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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, John F X Diffley, Julia Locke, Andrea Nans, 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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atpase dependent quality control of dna Replication Origin licensing
Nature, 2013Co-Authors: Jordi Frigola, Dirk Remus, Amina Mehanna, John F X DiffleyAbstract:The regulated loading of the Mcm2–7 DNA helicase (comprising six related subunits, Mcm2 to Mcm7) into pre-replicative complexes at multiple Replication Origins ensures precise once per cell cycle Replication in eukaryotic cells. The Origin recognition complex (ORC), Cdc6 and Cdt1 load Mcm2–7 into a double hexamer bound around duplex DNA in an ATP-dependent reaction, but the molecular mechanism of this Origin ‘licensing’ is still poorly understood. Here we show that both Mcm2–7 hexamers in Saccharomyces cerevisiae are recruited to Origins by an essential, conserved carboxy-terminal domain of Mcm3 that interacts with and stimulates the ATPase activity of ORC–Cdc6. ATP hydrolysis can promote Mcm2–7 loading, but can also promote Mcm2–7 release if components are missing or if ORC has been inactivated by cyclin-dependent kinase phosphorylation. Our work provides new insights into how Origins are licensed and reveals a novel ATPase-dependent mechanism contributing to precise once per cell cycle Replication. The authors describe how the eukaryotic replicative helicase is recruited to Origins and reveal a novel ATPase-dependent quality control mechanism. Eukaryotes strictly regulate DNA Replication so that it occurs once per cell cycle. This condition is maintained by a 'licensing' process, involving the ORC, Cdc6, Cdt1 and Mcm2–7 proteins. The stability of this process is essential for stable inheritance of large genomes in the eukaryotic cell cycle. In this study, John Diffley and colleagues show that in budding yeast, ORC–Cdc6 recruits the Mcm2–7 double hexamer through the Mcm3 subunit. The interaction stimulates the ATPase activity of ORC–Cdc6. However, this hydrolysis can promote either Mcm loading or its release, depending on whether the pre-Replication complex is complete and the stage of the cell cycle is appropriate for activation of the Origin. This kinetic proofreading mechanism prevents the accumulation of partially assembled, dead-end complexes.
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cdks promote dna Replication Origin licensing in human cells by protecting cdc6 from apc c dependent proteolysis
Cell, 2005Co-Authors: Niels Mailand, John F X DiffleyAbstract:Cyclin-dependent kinases (CDKs) restrict DNA Replication Origin firing to once per cell cycle by preventing the assembly of prereplicative complexes (pre-RCs; licensing) outside of G1 phase. Paradoxically, under certain circumstances, CDKs such as cyclin E-cdk2 are also required to promote licensing. Here, we show that CDK phosphorylation of the essential licensing factor Cdc6 stabilizes it by preventing its association with the anaphase promoting complex/cyclosome (APC/C). APC/C-dependent Cdc6 proteolysis prevents pre-RC assembly in quiescent cells and, when cells reenter the cell cycle from quiescence, CDK-dependent Cdc6 stabilization allows Cdc6 to accumulate before the licensing inhibitors geminin and cyclin A which are also APC/C substrates. This novel mechanism for regulating protein stability establishes a window of time prior to S phase when pre-RCs can assemble which we propose represents a critical function of cyclin E.
Marcel Méchali - One of the best experts on this subject based on the ideXlab platform.
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The ORC ubiquitin ligase OBI1 promotes DNA Replication Origin firing
Nature Communications, 2019Co-Authors: Philippe Coulombe, Joelle Nassar, Isabelle Peiffer, Slavica Stanojcic, Yvon Sterkers, Axel Delamarre, Stéphane Bocquet, 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, Philippe Coulombe, Benoit Ballester, Romain Fenouil, Jeanchristophe Andrau, Jacques Van Helden, 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.
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dna Replication Origin activation in space and time
Nature Reviews Molecular Cell Biology, 2015Co-Authors: Michalis Fragkos, Philippe Coulombe, Olivier Ganier, Marcel MéchaliAbstract:During the G1–S phase transition of the cell cycle, a variable subset of previously 'licensed' Origins of Replication is activated to initiate DNA synthesis. Insight is being gained into the mechanisms underlying which Origins are activated and when; these mechanisms are associated with nuclear organization, cell differentiation and Replication stress.
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new insights into Replication Origin characteristics in metazoans
Cell Cycle, 2012Co-Authors: Christelle Cayrou, Philippe Coulombe, Stephanie Rialle, N Kaplan, Eran Segal, Marcel MéchaliAbstract:We recently reported the identification and characterization of DNA Replication Origins (Oris) in metazoan cell lines. Here, we describe additional bioinformatic analyses showing that the previously identified GC-rich sequence elements form Origin G-rich repeated elements (OGREs) that are present in 67% to 90% of the DNA Replication Origins from Drosophila to human cells, respectively. Our analyses also show that initiation of DNA synthesis takes place precisely at 160 bp (Drosophila) and 280 bp (mouse) from the OGRE. We also found that in most CpG islands, an OGRE is positioned in opposite orientation on each of the two DNA strands and detected two sites of initiation of DNA synthesis upstream or downstream of each OGRE. Conversely, Oris not associated with CpG islands have a single initiation site. OGRE density along chromosomes correlated with previously published Replication timing data. Ori sequences centered on the OGRE are also predicted to have high intrinsic nucleosome occupancy. Finally, OGREs predict G-quadruplex structures at Oris that might be structural elements controlling the choice or activation of Replication Origins.
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cdk1 and cdk2 activity levels determine the efficiency of Replication Origin firing in xenopus
The EMBO Journal, 2008Co-Authors: Marcel Méchali, Liliana Krasinska, Emilie Besnard, Christiane Dohet, Jeanmarc Lemaitre, Daniel FisherAbstract:In this paper, we describe how, in a model embryonic system, cyclin-dependent kinase (Cdk) activity controls the efficiency of DNA Replication by determining the frequency of Origin activation. Using independent approaches of protein depletion and selective chemical inhibition of a single Cdk, we find that both Cdk1 and Cdk2 are necessary for efficient DNA Replication in Xenopus egg extracts. Eliminating Cdk1, Cdk2 or their associated cyclins changes Replication Origin spacing, mainly by decreasing frequency of activation of Origin clusters. Although there is no absolute requirement for a specific Cdk or cyclin, Cdk2 and cyclin E contribute more to Origin cluster efficiency than Cdk1 and cyclin A. Relative Cdk activity required for DNA Replication is very low, and even when both Cdk1 and Cdk2 are strongly inhibited, some Origins are activated. However, at low levels, Cdk activity is limiting for the pre-Replication complex to pre-initiation complex transition, Origin activation and Replication efficiency. As such, unlike mitosis, initiation of DNA Replication responds progressively to changes in Cdk activity at low activity levels.
Conrad A Nieduszynski - One of the best experts on this subject based on the ideXlab platform.
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conservation of Replication timing reveals global and local regulation of Replication Origin activity
Genome Research, 2012Co-Authors: Carolin A Muller, Conrad A NieduszynskiAbstract:DNA Replication initiates from defined locations called Replication Origins; some Origins are highly active, whereas others are dormant and rarely used. Origins also differ in their activation time, resulting in particular genomic regions replicating at characteristic times and in a defined temporal order. Here we report the comparison of genome Replication in four budding yeast species: Saccharomyces cerevisiae, S. paradoxus, S. arboricolus, and S. bayanus. First, we find that the locations of active Origins are predominantly conserved between species, whereas dormant Origins are poorly conserved. Second, we generated genome-wide Replication profiles for each of these species and discovered that the temporal order of genome Replication is highly conserved. Therefore, active Origins are not only conserved in location, but also in activation time. Only a minority of these conserved Origins show differences in activation time between these species. To gain insight as to the mechanisms by which Origin activation time is regulated we generated Replication profiles for a S. cerevisiae/S. bayanus hybrid strain and find that there are both local and global regulators of Origin function.
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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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Detection of Replication Origins using comparative genomics and recombinational ARS assay.
Methods of Molecular Biology, 2009Co-Authors: Conrad A Nieduszynski, Anne D DonaldsonAbstract:Effective experimental techniques are available to identify Replication Origin regions in eukaryotic cells. Genome-wide identification of the precise sequence elements that direct Origin activity is however still not straightforward, even in the yeast Saccharomyces cerevisiae which has the best characterised eukaryotic Replication Origins. The availability of genome sequences for a series of closely related (sensu stricto) budding yeasts has allowed us to take a 'comparative genomics' approach to this problem. Since they represent functional protein-binding sites, Origin sequences are conserved better than the surrounding intergenic sequence within the genomes of closely related yeasts. We describe here how phylogenetic comparison data can be used to identify candidate Replication Origin sequences in the S. cerevisiae genome, and how large numbers of such candidate sites can simultaneously be assayed for ability to initiate Replication. Similar approaches could potentially be used to identify protein-binding sequence elements having other functions, as well as Replication Origin sites in other species.
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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.
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Genome-wide identification of Replication Origins in yeast by comparative genomics
Genes & Development, 2006Co-Authors: Conrad A Nieduszynski, Yvonne Knox, Anne D DonaldsonAbstract:We discovered that sequences essential for Replication Origin function are frequently conserved in sensu stricto Saccharomyces species. Here we use analysis of phylogenetic conservation to identify Replication Origin sequences throughout the Saccharomyces cerevisiae genome at base pair resolution. Origin activity was confirmed for each of 228 predicted sites—representing 86% of apparent Origin regions. This is the first study to determine the genome-wide location of Replication Origins at a resolution sufficient to identify the sequence elements bound by Replication proteins. Our results demonstrate that phylogenetic conservation can be used to identify the Origin sequences responsible for replicating a eukaryotic genome.
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, Weili Chen, Kirk Twaroski, Beau R Webber, Jakub Tolar, Anjakatrin Bielinsky, 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, Weili Chen, Kirk Twaroski, Beau R Webber, Jakub Tolar, Anjakatrin Bielinsky, 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.
James M Berger - One of the best experts on this subject based on the ideXlab platform.
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dna stretching by bacterial initiators promotes Replication Origin opening
Nature, 2011Co-Authors: Karl E Duderstadt, Kevin Chuang, James M BergerAbstract:Many Replication initiators form higher-order oligomers that process host Replication Origins to promote replisome formation. In addition to dedicated duplex-DNA-binding domains, cellular initiators possess AAA+ (ATPases associated with various cellular activities) elements that drive functions ranging from protein assembly to Origin recognition. In bacteria, the AAA+ domain of the initiator DnaA has been proposed to assist in single-stranded DNA formation during Origin melting. Here we show crystallographically and in solution that the ATP-dependent assembly of Aquifex aeolicus DnaA into a spiral oligomer creates a continuous surface that allows successive AAA+ domains to bind and extend single-stranded DNA segments. The mechanism of binding is unexpectedly similar to that of RecA, a homologous recombination factor, but it differs in that DnaA promotes a nucleic acid conformation that prevents pairing of a complementary strand. These findings, combined with strand-displacement assays, indicate that DnaA opens Replication Origins by a direct ATP-dependent stretching mechanism. Comparative studies reveal notable commonalities between the approach used by DnaA to engage DNA substrates and other, nucleic-acid-dependent, AAA+ systems. DNA Replication initiates locations known as Origins. One feature of bacterial Origins is an AT-rich sequence known as a DNA unwinding element (DUE) that is melted to allow assembly of the replisome. DnaA is an AAA+ ATPase involved in the initiation of Replication. Although it was thought that the energy of ATP hydrolysis was used to disrupt base pairing of the DUE, Berger and colleagues now show that the ATPase activity of DnaA helps it to assemble as a spiral filament that opens and extends single-stranded DNA. Although this extension of DNA by a filament is surprisingly similar to the early steps in homologous pairing by RecA protein, the DNA stretched by DnaA is inaccessible to base pairing with a complementary strand.
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Replication Origin recognition and deformation by a heterodimeric archaeal orc1 complex
Science, 2007Co-Authors: Erin Cunningham L Dueber, Jacob E Corn, Stephen D Bell, James M BergerAbstract:The faithful duplication of genetic material depends on essential DNA Replication initiation factors. Cellular initiators form higher-order assemblies on Replication Origins, using adenosine triphosphate (ATP) to locally remodel duplex DNA and facilitate proper loading of synthetic replisomal components. To better understand initiator function, we determined the 3.4 angstrom-resolution structure of an archaeal Cdc6/Orc1 heterodimer bound to Origin DNA. The structure demonstrates that, in addition to conventional DNA binding elements, initiators use their AAA+ ATPase domains to recognize Origin DNA. Together these interactions establish the polarity of initiator assembly on the Origin and induce substantial distortions into Origin DNA strands. Biochemical and comparative analyses indicate that AAA+/DNA contacts observed in the structure are dynamic and evolutionarily conserved, suggesting that the complex forms a core component of the basal initiation machinery.
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structural basis for atp dependent dnaa assembly and Replication Origin remodeling
Nature Structural & Molecular Biology, 2006Co-Authors: Jan P Erzberger, Melissa L Mott, James M BergerAbstract:In bacteria, the initiation of Replication is controlled by DnaA, a member of the ATPases associated with various cellular activities (AAA+) protein superfamily. ATP binding allows DnaA to transition from a monomeric state into a large oligomeric complex that remodels Replication Origins, triggers duplex melting and facilitates replisome assembly. The crystal structure of AMP-PCP–bound DnaA reveals a right-handed superhelix defined by specific protein-ATP interactions. The observed quaternary structure of DnaA, along with topology footprint assays, indicates that a right-handed DNA wrap is formed around the initiation nucleoprotein complex. This model clarifies how DnaA engages and unwinds bacterial Origins and suggests that additional, regulatory AAA+ proteins engage DnaA at filament ends. Eukaryotic and archaeal initiators also have the structural elements that promote open-helix formation, indicating that a spiral, open-ring AAA+ assembly forms the core element of initiators in all domains of life.
