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Diversity of Eukaryotic DNA Replication Origins Revealed by Genome-Wide Analysis of Chromatin Structure  [PDF]
Nicolas M. Berbenetz,Corey Nislow ,Grant W. Brown
PLOS Genetics , 2010, DOI: 10.1371/journal.pgen.1001092
Abstract: Eukaryotic DNA replication origins differ both in their efficiency and in the characteristic time during S phase when they become active. The biological basis for these differences remains unknown, but they could be a consequence of chromatin structure. The availability of genome-wide maps of nucleosome positions has led to an explosion of information about how nucleosomes are assembled at transcription start sites, but no similar maps exist for DNA replication origins. Here we combine high-resolution genome-wide nucleosome maps with comprehensive annotations of DNA replication origins to identify patterns of nucleosome occupancy at eukaryotic replication origins. On average, replication origins contain a nucleosome depleted region centered next to the ACS element, flanked on both sides by arrays of well-positioned nucleosomes. Our analysis identified DNA sequence properties that correlate with nucleosome occupancy at replication origins genome-wide and that are correlated with the nucleosome-depleted region. Clustering analysis of all annotated replication origins revealed a surprising diversity of nucleosome occupancy patterns. We provide evidence that the origin recognition complex, which binds to the origin, acts as a barrier element to position and phase nucleosomes on both sides of the origin. Finally, analysis of chromatin reconstituted in vitro reveals that origins are inherently nucleosome depleted. Together our data provide a comprehensive, genome-wide view of chromatin structure at replication origins and suggest a model of nucleosome positioning at replication origins in which the underlying sequence occludes nucleosomes to permit binding of the origin recognition complex, which then (likely in concert with nucleosome modifiers and remodelers) positions nucleosomes adjacent to the origin to promote replication origin function.
The Chromatin Assembly Factor 1 Promotes Rad51-Dependent Template Switches at Replication Forks by Counteracting D-Loop Disassembly by the RecQ-Type Helicase Rqh1  [PDF]
Violena Pietrobon equal contributor,Karine Fréon equal contributor,Julien Hardy,Audrey Costes,Is Iraqui,Fran?oise Ochsenbein,Sarah A.E. Lambert
PLOS Biology , 2014, DOI: 10.1371/journal.pbio.1001968
Abstract: At blocked replication forks, homologous recombination mediates the nascent strands to switch template in order to ensure replication restart, but faulty template switches underlie genome rearrangements in cancer cells and genomic disorders. Recombination occurs within DNA packaged into chromatin that must first be relaxed and then restored when recombination is completed. The chromatin assembly factor 1, CAF-1, is a histone H3-H4 chaperone involved in DNA synthesis-coupled chromatin assembly during DNA replication and DNA repair. We reveal a novel chromatin factor-dependent step during replication-coupled DNA repair: Fission yeast CAF-1 promotes Rad51-dependent template switches at replication forks, independently of the postreplication repair pathway. We used a physical assay that allows the analysis of the individual steps of template switch, from the recruitment of recombination factors to the formation of joint molecules, combined with a quantitative measure of the resulting rearrangements. We reveal functional and physical interplays between CAF-1 and the RecQ-helicase Rqh1, the BLM homologue, mutations in which cause Bloom's syndrome, a human disease associating genome instability with cancer predisposition. We establish that CAF-1 promotes template switch by counteracting D-loop disassembly by Rqh1. Consequently, the likelihood of faulty template switches is controlled by antagonistic activities of CAF-1 and Rqh1 in the stability of the D-loop. D-loop stabilization requires the ability of CAF-1 to interact with PCNA and is thus linked to the DNA synthesis step. We propose that CAF-1 plays a regulatory role during template switch by assembling chromatin on the D-loop and thereby impacting the resolution of the D-loop.
Evidence for Sequential and Increasing Activation of Replication Origins along Replication Timing Gradients in the Human Genome  [PDF]
Guillaume Guilbaud ?,Aurélien Rappailles ?,Antoine Baker,Chun-Long Chen,Alain Arneodo,Arach Goldar,Yves d'Aubenton-Carafa,Claude Thermes,Benjamin Audit,Olivier Hyrien
PLOS Computational Biology , 2011, DOI: 10.1371/journal.pcbi.1002322
Abstract: Genome-wide replication timing studies have suggested that mammalian chromosomes consist of megabase-scale domains of coordinated origin firing separated by large originless transition regions. Here, we report a quantitative genome-wide analysis of DNA replication kinetics in several human cell types that contradicts this view. DNA combing in HeLa cells sorted into four temporal compartments of S phase shows that replication origins are spaced at 40 kb intervals and fire as small clusters whose synchrony increases during S phase and that replication fork velocity (mean 0.7 kb/min, maximum 2.0 kb/min) remains constant and narrowly distributed through S phase. However, multi-scale analysis of a genome-wide replication timing profile shows a broad distribution of replication timing gradients with practically no regions larger than 100 kb replicating at less than 2 kb/min. Therefore, HeLa cells lack large regions of unidirectional fork progression. Temporal transition regions are replicated by sequential activation of origins at a rate that increases during S phase and replication timing gradients are set by the delay and the spacing between successive origin firings rather than by the velocity of single forks. Activation of internal origins in a specific temporal transition region is directly demonstrated by DNA combing of the IGH locus in HeLa cells. Analysis of published origin maps in HeLa cells and published replication timing and DNA combing data in several other cell types corroborate these findings, with the interesting exception of embryonic stem cells where regions of unidirectional fork progression seem more abundant. These results can be explained if origins fire independently of each other but under the control of long-range chromatin structure, or if replication forks progressing from early origins stimulate initiation in nearby unreplicated DNA. These findings shed a new light on the replication timing program of mammalian genomes and provide a general model for their replication kinetics.
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
Rif1 Regulates Initiation Timing of Late Replication Origins throughout the S. cerevisiae Genome  [PDF]
Jared M. Peace, Anna Ter-Zakarian, Oscar M. Aparicio
PLOS ONE , 2014, DOI: 10.1371/journal.pone.0098501
Abstract: Chromosomal DNA replication involves the coordinated activity of hundreds to thousands of replication origins. Individual replication origins are subject to epigenetic regulation of their activity during S-phase, resulting in differential efficiencies and timings of replication initiation during S-phase. This regulation is thought to involve chromatin structure and organization into timing domains with differential ability to recruit limiting replication factors. Rif1 has recently been identified as a genome-wide regulator of replication timing in fission yeast and in mammalian cells. However, previous studies in budding yeast have suggested that Rif1’s role in controlling replication timing may be limited to subtelomeric domains and derives from its established role in telomere length regulation. We have analyzed replication timing by analyzing BrdU incorporation genome-wide, and report that Rif1 regulates the timing of late/dormant replication origins throughout the S. cerevisiae genome. Analysis of pfa4Δ cells, which are defective in palmitoylation and membrane association of Rif1, suggests that replication timing regulation by Rif1 is independent of its role in localizing telomeres to the nuclear periphery. Intra-S checkpoint signaling is intact in rif1Δ cells, and checkpoint-defective mec1Δ cells do not comparably deregulate replication timing, together indicating that Rif1 regulates replication timing through a mechanism independent of this checkpoint. Our results indicate that the Rif1 mechanism regulates origin timing irrespective of proximity to a chromosome end, and suggest instead that telomere sequences merely provide abundant binding sites for proteins that recruit Rif1. Still, the abundance of Rif1 binding in telomeric domains may facilitate Rif1-mediated repression of non-telomeric origins that are more distal from centromeres.
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
Surveying genome replication
Stephen Kearsey
Genome Biology , 2002, DOI: 10.1186/gb-2002-3-6-reviews1016
Abstract: Eukaryotes differ fundamentally from prokaryotes in the chromosomal organization of the sites at which DNA replication is initiated. In Escherichia coli, for instance, two replication forks originating from a single origin of replication are responsible for replicating the entire genome, whereas the replication of eukaryotic chromosomes during S phase of the cell cycle starts from many origins. Increasing the number of origins allows DNA polymerases to work at many sites simultaneously, speeding up replication, and this was presumably a prerequisite for the evolution of large genomes. Working out how origins are distributed in eukaryotic chromosomes is therefore addressing a fundamental question about replication. Origins have been most extensively studied in the budding yeast Saccharomyces cerevisiae, where they consist of short sequences (less than 100-150 base-pairs). Although budding yeast origins share some consensus sequence similarities, genetic and biochemical assays are required to map them precisely. Over the years, detailed work has systematically mapped the origins on two yeast chromosomes [1,2,3], but two recent papers [4,5] have used microarray technology to provide a whole-genome view of how replication origins are distributed and when they function only in S phase.In the first paper, Wyrick et al. [4] mapped the chromosomal binding sites of the proteins associated with initiation. Yeast replication origins are bound by the proteins that make up the origin recognition complex (ORC) throughout the cell cycle, and additional proteins associate with ORC before S phase and form a pre-replicative complex (pre-RC) which is competent to initiate replication. Six MCM (mini-chromosome maintenance) proteins are key components of the pre-RC, and these factors are likely to provide the DNA helicase function required for DNA synthesis [6]. Wyrick et al. [4] used the chromatin immunoprecipitation (CHIP) technique in combination with microarrays to determine the chr
Controlling origins of replication  [cached]
Cathy Holding
Genome Biology , 2003, DOI: 10.1186/gb-spotlight-20030812-01
Abstract: Using the dynamic molecular combing (DMC) procedure, Anglana et al. studied the replication pattern of three cell lines that acquired resistance to the inhibitor coformycin through amplification of a multigene locus including an origin already under study, oriGNAI3. Replication forks traveled across the amplified region at different speeds in different DNA fibers in the same cell. Seven repeatedly firing loci were visualized using the DMC technique, four of which were in an extensively analyzed region - including highly AT-rich matrix attachment regions - suggesting a role for these regions in DNA replication. The efficiency of firing of these four origins depended on culture conditions. A slower fork speed was compensated for by firing additional replication origins, while firing the predominant oriGNAI3 origin in this region resulted in the silencing of surrounding weaker origins."In mammalian cells, as in yeast models, not all the potential replication origins fire during S phase and the efficiency of some origins relies more on nucleotide availability and/or fork progression rate than on specific cis-sequences. Interestingly, the initiation sites identified within the sequenced part of the domain lie in intergenic regions and precisely co-map with previously identified A+T rich matrix attachment regions," conclude the authors.
Bright days for yeast research
Martin P?evorovsky, Charalampos Rallis
Genome Biology , 2011, DOI: 10.1186/gb-2011-12-5-305
Abstract: The annual British Yeast Group Meetings form a platform for high quality scientific interactions and have significant impact on the research directions followed. Here, we summarize talks and poster presentations on DNA replication dynamics and evolution, high-throughput screens and phenomics, chromatin biology and DNA damage presented at the meeting.Chromosomes must be correctly replicated before cell division, as aberrant replication can cause genome instability. Therefore, origins of replication must be tightly regulated and appropriately distributed in the genome. Michelle Hawkins (University of Nottingham, UK) discussed the genome-wide measurement of DNA copy numbers from synchronized budding yeast cell populations as they progress through S phase. Using next-generation sequencing, replication profiles were generated. The data obtained were then combined with a mathematical model of DNA replication, which enabled predictions of individual origin properties that largely agree with independent experimental data. The model predicts cell-to-cell variations, the distribution of distances between active origins and the number of replication forks. These studies resulted in a data-rich platform that will be used to understand the mechanisms involved in faithful and precise DNA replication.Origins of replication fall into three categories - early, late and dormant - because for unknown reasons they do not initiate simultaneously at the beginning of the S phase. Two kinase activities are required for replication initiation throughout S phase: cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK). Philip Zegerman (Gurdon Institute, University of Cambridge, UK) showed that substrates of CDK (Sld2, Sld3 and their binding partner Dpb11) and the DDK subunit Dbf4 are limiting factors for replication initiation in budding yeast. Overexpression of these factors led to early initiation of late origins. When such overexpression was combined with deletion of the histone dea
Proteins of the origin recognition complex (ORC) and DNA topoisomerases on mammalian chromatin
Hong-gang Hu, Martina Baack, Rolf Knippers
BMC Molecular Biology , 2009, DOI: 10.1186/1471-2199-10-36
Abstract: We have used different cell fractionation procedures, namely salt and nuclease treatment of isolated nuclei as well as formaldehyde-mediated cross-linking of chromatin, to investigate the distribution of topoisomerases and proteins of the origin recognition complex (ORC) in the chromatin of human HeLa cells. First we obtained no evidence for a physical interaction of either topoisomerase I or topoisomerase II with ORC. Then we found, however, that (Orc1-5) and topo II occurred together on chromatin fragments of 600 and more bp lengths. At last we showed that both topo II and Orc2 protein are enriched near the origin at the human MCM4 gene, and at least some of the topo II at the origin is active in proliferating HeLa cells. So taken together, topoisomerase II, but not topoisomerase I, is located close to ORC on chromatin.Topoisomerase II is more highly expressed than ORC proteins in mammalian cells, so only a small fraction of total chromatin-bound topoisomerase II was found in the vicinity of ORC. The precise position of topo II relative to ORC may differ among origins.DNA replication requires the separation of complementary DNA strands. This process begins at the origin of replication under the direction of initiator proteins such as the T antigen of Simian Virus 40 (SV40) or the eukaryotic preinitiation complex including ORC (origin recognition complex). The unwinding of circularly closed or long DNA duplices leads to torsional tensions which must be released by topoisomerases. According to earlier biochemical work, high local concentrations of the eukaryotic type IB topoisomerase (topo I) and a type II topoisomerase (topo II) are required to release the torsional stress that accompanies the initiation and propagation of replication forks on closed circular SV40 viral DNA in vitro [1-3]. Further, topo I was shown to be mainly located in vivo at regions ahead of the replication forks on replicating SV40 DNA molecules, while topo II also occurs in pre-fork regions,

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