Dephosphorylation of the pre-initiation complex during S-phase is critical for origin firing dynamics
Ontology highlight
ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE186490 | GEO | 2021/11/01
REPOSITORIES: GEO
Similar Datasets
Project description:Initiation of eukaryotic DNA replication requires temporal separation of helicase loading from helicase activation and replisome assembly. Using an in vitro assay for eukaryotic origin-dependent replication initiation, we investigated the control of these events. After helicase loading, we found that the Dbf4-dependent Cdc7 kinase (DDK) initially drives origin recruitment of Sld3 and the Cdc45 helicase-activating protein. Corresponding in vivo studies found that DDK was required for Cdc45 binding at early origins during G1. Upon activation of S-phase cyclin-dependent kinases (S-CDK), a second helicase-activating protein (GINS) and the remainder of the replisome are recruited to the origin. Investigation of DNA polymerase recruitment showed that Mcm10 and DNA unwinding both were critical for recruitment of the lagging but not leading strand DNA polymerases. Our studies identify distinct roles for DDK and S-CDK during helicase activation and support a model in which the leading strand DNA polymerase is recruited prior to DNA unwinding and initial RNA primer synthesis. We performed chromatin immunoprecipitation (ChIP) against Cdc45 in CDC7 and cdc7-4 cells arrested in G1 phase to assess the requirement of the Dbf4-dependent kinase on the recruitment of Cdc45 to origin DNA during G1.
2011-07-21 | E-GEOD-29646 | biostudies-arrayexpress
Project description:Initiation of eukaryotic chromosome replication follows a spatiotemporal program. Current model suggests that replication origins compete for a limited pool of initiation factors. However, it remains to be answered how these limiting factors are preferentially recruited to early origins. Here, we report that Dbf4 is enriched at early origins through its interaction with forkhead transcription factors Fkh1 and Fkh2. This interaction is mediated by Dbf4 C-terminus and was successfully reconstituted in vitro. An interaction defective mutant dbf4ΔC phenocopies fkh alleles in terms of origin firing. Remarkably, genome-wide replication profiles reveal that the direct fusion of the DNA-binding domain of Fkh1 to Dbf4 restores the Fkh-dependent origin firing, but specifically interferes with the pericentromeric origin activation. Furthermore, Dbf4 directly interacts with Sld3 and promotes the recruitment of downstream limiting factors. These data suggest that Fkh1 targets Dbf4 to a subset of non-centromeric origins to promote early replication, in a manner that is reminiscent to the recruitment of Dbf4 to pericentromeric origins by Ctf19.
2018-01-19 | GSE103073 | GEO
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:The minichromosome maintenance complex (MCM) DNA helicase is an important replicative factor during DNA replication. The proper chromatin loading of MCM is a key step to ensure replication initiation during G1/S phase. Because replication initiation is regulated by multiple biological cues, additional changes to MCM may provide deeper understanding towards this event. Here, we uncover that the histidine methyltransferase SETD3 promotes DNA replication in an enzymatic activity dependent manner. Nascent-strand sequencing (NS-seq) shows that SETD3 regulates replication initiation, as depletion of SETD3 attenuates early replication origins firing. Mechanistically, biochemical experiments reveal that SETD3 binds MCM mainly during G1/S phase, which is required for CDT1-mediated chromatin loading of MCM. The MCM loading relies on the histidine-459 methylation (H459me) on MCM7 that is catalyzed by SETD3. Impairment of H459 methylation attenuates DNA synthesis and chromatin loading of MCM. Furthermore, we show that CDK2 phosphorylates SETD3 at Serine-21 during the G1/S phase, which is required for DNA replication and cell cycle progression. These findings demonstrate a novel mechanism by which SETD3 methylates MCM to regulate replication initiation.
2024-03-12 | GSE243937 | GEO
Project description:How early- and late-firing origins are selected on eukaryotic chromosomes is largely unknown. Here we show that Mrc1, a conserved factor required for stabilization of stalled replication forks, selectively binds to the early-firing origins in a manner independent of Cdc45 and Hsk1 kinase in fission yeast. In mrc1∆ (and in swi1∆ to some extent), efficiency of firing is stimulated and its timing is advanced selectively at those origins that are normally bound by Mrc1. In contrast, the late or inefficient origins which are not bound by Mrc1 are not activated in mrc1∆. The enhanced firing and precocious Cdc45 loading at Mrc1-bound early-firing origins are not observed in a checkpoint mutant of mrc1, suggesting that non-checkpoint function is involved in maintaining the normal program of early-firing origins. We propose that pre-firing binding of Mrc1 is an important marker of early-firing origins which are precociously activated by the absence this protein. Mrc1 binding profiles at G1/S boundary or early S-phase in wild type vs hsk1-89 mutant.
2011-03-26 | GSE28182 | GEO
Project description:To achieve faithful replication of the genome once in each cell cycle, re-initiation of S-phase is prevented in G2 and origins are restricted from re-firing within S-phase. We have investigated the block to re-replication during G2 in fission yeast. The DNA synthesis that occurs when G2/M cyclin dependent kinase (CDK) activity is depleted has been assumed to be repeated rounds of S-phase without mitosis but this has not been demonstrated to be the case. In a normal mitotic S-phase, the genome is uniformly replicated with no areas amplified relative to other regions, so there is an equal copy number of each portion of the genome. To determine whether equal rounds of replication or local amplification occurred in the cdc13 s/o strain we assayed genomic DNA from cells which had increased their DNA content to 16C-32C. Using microarrays, we measured the relative DNA content across the genome, using DNA from control G1-arrested cells as a reference. This experiment revealed that replication was essentially equal across the genome, with no region becoming significantly amplified to a higher copy number than any other. Since the genome was found to be essentially evenly replicated as would be expected if each round of replication corresponded to a normal S-phase, we investigated whether this was the result of a normal replication program at the level of origin firing. We asked whether S-phase origins are used to reduplicate the genome, and whether origins are used with the same efficiency as in wild type cells. We identified the origins utilized in the first endoreduplication cycle of cdc13 s/o using microarray analyses of cells treated with 11 mM HU. Samples were taken at 5 hours when most cells of the culture not treated with HU had undergone a doubling in DNA content. A total of 799 origins were identified, a number roughly similar to the 904 identified in a normal S-phase. We show here that on G2/M CDK depletion in G2, repeated S-phases are induced. Mostly normal mitotic S-phase origins were utilized although at different efficiencies, and replication was essentially equal across the genome. We conclude that CDK inhibits re-initiation of S-phase during G2, and if G2/M CDK is depleted replication results from induction of a largely normal S-phase program with only small differences in origin usage and efficiency.
2009-05-13 | E-MTAB-105 | biostudies-arrayexpress
Project description:Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of inactive double hexamers of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic origins are not irrevocably defined by selection of the loading site for the replicative helicase, but can shift in position after helicase loading. Using purified proteins, we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. In yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a shift in origin positions that correlates with the direction of transcription. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict. 4 samples: one replicate for WT at 37C, two replicates for rat1-1 at 37C, and one replicate for rat1-1 at 24C. All are single-end sequenced via Ion Torrent PGM methodology. rat1 = Nuclear 5' to 3' single-stranded RNA exonuclease; involved in RNA metabolism (http://www.yeastgenome.org/locus/S000005574/overview). 4 ChIP seq samples and their duplicates are submitted. rat1-1 ORC ChIP at 24C and 37C; rat1-1 MCM ChIP at 24C and 37C.
2016-01-07 | E-GEOD-69065 | biostudies-arrayexpress
Project description:RECQL4, a member of the RecQ helicase family, plays a role in maintaining genomic stability, but its precise function remains unclear. The N-terminus of RECQL4 has similarity to Sld2, a protein required for the firing of DNA replication origins in budding yeast. Consistent with this sequence similarity, Xenopus RECQL4 has been implicated in initiating DNA replication in oocyte extracts. To determine whether human RECQL4 is required for firing of DNA replication origins, we generated cells in which both RECQL4 alleles were targeted, resulting in either lack of protein expression (Knock-Out) or expression of a full-length, mutant protein lacking helicase activity (Helicase Dead). Surprisingly, both the RECQL4 Knock-Out and Helicase Dead cells were viable and exhibited essentially identical origin firing profiles as the parental cells. Analysis of the rate of fork progression revealed normal rates in the RECQL4 Knock Out cells, but decreased rates in the RECQL4 inactive cells. Thus, while budding yeast Sld2 is required for DNA replication origin firing, our evidence suggest that human RECQL4 has assumed a role in the regulation of replication fork progression.
2024-04-17 | GSE225532 | GEO
Project description:Eukaryotic genomes are replicated from many origin sites that are licensed by the loading of inactive double hexamers of the replicative DNA helicase, Mcm2-7. How eukaryotic origin positions are specified remains elusive. Here we show that, contrary to the bacterial paradigm, eukaryotic origins are not irrevocably defined by selection of the loading site for the replicative helicase, but can shift in position after helicase loading. Using purified proteins, we show that DNA translocases, including RNA polymerase, can push budding yeast Mcm2-7 double hexamers along DNA. Displaced Mcm2-7 double hexamers support DNA replication initiation distal to the loading site in vitro. In yeast cells that are defective for transcription termination, collisions with RNA polymerase induce a shift in origin positions that correlates with the direction of transcription. These results reveal a eukaryotic origin specification mechanism that departs from the classical replicon model, helping eukaryotic cells to negotiate transcription-replication conflict.
2016-01-07 | GSE69065 | GEO
Project description:Across eukaryotes, inhibition of the progression of the DNA replication machinery causes an S-phase checkpoint response. This response regulates multiple processes, including inhibition of replication initiation and fork stabilisation. How these events are coordinated remains poorly understood. Here we show that the replicative helicase component Cdc45 targets the checkpoint kinase Rad53 to distinct replication complexes in the budding yeast Saccharomyces cerevisiae. Rad53 binds to FHA-interaction motifs in an unstructured loop region of Cdc45, which is phosphorylated by Rad53 itself, and this interaction is necessary for the inhibition of origin firing through Sld3. In addition to regulating replication initiation, Cdc45 also recruits Rad53 to stalled replication forks, which we demonstrate is important for the response to replication stress. Finally we show that a Cdc45 mutation that is orthologous to an allele found in patients with Meier-Gorlin Syndrome disrupts the functional interaction with Rad53. Together we present a single mechanism by which Rad53 targets replication initiation and elongation complexes, which may be relevant to human disease.
2019-02-26 | GSE122110 | GEO