstituted in vitro with eight purified components. DNA polymerase α-primase complex first initiates DNA synthesis at the Replication Origin and continues as the lagging strand polymerase. Subsequently, the DNA polymerase δ complex initiates Replication on the leading strand template. Some prokaryotic DNA polymerase complexes can replace the eukaryotic polymerase δ complex. A model for polymerase switching during initiation of DNA Replication is presented.
Jeremy E Purvis - One of the best experts on this subject based on the ideXlab platform.
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rapid DNA Replication Origin licensing protects stem cell pluripotency
eLife, 2017Co-Authors: Jacob Peter Matson, Raluca Dumitru, Philip Coryell, Ryan M Baxley, Kirk Twaroski, Beau R Webber, Jakub Tolar, Anjakatrin Bielinsky, Weili Chen, Jeremy E PurvisAbstract:From red blood cells to nerve cells, animals’ bodies contain many different types of specialized cells. These all begin as stem cells, which have the potential to divide and make more stem cells or to specialize. All dividing cells must first unwind their DNA so that it can be copied. To achieve this, cells load DNA-unwinding enzymes called helicases onto their DNA during the part of the cell cycle known as G1 phase. Cells must load enough helicase enzymes to ensure that their DNA is copied completely and in time. Stem cells divide faster than their specialized descendants, and have a much shorter G1 phase too. Yet these cells still manage to load enough helicases to copy their DNA. Little is known about how the amount, rate and timing of helicase loading varies between cells that divide at different speeds. Now Matson et al. have measured how quickly helicase enzymes are loaded onto DNA in individual human cells, including stem cells and specialized or “differentiated” cells. Stem cells loaded helicases rapidly to make up for the short time they spent in G1 phase, while differentiated cells loaded the enzymes more slowly. Measuring how the loading rate changed when stem cells were triggered to specialize showed that helicase loading slowed as the G1 phase got longer. Matson et al. found that the levels of key proteins required for helicase loading correlated with the rates of loading. Altering the levels of the proteins changed how quickly the enzymes were loaded and how the cells behaved – for example, slowing down the loading of helicases made the stem cells specialize quicker. These findings show that the processes of cell differentiation and DNA Replication are closely linked. This study and future ones will help scientists understand what is happening during early animal development, when specialization first takes place, as well as what has gone wrong in cancer cells, which also divide quickly. A better understanding of this process also helps in regenerative medicine – where one of the challenges is to make enough specialized cells to transplant into a patient with tissue damage without those cells becoming cancerous.
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rapid DNA Replication Origin licensing protects stem cell pluripotency
bioRxiv, 2017Co-Authors: Jacob Peter Matson, Raluca Dumitru, Philip Coryell, Ryan M Baxley, Kirk Twaroski, Beau R Webber, Jakub Tolar, Anjakatrin Bielinsky, Weili Chen, Jeremy E PurvisAbstract:Complete and robust human genome duplication requires loading MCM helicase complexes at many DNA Replication Origins, an essential process termed Origin licensing. Licensing is restricted to G1 phase of the cell cycle, but G1 length varies widely among cell types. Using quantitative single cell analyses we found that pluripotent stem cells with naturally short G1 phases load MCM much faster than their isogenic differentiated counterparts with long G1 phases. During the earliest stages of differentiation towards all lineages, MCM loading slows concurrently with G1 lengthening, revealing developmental control of MCM loading. In contrast, ectopic Cyclin E overproduction uncouples short G1 from fast MCM loading. Rapid licensing in stem cells is caused by accumulation of the MCM loading protein, Cdt1. Prematurely slowing MCM loading in pluripotent cells not only lengthens G1 but also accelerates differentiation. Thus, rapid Origin licensing is an intrinsic characteristic of stem cells that contributes to pluripotency maintenance.
Conrad A Nieduszynski - One of the best experts on this subject based on the ideXlab platform.
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oridb the DNA Replication Origin database updated and extended
Nucleic Acids Research, 2012Co-Authors: Cheuk C Siow, Sian R Nieduszynska, Carolin A Muller, Conrad A NieduszynskiAbstract:OriDB (http://www.oridb.org/) is a database containing collated genome-wide mapping studies of confirmed and predicted Replication Origin sites. The Original database collated and curated Saccharomyces cerevisiae Origin mapping studies. Here, we report that the OriDB database and web site have been revamped to improve user accessibility to curated data sets, to greatly increase the number of curated Origin mapping studies, and to include the collation of Replication Origin sites in the fission yeast Schizosaccharomyces pombe. The revised database structure underlies these improvements and will facilitate further expansion in the future. The updated OriDB for S. cerevisiae is available at http://cerevisiae.oridb.org/ and for S. pombe at http://pombe.oridb.org/.
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oridb a DNA Replication Origin database
Nucleic Acids Research, 2007Co-Authors: Conrad A Nieduszynski, Shin-ichiro Hiraga, Prashanth Ak, Craig J Benham, Anne D DonaldsonAbstract:Replication of eukaryotic chromosomes initiates at multiple sites called Replication Origins. Replication Origins are best understood in the budding yeast Saccharomyces cerevisiae, where several complementary studies have mapped their locations genome-wide. We have collated these datasets, taking account of the resolution of each study, to generate a single list of distinct Origin sites. OriDB provides a web-based catalogue of these confirmed and predicted S.cerevisiae DNA Replication Origin sites. Each proposed or confirmed Origin site appears as a record in OriDB, with each record comprising seven pages. These pages provide, in text and graphical formats, the following information: genomic location and chromosome context of the Origin site; time of Origin Replication; DNA sequence of proposed or experimentally confirmed Origin elements; free energy required to open the DNA duplex (stress-induced DNA duplex destabilization or SIDD); and phylogenetic conservation of sequence elements. In addition, OriDB encourages community submission of additional information for each Origin site through a User Notes facility. Origin sites are linked to several external resources, including the Saccharomyces Genome Database (SGD) and relevant publications at PubMed. Finally, a Chromosome Viewer utility allows users to interactively generate graphical representations of DNA Replication data genome-wide. OriDB is available at www.oridb.org.