Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

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

ABSTRACT: 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.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Xenopus Laevis (african Clawed Frog)

TISSUE(S): Cell Culture

SUBMITTER: Markus Räschle

LAB HEAD: Markus Räschle

PROVIDER: PXD040024 | Pride | 2024-03-26

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Similar Datasets

Protein recruitment on chromatin in the presence or absence of Plk1

Project description:Polo-like kinase 1 (Plk1) is an important protein kinase for checkpoint recovery and adaptation in response to DNA damage and replication stress. However, although Plk1 is present in S phase, little is known about its localization and function during unperturbed DNA replication. Here we used a previously described Chromatin Mass spectrometry (Chromass) protocol (Project PXD000490) to compare the protein recruitment on chromatin in Xenopus egg extracts during unperturbed DNA replication in the presence or after Plk1 depletion from the extract.

2021-08-25 | PXD022572 | Pride

Proteomics reveals dynamic assembly of repair complexes during bypass of DNA cross-links, part 1 of 2: CHROMASS DATA

Project description:DNA interstrand crosslinks (ICLs) block replication fork progression by inhibiting DNA strand separation. Repair of ICLs requires sequential incisions, translesion DNA synthesis, and homologous recombination, but the full set of factors involved in these transactions remains unknown. Here we established CHROmatin MASS spectrometry (CHROMASS) to study protein recruitment dynamics during perturbed DNA replication in Xenopus egg extracts. Using CHROMASS, we systematically monitored protein assembly and disassembly at ICL-containing chromatin. Among numerous prospective new DNA repair factors we identified SLF1 and SLF2, which form a complex with RAD18 and together define a new pathway that suppresses genome instability by recruiting the SMC5/6 cohesion complex to DNA lesions. Our study provides the first global analysis of an entire DNA repair pathway and reveals the mechanism of SMC5/6 relocalization to damaged DNA in vertebrate cells.

2015-05-07 | PXD000490 | Pride

DNMT1 IP-MS alalysis to identify ubiquitin-dependent interacting partners

Project description:Stable inheritance of DNA methylation is critical for maintaining the differentiated phenotypes in multicellular organisms. However, the molecular basis ensuring high fidelity of maintenance DNA methylation is largely unknown. Here, we demonstrate that two distinct modes of DNMT1 recruitment, one is DNA replication-coupled and the other is uncoupled mechanism, regulate the stable inheritance of DNA methylation. PCNA-associated factor 15 (PAF15) represents a primary target of UHRF1 and undergoes dual mono-ubiquitylation (PAF15Ub2) on chromatin. PAF15Ub2 specifically interacts with DNMT1 and controls the recruitment of DNMT1 in a DNA replication-coupled manner. Thus, loss of PAF15Ub2 results in impaired DNA methylation at sites replicating during early S phase. In contrast, outside of S phase or when PAF15 ubiquitylation is perturbed, UHRF1 ubiquitylates histone H3 to promote DNMT1 recruitment. Together, we identify replication-coupled and uncoupled mechanisms of maintenance DNA methylation, both of which collaboratively ensure the stable DNA methylation.

2020-02-25 | PXD015282 | Pride

Activation of the ATR kinase by the RPA-binding protein ETAA1

Project description:The ATR kinase is a master regulator of cellular responses to DNA damage and replication stress that is activated by a complex mechanism involving its binding partner ATRIP, RPA-coated ssDNA, the 9-1-1 complex and TopBP1. Here, we discovered a new ATR activation mechanism in human cells, mediated by the uncharacterized protein ETAA1 (Ewing’s tumor-associated antigen 1). From a comprehensive proteomic survey of protein recruitment to DNA double-strand break-modified chromatin, we identified ETAA1 as a factor that accumulates at DNA damage sites via an RPA-binding motif, and which has an important role in promoting cell survival and preventing replication fork breakage after genotoxic insults. Mechanistically, we show that ETAA1 harbors a conserved domain that potently and directly stimulates ATR kinase activity independent of TopBP1 and 9-1-1, and which is essential for the function of ETAA1 in the DNA damage response. Consistently, ablation of ETAA1 shows profound synthetic lethality with TopBP1 knockdown, resulting from massive replication fork collapse and abrogation of ATR-dependent signaling. Finally, we show that ETAA1 levels in cancer cell lines inversely correlate with their dependency on TopBP1 for ATR activation, and that overexpression of ETAA1 partially restores ATR-dependent signaling after loss of TopBP1. Together, these findings establish a new mechanism of ATR activation in human cells that operates in parallel with, but independent of, the canonical TopBP1-mediated pathway.

2016-09-30 | PXD003529 | Pride

ChIP-chip from Drosophila egg chambers using MCM2-7 antibody

Project description:Chromatin immunoprecipitation of the MCM2-7 replicative helicase followed by hybridization to genome-wide tiling microarrays demonstrated that MCMs localize to the follicle cell amplicons at stages of replication initiation Comparison of MCM2-7 ChIP sample compared to input DNA for Drosophila egg chambers

2011-09-06 | E-GEOD-31889 | biostudies-arrayexpress

CRL2LRR1 is required for CMG unloading during vertebrate replication termination

Project description:A key event during eukaryotic replication termination is the removal of the CMG helicase from chromatin. CMG unloading involves ubiquitylation of its MCM7 subunit and the action of the p97 ATPase. Using a proteomic screen in Xenopus egg extracts, we identified factors that are retained on chromatin when CMG unloading is blocked and identified the E3 ubiquitin ligase CRL2LRR1 and several other potential regulators of termination including a specific p97 complex. We show that CRL2LRR1 recruitment coincides with MCM7 ubiquitylation and occurs only when replisomes converge. In the absence of CRL2LRR1, MCM7 is not ubiquitylated, CMG unloading is inhibited, and many replisome components including DNA pol are retained on DNA. Our data identify CRL2LRR1 as a master regulator of replisome disassembly during vertebrate DNA replication termination.

2017-02-02 | PXD004828 | Pride

Analysis of gene amplification at DAFC-34B

Project description:This SuperSeries is composed of the following subset Series: GSE31889: ChIP-chip from Drosophila egg chambers using MCM2-7 antibody GSE31890: ChIP-chip from Drosophila egg chambers using ORC2 antibody Refer to individual Series

2011-09-06 | E-GEOD-31891 | biostudies-arrayexpress

Immunoprecipitation of p97 from X.laevis S-phase chromatin

Project description:Complex cellular processes are driven by the regulated assembly and disassembly of large multi-protein complexes. In eukaryotic DNA replication, whilst we are beginning to understand the molecular mechanism for assembly of the replication machinery (replisome), we still know little about the regulation of its disassembly at replication termination. Over recent years, the first elements of this process emerged: the replicative helicase at the heart of the replisome is polyubiquitylated prior to unloading and that this unloading requires p97 segregase activity. Two different E3 ubiquitin ligases are now known to ubiquitylate the helicase under different conditions: Cul2Lrr1 and TRAIP. To understand how p97 is targeted to the terminated replisome, we immunoprecipitated p97 from chromatin replicating in Xenopus laevis egg extract. We have found a p97 cofactor, Ubxn7, which can interact with active Cul2Lrr1 and with p97, facilitating efficient recognition and processing of terminated helicases ubiquitylated by Cul2.

2022-10-14 | PXD029705 | Pride

SCAI orchestrates DNA interstrand crosslink repair downstream of FANCI-FANCD2 ubiquitylation

Project description:By covalently linking Watson and Crick strands, DNA interstrand crosslinks (ICLs) are highly toxic lesions that block DNA replication and threaten genome integrity. The Fanconi anemia (FA) pathway orchestrates ICL repair during DNA replication, with ubiquitylated FANCI-FANCD2 (ID2) marking the activation step that triggers incisions on one DNA strand to unhook the ICL. ICL unhooking generates a two-ended double-strand break (DSB) on one of the daughter molecules, while leaving a gap comprising the ICL-adduct on the other. Restoration of the adducted molecule by DNA polymerase zeta-mediated translesion synthesis (TLS) creates a template required for repair of the DSB via homologous recombination (HR), but how these processes are coordinated to ensure faithful ICL repair is not known. Here, we uncover a crucial role for SCAI in promoting ICL repair downstream of ID2 activation. Using Xenopus egg extracts and human cells, we show that SCAI forms a complex with DNA polymerase zeta and localizes to ICLs during DNA replication. SCAI-deficient cells are exquisitely sensitive to ICL-inducing drugs and display principal hallmarks of FA gene inactivation, including broken and radial chromosome accumulation. In the absence of SCAI, DSB intermediates are preferentially re-ligated by illegitimate DNA polymerase theta-dependent microhomology-mediated end-joining (MMEJ). Consistently, the hypersensitivity of SCAI-deficient cells to ICLs can be reverted by suppressing FA pathway activation. Our work establishes SCAI as a critical mediator of ICL resolution via the FA pathway, acting at the interphase of the TLS and HR steps to prevent erroneous repair and chromosomal instability.

2022-02-18 | PXD024280 | Pride

Mechanism and function of DNA replication-independent DNA-protein crosslink repair via the SUMO pathway

Project description:DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN or the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair pathways remain unknown. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in a DNA replication-independent manner. In addition, we provide evidence that a subset of DPCs can be SUMOylated and processed via transcription, independently of RNF4. SUMO modification of DPCs is mediated by PIAS4 and neither stimulates nor inhibits their rapid DNA replication-dependent proteolysis. Instead, SUMOylation provides a critical salvage mechanism to remove DPCs formed or persisting after replication, as we demonstrate that DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells can enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.

2021-08-06 | PXD021947 | Pride

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data