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Confirmation of replication interactome of TopBP1 using proximity-dependent biotinylation MS

ABSTRACT: Goal of the Experiment is to confirm the replication interactome of TopBP1 using stabile 293 FlpIn cell lines expressing TopBP1 adapter fused to APEX2. Proteins in its close proximity will become biotinylated by APEX2, thereby enabling the pulldown of biotinylated proteins and thus, replication involved substrates of TopBP1

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Suspension Culture

SUBMITTER: Farnusch Kaschani

LAB HEAD: Dr Farnusch Kaschani

PROVIDER: PXD040000 | Pride | 2024-05-21

REPOSITORIES: Pride

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			Action	DRS
	ACE_0727_MP01.raw	Raw
	ACE_0727_MP02.raw	Raw
	ACE_0727_MP03.raw	Raw
	ACE_0727_MP04.raw	Raw
	ACE_0727_MP05.raw	Raw

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Publications

TopBP1 utilises a bipartite GINS binding mode to support genome replication.

Day Matthew M Tetik Bilal B Parlak Milena M Almeida-Hernández Yasser Y Räschle Markus M Kaschani Farnusch F Siegert Heike H Marko Anika A Sanchez-Garcia Elsa E Kaiser Markus M Barker Isabel A IA Pearl Laurence H LH Oliver Antony W AW Boos Dominik D

Nature communications 20240227 1

Activation of the replicative Mcm2-7 helicase by loading GINS and Cdc45 is crucial for replication origin firing, and as such for faithful genetic inheritance. Our biochemical and structural studies demonstrate that the helicase activator GINS interacts with TopBP1 through two separate binding surfaces, the first involving a stretch of highly conserved amino acids in the TopBP1-GINI region, the second a surface on TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS s ...[more]

PMID: 38413589

Similar Datasets

Deciphering the interaction between TopBP1 and GINS using crosslinking mass spectrometry

Project description:Recombinant TopBP1 and GINS were cross linked using the MS non-cleavable crosslinker BS3. Crosslinked proteins were then subsequently either digested directly using the SP3 strategy or first seperated on SDS-PAGE and then in-gel digested. cross-linked peptides were identified by LC-MS. The information from this experiment was used to refine cryo EM structures.

2024-05-24 | PXD040156 | Pride

Proteogenomics uncovers critical elements of host response in bovine soft palate epithelial cells following in vitro infection with foot-and-mouth disease virus (FMDV)

Project description:The pathogen and host factors that contribute to the establishment of foot-and-mouth disease virus (FMDV) persistence are currently not understood. Using primary bovine soft palate multilayers in combination with RNA sequencing, we analyzed the transcriptional responses during acute and persistent FMDV infection.

2019-01-18 | E-MTAB-7605 | biostudies-arrayexpress

TOPBP1 Shapes XY Silencing Dynamics Independently of Sex Body Formation to Ensure Male Fertility

Project description:In mammals and several other taxa, the ability of males to cope with the limited synapsis of the X and Y chromosomes during prophase I of meiosis relies on the process of meiotic sex chromosome inactivation (MSCI). Components of the somatic DNA damage response machinery, including ATR, TOPBP1, MDC1 and BRCA1 play key roles in MSCI, although how they establish XY silencing remains incompletely understood. In particular, it remains unclear how DDR factors coordinate XY silencing with DNA repair, chromosome synapsis and the formation of the sex body, a distinct phase-separated sub-nuclear structure formed during prophase I to house the unsynapsed XY bivalent. Here we report a mutant mouse (Topbp1B5/B5), harboring mutations in the BRCT5 domain of Topbp1, that shows impaired XY silencing but grossly normal sex body formation. While Topbp1B5/B5 mice are viable, without detectable somatic defects, males are completely infertile. Distinct from mice lacking ATR or TOPBP1 specifically during meiosis, Topbp1B5/B5 males exhibit normal chromosome synapsis and canonical markers of DNA repair in early prophase I. ATR signaling is mostly intact in Topbp1B5/B5 spermatocytes, although specific ATR-dependent events are disrupted, including localization of the RNA:DNA helicase Senataxin to chromatin loops of the XY. Strikingly, while Topbp1B5/B5 spermatocytes are able to initiate MSCI the completion of gene silencing is defective, with a subset of X chromosome genes displaying distinct patterns of transcriptional deregulation. These findings suggest a non-canonical role for the ATR-TOPBP1 signaling axis in XY silencing dynamics at advanced stages in pachynema. This is the first DDR mutant that separates XY silencing from sex body formation, as well as TOPBP1’s role in spermatogenesis from its roles in organismal viability.

2024-02-09 | PXD042199 | Pride

Transcriptomic analysis of F. oxysporum M-NM-^Tcon7-1 mutant during in vitro growth

Project description:Previous studies have demonstrated the essential role of morphogenetic regulation in Fusarium oxysporum pathogenesis, including processes such as cell-wall biogenesis, cell division and differentiation of infection-like structures. We identified three F. oxysporum genes encoding predicted transcription factors showing significant identities to Magnaporthe oryzae Con7p, Con7-1, plus two identical copies of Con7-2. Targeted deletion of con7-1 produced non-pathogenic mutants with altered morphogenesis, including defects in cell wall structure, polar growth, hyphal branching and conidiation. By contrast, simultaneous inactivation of both con7-2 copies caused no detectable defects in the resulting mutants. Comparative microarray-based gene expression analysis indicated that Con7-1 modulates the expression of a large number of genes involved in different biological functions, including host-pathogen interactions, morphogenesis and development, signal perception and transduction, transcriptional regulation and primary and secondary metabolism. Taken together, our results points to Con7-1 as general regulator of morphogenesis and virulence in F. oxysporum. Con7-1 dependent gene expression in Fusarium oxysporum wt and M-NM-^Tcon7-1 mutant was measured after shifting the mycelia to PDB pH5.0 for 30 min and the transferred to PDB pH7.0 for 30 min. Three independent experiments were performed.

2014-07-10 | E-GEOD-59311 | biostudies-arrayexpress

Bipartite GINS binding mode of TopBP1 to activate the replicative helicase - Chromass Data Set

Project description:How the replicative Mcm2-7 helicase is activated during replication origin firing remains largely unknown. Our biochemical and structural studies reveal that the helicase activator GINS interacts with TopBP1 using two separate binding surfaces. One involves a stretch of highly conserved amino acids in the TopBP1-GINI domain. The other surface is located in TopBP1-BRCT4. The two surfaces bind to opposite ends of the A domain of the GINS subunit Psf1, and their cooperation is required for a biochemically stable TopBP1-GINS interaction. The GINI and BRCT4 domains also cooperate during replication origin firing, as immune-replacement experiments in Xenopus egg extract using different GINI and BRCT4 mutations suggest. The TopBP1-GINS interaction is incompatible with simultaneous binding of DNA polymerase epsilon to GINS when bound to Mcm2-7-Cdc45. Our TopBP1-GINS model predicts the coordination of three molecular processes, DNA polymerase epsilon arrival, TopBP1 ejection and GINS integration into Mcm2-7-Cdc45.

2024-03-26 | PXD040024 | Pride

MDC1 interacts with TOPBP1 to maintain chromosomal stability during mitosis

Project description:MDC1 is a large, modular phospho-protein scaffold that mediates recruitment of signaling and repair complexes to sites of DNA double-strand breaks (DSBs). MDC1 is anchored to damaged chromatin through interaction of its C-terminal BRCT-repeat domain with phosphorylated H2AX (γH2AX), where it recruits downstream factors via direct phosphorylation-dependent protein-protein interactions. Here, using unbiased proteomic analyses we identify a highly conserved protein interaction surface near the MDC1 N-terminus for the DNA damage response protein TOPBP1. We show that TOPBP1 directly binds to two residues in MDC1 that are phosphorylated by CK2. Interestingly, we find that TOPBP1 recruitment to DSBs depends on direct interaction with MDC1 only during mitosis. Furthermore, we demonstrate that disrupting MDC1-TOPBP1 binding causes hypersensitivity to ionizing radiation in mitosis, as well as increased micronuclei and chromosomal instability. Thus, our results highlight an important and hitherto unnoticed function for MDC1 and TOPBP1 in maintaining genome stability during cell division.

2019-03-25 | PXD011850 | Pride

Treacle controls the nucleolar response to rDNA breaks via TOPBP1 recruitment and ATR activation

Project description:Induction of DNA double-strand breaks (DSBs) in ribosomal DNA (rDNA) repeats is associated with ATM-dependent repression of ribosomal RNA synthesis and large-scale reorganization of nucleolar architecture, but the signaling events that regulate these responses are largely elusive. Here we show that the nucleolar response to rDNA breaks is dependent on both ATM and ATR activity. We further demonstrate that ATM- and NBS1-dependent recruitment of TOPBP1 in the nucleoli is required for inhibition of ribosomal RNA synthesis and nucleolar segregation in response to rDNA breaks. Mechanistically, TOPBP1 recruitment is mediated by phosphorylation-dependent interactions between three of its BRCT domains and conserved phosphorylated Ser/Thr residues at the C-terminus of the nucleolar phosphoprotein Treacle. Our data thus reveal an important cooperation between TOPBP1 and Treacle in the signaling cascade that triggers transcriptional inhibition and nucleolar segregation in response to rDNA breaks.

2019-12-06 | PXD015138 | Pride

Characterization of ultraID for PDB-MS

Project description:Comparison of BioID (24h labeling) and ultraID (10 min labeling) for PDB-MS (POI = Ago2, control protein = Rab11). 4 biological replicates per condition.

2022-07-05 | PXD026719 | Pride

The Transcriptional Landscape of Marek’s Disease Virus in Primary Chicken B Cells Reveals Novel Splice Variants and Genes

Project description:Marek's disease virus (MDV) belongs to the alphaherpesviruses and is the causative agent of fatal T cell lymphomas in chickens. Aftermaths of MDV infections lead to substantial losses in poultry industry and there is evidence that MDV constantly evolves towards higher virulence. This study takes a detailed look at the very first step of the viral life cycle in the host: the infection of B cells. Recently, it has become possible to culture and infect primary chicken B cells ex vivo, enabling us to apply those cells to RNAseq analysis. Here, we provide novel insights into MDV gene expression profiles and post-transcriptional modifications of this important poultry pathogen in its primary target cells for lytic replication. Remarkably enough, we could detect a novel hypothetical MDV gene, identify multicistronic transcripts and previously unannotated splice forms. These findings lay the foundation for future research on MDV replication and pathogenesis and the developed bioinformatics pipeline can be easily transferred to other herpesviruses or large DNA viruses to identify unknown transcript isoforms and associated motifs.

2019-03-19 | E-MTAB-7772 | biostudies-arrayexpress

SMARCAD1 and TOPBP1 contribute to heterochromatin maintenance at the transition from the 2C-like to the pluripotent state

Project description:Chromocenters are established after the 2-cell (2C) stage during mouse embryonic development, but the factors that mediate chromocenter formation remain largely unknown. To identify regulators of 2C heterochromatin establishment, we generated an inducible system to convert embryonic stem cells (ESCs) to 2C-like cells. This conversion is marked by a global reorganization and dispersion of H3K9me3-heterochromatin foci, which are then reversibly formed upon re-entry into pluripotency. Profiling the chromatin-bound proteome (chromatome) by genome capture of ESCs transitioning to 2C-like cells, we uncover chromatin regulators involved in de novo heterochromatin formation. We identified TOPBP1 and investigated its binding partner SMARCAD1. SMARCAD1 and TOPBP1 associate with H3K9me3-heterochromatin in ESCs. Interestingly, the nuclear localization of SMARCAD1 is lost in 2C-like cells. SMARCAD1 or TOPBP1 depletion in mouse embryos lead to developmental arrest, reduction of H3K9me3 and remodeling of heterochromatin foci. Collectively, our findings contribute to comprehending the maintenance of chromocenters during early development.

2023-04-18 | PXD019703 | Pride

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