Cross-linking MS of S. cerevisiae ORC bound to DNA and Cdc6
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Orbitrap Fusion Lumos
ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)
SUBMITTER: 
Jan Seebacher
LAB HEAD: Franziska Bleichert
PROVIDER: PXD031033 | Pride | 2022-02-28
REPOSITORIES: Pride
Dataset's files
Items per page: 5 1 - 5 of 44 |
Similar Datasets
Project description:Sumoylation is emerging as a post-translation modification important for chromosome duplication and stability. The origin recognition complex (ORC), which directs DNA replication initiation by loading the MCM replicative helicases onto origins, is sumoylated in both yeast and human cells. However, the biological consequences of ORC sumoylation are largely unclear. Here we report the effects of hyper- and hypo-sumoylation of yeast ORC using multiple approaches. We show that ORC hyper-sumoylation preferentially reduces the activity of a subset of early origins, while Orc2 hypo-sumoylation has an opposing effect. Mechanistically, ORC hyper-sumoylation leads to reduced MCM loading in vitro and diminished MCM chromatin association in vivo. The importance of an appropriate level of ORC sumoylation is suggested by the data that either hyper- or hypo-sumoylation of ORC results in genome instability and a dependence on other genome maintenance factors for cell fitness. Thus, yeast ORC sumoylation status needs to be fine-tuned to achieve optimal origin activity control and genome stability.
2023-03-11 | PXD032977 | Pride
Project description:The loading and activation of the replicative helicase MCM2-7 are key events during the G1-S phase transition.In budding yeast, the origin recognition complex (ORC) binds to the conserved DNA elements A and B1 of the autonomously replicating sequence (ARS).This is followed by the consecutive loading of two MCM2-7 hetero-hexamers into a MCM2-7 double-hexamer(DH).In S-phase the MCM2-7DH is activated, resulting in two Cdc45-MCM-GINS(CMG) helicases that bidirectionally unwind DNA ahead of the replication fork.Here,we show that MCM2-7 helicase loading across the B1 element displaces ORC fromo rigins.This allows ORC binding and helicase loading at lower affinity binding sites and origins throughout the genome.Furthermore, we mapped the sites of initial DNA unwinding genome-wide and show that these sites appear near the N-terminal domains of the MCM2-7 double-hexamer in proximity of the B1 element.Finally, employing a chemical-biology approach, we establish that during helicase activation the Mcm2/5 interface acts as the DNA exit gate for single-stranded-DNA extrusion.Our work identifies that helicase loading follows a distributive mechanism, allowing for equal MCM2-7 loading across the genome and surprisingly finds that DNA unwinding initiates from the helicase N-terminal interface in proximity to the ARS B1 element.
2024-06-21 | GSE240472 | GEO
Project description:The origin recognition complex (ORC) nucleates DNA replication initiation in eukaryotic cells. This six-protein complex binds replication origin DNA, recruits other initiation factors and facilitates loading of the DNA helicase. Studying the function of individual ORC subunits during pre-RC formation has been hampered by the requirement of most subunits for DNA binding. In this study, we investigate the function of the S. cerevisiae Orc6 subunit, the only subunit not required for DNA binding. In vivo, depletion of Orc6 inhibits pre-replicative complex (pre-RC) assembly and maintenance. In vitro, ORC lacking Orc6 fails to interact with Cdt1 and to load the Mcm2-7 helicase onto origin DNA. We demonstrate that two regions of Orc6 bind Cdt1 directly and that the extreme C-terminus of Orc6 (Orc6-CTD) interacts tightly with the remaining five ORC subunits. Replacing Orc6 with a fusion protein linking Cdt1 to the Orc6-CTD results in an ORC complex that loads Mcm2-7 onto DNA. Interestingly, this complex can only perform a single round of Mcm2-7 loading, suggesting that a dynamic association of Cdt1 with ORC is required for multiple rounds of pre-RC assembly. Keywords: ChIP-chip
2007-11-14 | GSE9318 | GEO
Project description:The loading and activation of the replicative helicase MCM2-7 are key events during the G1-S phase transition. In budding yeast, the origin recognition complex (ORC) binds to the conserved DNA elements A and B1 of the autonomously replicating sequence (ARS). This is followed by the consecutive loading of two MCM2-7 hetero-hexamers into a MCM2-7 double-hexamer (DH). In S-phase the MCM2-7 DH is activated, resulting in two Cdc45-MCM-GINS (CMG) helicases that bidirectionally unwind DNA ahead of the replication fork. Here, we show that MCM2-7 helicase loading across the B1 element displaces ORC from origins. This allows ORC binding and helicase loading at lower affinity binding sites and origins throughout the genome. Furthermore, we mapped the sites of initial DNA unwinding genome-wide and show that these sites appear near the N-terminal domains of the MCM2-7 double-hexamer in proximity of the B1 element. Finally, employing a chemical-biology approach, we establish that during helicase activation the Mcm2/5 interface acts as the DNA exit gate for single-stranded-DNA extrusion. Our work identifies that helicase loading follows a distributive mechanism, allowing for equal MCM2-7 loading across the genome and surprisingly finds that DNA unwinding initiates from the helicase N-terminal interface in proximity to the ARS B1 element.
2024-06-21 | GSE173527 | GEO
Project description:The Origin Recognition Complex (ORC) is essential for the initiation of eukaryotic chromosome replication as it loads the replicative helicase, the minichromosome maintenance (MCM) complex, at replication origins. Origins display a stereotypic chromatin structure with nucleosome depletion at ORC binding sites and flanking arrays of regularly spaced nucleosomes. Although discovered long ago, it is unknown how this chromatin struture is established and if it matters for replication. By genome-scale biochemical reconstitution we screened ~300 origins and 17 purified budding yeast chromatin factors and found that ORC established nucleosome depletion over origins and flanking nucleosome arrays by orchestrating the chromatin remodelers INO80, ISW1a, ISW2 and Chd1. The functional importance of ORC’s chromatin organizing activity was directly demonstrated by ORC mutations that maintained classical MCM loader activity but lost array generation activity. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, besides its canonical role as MCM loader, has a second essential function as master regulator of the origin chromatin structure that is a crucial prerequisite for efficient chromosome replication.
2023-03-30 | GSE209681 | GEO
Project description:The Origin Recognition Complex (ORC) seeds replication-fork formation by binding to DNA replication origins, which in budding yeast contain a 17bp DNA motif. High resolution structure of the ORC-DNA complex revealed two base-interacting elements: a disordered basic patch (Orc1-BP4) and an insertion helix (Orc4-IH). To define the ORC elements guiding its DNA binding in vivo, we mapped genomic locations of 38 designed ORC mutants, revealing that different ORC elements guide binding at different sites. At silencing-associated sites lacking the motif, ORC binding and activity were fully explained by a BAH domain. Within replication origins, we reveal two dominating motif variants showing differential binding modes and symmetry: a non-repetitive motif whose binding requires Orc1-BP4 and Orc4-IH, and a repetitive one where another basic patch, Orc1-BP3, can replace Orc4-IH. Disordered basic patches are therefore key for ORC-motif binding in vivo, and we discuss how these conserved, minor-groove interacting elements can guide specific ORC-DNA recognition.
2024-03-18 | GSE249817 | GEO
Project description:Eukaryotic DNA replication origins are selected in G1-phase when the origin recognition complex (ORC) binds chromosomal DNA, triggering a series of molecular events that culminate in the initiation of DNA replication (a.k.a. origin firing) during S-phase. Each chromosome requires multiple origins for its duplication, and each origin fires at a characteristic time during S-phase, creating a cell-type specific genome replication pattern with relevance to differentiation, genome stability and evolution. It is unclear whether ORC-origin interactions are relevant to the regulation of origin activation time. Here we applied a novel genome-wide strategy to classify origins in the model eukaryote Saccharomyces cerevisiae based on the types of molecular interactions used for ORC-origin binding. Specifically, origins were classified as DNA-dependent when the strength of ORC-origin binding in vivo could be explained by the affinity of ORC for origin DNA in vitro, and, conversely, as ‘chromatin-dependent’ when the ORC-DNA interaction in vitro was insufficient to explain the strength of ORC-origin binding in vivo. The two classes of origins were distinct in terms of local nucleosome architecture and dependence on origin-flanking sequences in plasmid replication assays, consistent with local features of chromatin promoting ORC binding at ‘chromatin-dependent’ origins. Importantly, origins that fired in late S-phase, but not those that fired in early S-phase, showed a significant dependence on the ORC-origin DNA interaction for ORC binding in vivo. Therefore we demonstrate for the first time a connection between ORC-origin binding mechanisms and the regulation of origin activation time at a genome-wide level. Analysis of ORC genomic DNA binding across three ORC concentrations.
2013-09-30 | E-GEOD-48440 | biostudies-arrayexpress
Project description:Mammalian DNA replication relies on various DNA helicases and nuclease activities to ensure accurate genetic duplication, but how different helicase and nuclease activities are properly directed remains unclear. Here, we identify the ubiquitin-specific protease, USP50, as a chromatin-associated protein required to promote ongoing replication, fork restart, telomere maintenance, cellular survival following hydroxyurea or pyridostatin treatment, and suppression of DNA breaks near GC-rich sequences. We find that USP50 supports proper WRN: FEN1 localisation at or near stalled replication forks. Nascent DNA in cells lacking USP50 shows increased association of the DNA2 nuclease and RECQL4 and RECQL5 helicases and replication defects in cells lacking USP50, or FEN1 are driven by these proteins. Consequently, suppression of DNA2 or RECQL4/5 improves USP50-depleted cell resistance to agents inducing replicative stress and restores telomere stability. These data define an unexpected regulatory protein that promotes the balance of helicase and nuclease use at ongoing and stalled replication forks..
2024-08-21 | GSE269605 | GEO
Project description:R-loops are formed when replicative forks collide with the transcriptional machinery and can cause genomic instability. However, it is unclear how R-loops are regulated at transcription-replication conflicts (TRC) sites and how replisome proteins are regulated to prevent R-loop formation or mediate R-loop tolerance. Here, we report that ATAD5, a PCNA unloader, plays dual functions to reduce R-loops both under normal and replication stress conditions. ATAD5 interacts with RNA helicases such as DDX1, DDX5, DDX21 and DHX9 and increases the abundance of these helicases at replication forks to facilitate R-loop resolution. Depletion of ATAD5 or RNA helicases consistently increases R-loops during the S phase and reduces the replication rate, both of which are enhanced by replication stress. In addition to R-loop resolution, ATAD5 prevents the generation of new R-loops behind the replication forks by unloading PCNA which, otherwise, accumulates and persists on DNA, causing a collision with the transcription machinery. Depletion of ATAD5 reduces transcription rates due to PCNA accumulation. Consistent with the role of ATAD5 and RNA helicases in maintaining genomic integrity by regulating R-loops, the corresponding genes were mutated or downregulated in several human tumors.
2021-09-09 | PXD018207 | Pride
Project description:The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell, such as in the orc2-1 mutant, only sites that bind ORC tightly, such as HMR-E, remain fully occupied by ORC, while lower affinity sites, including most origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC “affinity” genome-wide by performing an ORC ChIP-on-chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1-resistant ORC-interacting chromosomal sites (ORF-ORC sites) that did not function as replication origins or silencers. Instead, ORF-ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC-silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF-ORC genes were enriched in proximity to origins of replication, and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection between ORC, replication origins and cellular metabolism.
2009-11-07 | GSE18895 | GEO