Quantitative, genome-wide analysis of eukaryotic replication initiation and termination
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE40696 | GEO | 2013/02/28
SECONDARY ACCESSION(S): PRJNA174633
REPOSITORIES: GEO
Similar Datasets
Project description:Via the deep-sequencing of Okazaki fragments from Saccharomyces cerevisiae, we report the first comprehensive documentation of genome-wide replication directionality in any eukaryote; this permits the systematic analysis of both replication initiation and termination. We conduct a genome-wide analysis of origin competence and efficiency, and conclude that the majority of origins are competent to fire in each cell cycle and generally do so with high efficiency. Additionally, the spatial resolution of our data allow us to determine that leading-strand initiation generally occurs within the nucleosome-free region at origins. Using a strain in which late origins can be induced to fire early, we show that replication termination is a largely passive phenomenon that does not rely on cis-acting sequences or replication fork pausing and that the replication profile is determined largely by the kinetics of origin firing, allowing us to reconstruct chromosome-wide timing profiles from an asynchronous culture. 5 samples are included. Two are replicate, paired-end, wild-type samples sequenced via Illumina methodology. The raw data for these two are also deposited in the GEO repository under accession numbers GSM835650 and GSM835651. Two replicate, single-end, Sld2, Sld3, Dbf4, Dpb11, Cdc45 and Sld7 (SSDDCS) overexpression via galactose induction experiments are reported sequenced via Ion Torrent methodology. One single-end, Sld2, Sld3, Dbf4, Dpb11, Cdc45 and Sld7 (SSDDCS) normal expression control via glucose media experiment is reported sequenced via Ion Torrent methodology.
2013-02-28 | E-GEOD-40696 | biostudies-arrayexpress
Project description:Eukaryotic genome replication is stochastic with each cell using a different cohort of replication origins. Interpreting high-resolution genome replication profiles with a mathematical model allowed us to quantify the stochastic nature of genome replication. This approach included estimation of the activity of every replication origin and the genome-wide location of replication termination events. Single-cell measurements verified the inferred values for stochastic origin replication time. Strains in which multiple origins had been inactivated, confirmed that the location of termination events is primarily dictated by the stochastic activation time of origins. Cell-to-cell variability in origin activity ensures that termination events are widely distributed across virtually the whole genome. We propose that the heterogeneity in origin usage contributes to genome stability by limiting potentially deleterious events from accumulating at particular loci. Measurement of genome replication time for two S. cerevisiae strains. For each strain six S phase samples werecompared with a non-replicating sample.
2013-11-07 | E-GEOD-48561 | biostudies-arrayexpress
Project description:During S-phase of the eukaryotic cell cycle, controls exist to ensure that replication origins fire only once and become inactivated after traverse of a replication fork. Failure of these controls results in local amplification of the genome which can lead to gene copy increases important for both evolutionary change and for somatic genetic diseases such as cancer. It is not known what characteristics of replication origins might predispose them to escape from these controls. We have investigated this problem in the fission yeast Schizosaccharomyces pombe by characterizing regions of the genome which become amplified when the DNA synthesis initiation factors cdc18 (cdc6) and cdt1 are co-overexpressed and by defining origins of replication responsible for local amplification. We find that a single origin can be necessary but not sufficient to induce ectopic local amplification and that origins likely to escape the regulation of one firing per cell cycle are among the most AT-rich, efficient and early firing in the genome, and are embedded in long intergenes. Replication origins with these features may be more prone to re-fire within a round of replication potentiating genome instability.
2010-08-13 | E-MTAB-139 | biostudies-arrayexpress
Project description:The chromatin at origins of replication is the template for DNA replication initiation in eukaryotic genomes. However, whether the chromatin composition instructs individual origins to fire early-efficiently (EE) or late-inefficiently (LI) remains to be investigated. Here, we used site-specific recombination and a single-locus chromatin isolation approach to purify selected EE and LI replication origins from chromosome III of Saccharomyces cerevisiae. Using mass spectrometry, we define the histone modification landscape and identify the protein composition of defined ~1kb native chromatin regions surrounding the EE and LI replication start sites. We confirm expected origin interactors but also find unexpected interactions, such as the Ask1 subunit of the kinetochore-associated DASH complex. Strikingly, we show that Ask1 is a critical factor in the replication timing control of specific origins in yeast. These results highlight that our unbiased approach can specifically identify the functionally-relevant proteome at a single-copy locus of interest and provide a detailed view of histone modification and protein interaction networks on replication origin chromatin. The current dataset contains proteomic data used for the histone PTM analysis.
2023-05-10 | PXD031984 | Pride
Project description:Eukaryotic genome replication is stochastic with each cell using a different cohort of replication origins. Interpreting high-resolution genome replication profiles with a mathematical model allowed us to quantify the stochastic nature of genome replication. This approach included estimation of the activity of every replication origin and the genome-wide location of replication termination events. Single-cell measurements verified the inferred values for stochastic origin replication time. Strains in which multiple origins had been inactivated, confirmed that the location of termination events is primarily dictated by the stochastic activation time of origins. Cell-to-cell variability in origin activity ensures that termination events are widely distributed across virtually the whole genome. We propose that the heterogeneity in origin usage contributes to genome stability by limiting potentially deleterious events from accumulating at particular loci.
2013-11-07 | GSE48561 | GEO
Project description:A form of dwarfism known as Meier-Gorlin syndrome (MGS) is caused by recessive mutations in one of six different genes (ORC1, ORC4, ORC6, CDC6, CDT1, and MCM5). These genes encode components of the pre-replication complex, which assembles at origins of replication prior to S phase. Also, variants in two additional replication initiation genes have joined the list of causative mutations for MGS (Geminin and CDC45). The identity of the causative MGS genetic variants strongly suggests that some aspect of replication is amiss in MGS patients; however, little evidence has been obtained regarding what aspect of chromosome replication is faulty. Since the site of one of the missense mutations in the human ORC4 alleles is conserved between humans and yeast, we sought to determine in what way this single amino acid change affects the process of chromosome replication, by introducing the comparable mutation into yeast (orc4Y232C). To examine early replication dynamics on a genome-wide scale in orc4Y232C cells, we utilized an assay that was previously developed in our lab. This assay uses microarray hybridization to measure the levels of single stranded DNA exposed at replication forks. While we do not fully understand the molecular processes that give rise to peaks of different amplitudes—e.g., number of cells that have activated a particular origin vs. amount of ssDNA revealed at different forks—the results from different replicates of the experiment are highly reproducible. We find that origins that are known to fire early and are efficient produce the peaks of greatest magnitude, while later firing and less efficient origins produce smaller or no peaks in this assay. The characteristic time and/or efficiency of origin firing within the S phase is altered for at least 15% of the 300 yeast origins. Among the origins with delayed/reduced origin firing are normally early-firing origins adjacent to centromeres.
2017-10-06 | GSE104671 | GEO
Project description:Background: Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation events can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, transiently disabling these mechanisms causes replication origins to re-initiate with diverse efficiencies, which do not correlate with known differences in the efficiency or timing of origin initiation during normal DNA replication. These observations suggest an additional local layer of replication control that can differentially influence how re-initiation is regulated at distinct origins. Principal Findings: We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ~60bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 re-associates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Conclusions/Significance: We have uncovered a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are peeled away. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome may be more susceptible to these alterations than others. 142 array CGH experiments are presented. Each experiment is done in experimental replicate. Cells grown as described in Richardson CD and Li JJ PLoS Genetics 2014. Ch1 samples taken from replicating (S phase) or re-replicating (M phase + 3/6 hour induced). Ch2 samples taken from M phase arrested DNA.
2014-03-15 | E-GEOD-55420 | biostudies-arrayexpress
Project description: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 nucleosomes 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.
2015-10-14 | GSE68347 | GEO
Project description:Eukaryotic cells must inhibit re-initiation of DNA replication at each of the thousands of origins in their genome because re-initiation can generate genomic alterations with extraordinary frequency. To minimize the probability of re-initiation from so many origins, cells use a battery of regulatory mechanisms that reduce the activity of replication initiation proteins. Given the global nature of these mechanisms, it has been presumed that all origins are inhibited identically. However, origins re-initiate with diverse efficiencies when these mechanisms are disabled, and this diversity cannot be explained by differences in the efficiency or timing of origin initiation during normal S phase replication. This observation raises the possibility of an additional layer of replication control that can differentially regulate re-initiation at distinct origins. We have identified novel genetic elements that are necessary for preferential re-initiation of two origins and sufficient to confer preferential re-initiation on heterologous origins when the control of re-initiation is partially deregulated. The elements do not enhance the S phase timing or efficiency of adjacent origins and thus are specifically acting as re-initiation promoters (RIPs). We have mapped the two RIPs to ~60 bp AT rich sequences that act in a distance- and sequence-dependent manner. During the induction of re-replication, Mcm2-7 reassociates both with origins that preferentially re-initiate and origins that do not, suggesting that the RIP elements can overcome a block to re-initiation imposed after Mcm2-7 associates with origins. Our findings identify a local level of control in the block to re-initiation. This local control creates a complex genomic landscape of re-replication potential that is revealed when global mechanisms preventing re-replication are compromised. Hence, if re-replication does contribute to genomic alterations, as has been speculated for cancer cells, some regions of the genome may be more susceptible to these alterations than others.
2014-03-15 | GSE55420 | GEO
Project description:The S. cerevisiae Forkhead Box (FOX) proteins, Fkh1 and Fkh2, regulate diverse cellular processes including transcription, long-range DNA interactions during homologous recombination, and replication origin timing and long-range origin clustering. As stimulators of early origin activation, we hypothesized that Fkh1 and Fkh2 abundance limits the rate of origin activation genome-wide. Existing methods, however, were not well suited to quantitative, genome-wide measurements of origin firing between strains and conditions. To overcome this limitation, we developed qBrdU-seq, a quantitative method for BrdU incorporation analysis of replication dynamics, and applied it to show that overexpression of Fkh1 and Fkh2 advance the initiation timing of many origins throughout the genome resulting in a higher total level of origin initiations in early S phase. The higher initiation rate is accompanied by slower replication fork progression, thereby maintaining a normal length of S phase without causing detectable Rad53 checkpoint kinase activation. The advancement of origin firing time, including that of origins in heterochromatic domains, was established in late G1 phase, indicating that origin timing can be reset subsequently to origin licensing. These results provide novel insights into the mechanisms of origin timing regulation by identifying Fkh1 and Fkh2 as rate-limiting factors for origin firing that determine the ability of replication origins to accrue limiting factors and have the potential to reprogram replication timing late in G1 phase. 5 total experiments with replicates
2015-12-18 | E-GEOD-71050 | biostudies-arrayexpress