Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

The MCM helicase displays distinct roles controlling dNTP synthesis and preventing genetic instability through physical interactions with the RNR complex

ABSTRACT: The helicase MCM and the ribonucleotide reductase RNR are the complexes that provide the substrates (ssDNA templates and dNTPs, respectively) for DNA replication. Here, we demonstrate that MCM interacts physically with RNR and some of its regulators, including the kinases Rad53 and Dun1. Partial disruption of the MCM/RNR interactions increases the levels of dNTPs under unperturbed conditions, pointing to a role for MCM counteracting excessive RNR activity. In response to DNA damage, most of these physical interactions augment and this increase depends on Dun1. Partial disruption of the MCM/RNR interactions does not affect the pool of dNTPs under these conditions. However, this disruption impairs the release of Rad52 from the DNA repair centers despite the lesions are repaired, leading to hypermutagenesis and sensitivity to genotoxic agents. Therefore, the MCM/RNR interactions play non-canonical roles preventing by distinct mechanisms excessive dNTP synthesis and persistent Rad52 centers.

INSTRUMENT(S): Q Exactive

ORGANISM(S): Saccharomyces Cerevisiae (baker's Yeast)

SUBMITTER: Román González-Prieto

LAB HEAD: Román González Prieto

PROVIDER: PXD045914 | Pride | 2024-05-15

REPOSITORIES: Pride

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
	MaxQuant_1.5.3.30.zip	Other
	YEAST.fasta	Fasta
	mqpar.xml	Xml
	q104320a.raw	Raw
	q104321a.raw	Raw

Items per page:

1 - 5 of 19

Similar Datasets

PfDOZI protein-protein interaction in plasmodium falciparum

Project description:Here, we characterized an RNA binding protein (RBP), PfDOZI, which is an integral cytoplasmic component of RNP granule. We observed that deleting pfdozi led to an asexual growth advantage due to the escalated merozoite invasion capacity. Also, pfdozi disruption resulted in a significant impairment in gametocyte commitment and morphology development. Through high-throughput RNA-seq, we revealed that PfDOZI affected the transcriptome in both asexual and sexual stages when pfdozi was deleted. Mechanically, we confirmed that PfDOZI could interact with P body like mRNP in the schizont stage, while stress granule-like granule in gametocyte stage. The identification of PfDOZI protein complex in stressed schizont further indicate PfDOZI could shift its function from mRNA degradation to translation repression during stage conversion.

2024-04-16 | PXD020862 | Pride

Ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR/Cas9 genome editing

Project description:Gene disruption by CRISPR/Cas9 is highly efficient and relies on the error-prone non-homologous end-joining (NHEJ) pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than NHEJ in mammalian cells. Here, by testing whether manipulation of DNA repair factors would improve HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative 53BP1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem (iPS) cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of NHEJ-mediated DSB repair in the presence of these two factors is not suppressed, and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.

2017-11-23 | GSE103241 | GEO

MEF2A interactome in striated muscle by an affinity-purification followed by LC-MS-MS

Project description:Myocyte Enhancer Factor 2 (MEF2) proteins are involved in multiple developmental, physiological, and pathological processes in vertebrates. Protein:protein interactions underlie the plethora of biological processes impacted by MEF2A, necessitating a detailed characterization of the MEF2A interactome. A nanobody based affinity-purification/mass spectrometry strategy was employed to achieve this goal. Specifically, the MEF2A protein complexes were captured from myogenic lysates using a GFP-tagged MEF2A protein immobilized with a GBP-nanobody followed by LC-MS/MS proteomic analysis to identify MEF2A interactors.

2024-02-27 | PXD050042 | Pride

H3K4me3 ChIPseq in chicken liver

Project description:This dataset has been generated to identify promoter regions in the chicken genome to distinguish active and inactive genes. We focussed our analyses on actively transcribed tRNA and mRNAs genes. Chicken liver was cross-linked to capture histone-DNA interactions. Sequencing libraries were prepared from H3K4me3-precipitated DNA and input control.

2021-02-04 | E-MTAB-8106 | biostudies-arrayexpress

Pan-claudin family interactome analysis reveals shared and specific interactions

Project description:Interactome study of the pan-claudin family by combining two complementary pull-down techniques followed by mass spectrometry to create an interaction landscape of the claudin family. Co-immunoprecipitation (CoIP) of recombinant claudins expressed in MDCK-C7 cells provided information about interactions beyond the already known TJ proteins. A protein interaction screen on a peptide matrix (PRISMA) allowed mapping interactions along the disordered cytosolic C-terminal region of claudins and studying the effect of post-translational modifications (PTMs) on these interactions. The new interaction partners of the C-terminus of several claudins were confirmed by proximity ligation assays (PLA) and revealed their possible implications in localized biological processes in epithelial cells. By combining these approaches, we created an extended interaction map of the claudin protein family, expanding the current knowledge in the field.

2023-10-24 | PXD031094 | Pride

A discrete chromatin loop in the murine Tcra/Tcrd locus shapes the TCRδ and TCRα repertoires

Project description:The Tcra/Tcrd locus undergoes V(D)J recombination in CD4−CD8− double-negative (DN) thymocytes and CD4+CD8+ double-positive (DP) thymocytes to generate diverse TCRδ and TCRα repertoires, respectively. Here we reveal a Tcra/Tcrd locus chromatin interaction network in DN thymocytes that is formed by interactions between CTCF-binding elements (CBEs). Disruption of a discrete chromatin loop encompassing the Dδ, Jδ and Cδ gene segments allows a single Vδ segment to frequently contact and rearrange to Dδ and Jδ segments and dominate the adult TCRδ repertoire. Disruption of this loop also narrows the TCRα repertoire, which, we believe, follows as a consequence of the restricted TCRδ repertoire. Hence, a single CTCF-mediated chromatin loop directly regulates TCRδ diversity and indirectly regulates TCRα diversity. Examination of chromatin loops by 4C-seq from 4 viewpoints in two lymphoid cell compartments: CD4-CD8- thymocytes and naïve B splenocytes.

2015-08-10 | E-GEOD-67442 | biostudies-arrayexpress

Comprehensive characterization of Minichromosome Maintenance Complex (MCM) protein interactions using affinity and proximity purifications coupled to mass spectrometry.

Project description:This project is the complete interactome of the Minichromosome Maintenance (MCM) complex by using AP-MS and BioID approaches for all the different MCM proteins. Overall, our analysis identified unique and shared interaction partners and proteins enriched for distinct biological processes including DNA replication, DNA repair and cell cycle regulation. Furthermore, we mapped the changes in protein interactions of the MCM complex in response to DNA damage, identifying a new role for this complex in DNA repair. In summary, we demonstrate the complementarity of these approaches for the characterization of protein interactions within the MCM complex.

2016-08-09 | PXD004089 | Pride

Accumulation of sumoylated Rad52 in checkpoint mutants perturbed in DNA replication

Project description:Checkpoints are cellular surveillance and signaling pathways that regulate responses to DNA damage and perturbations of DNA replication. Here we show that high levels of sumoylated Rad52 are present in the mec1 sml1 and rad53 sml1 checkpoint mutants exposed to DNA damaging agents such as methyl methanesulfonate (MMS) or the DNA replication inhibitor hydroxyurea (HU). The kinase-defective mutant rad53-K227A also showed high levels of Rad52 sumoylation. Elevated levels of Rad52 sumoylation occur in checkpoint mutants proceeding S phase being exposed DNA-damaging agent. Interestingly, ChIP on chip analyses revealed non-canonical chromosomal localization of Rad52 in the HU-treated rad53-K227A cells arrested in early S phase: Rad52 localization at dormant and early DNA replication origins. However, such unusual localization was not dependent on the sumoylation of Rad52. In addition, we also found that Rad52 could be highly sumoylated in the absence of Rad51. Double deletion of RAD51 and RAD53 exhibited the similar levels of Rad52 sumoylation to RAD51 single deletion. The significance and regulation mechanism of Rad52 sumoylation by checkpoint pathways will be discussed. Keywords: ChIP-chip

2009-05-26 | GSE14761 | GEO

Cohesin and CTCF Differentially Affect the Chromatin Architecture and Gene Expression in Human Cells

Project description:Recent studies of genome-wide chromatin interactions have revealed that the human genome is partitioned into many self-associating topological domains. The boundary sequences are enriched for binding sites of CTCF and the cohesin complex, implicating these two factors in the establishment or maintenance of topological domains. To determine the role of cohesin and CTCF in higher order chromatin architecture in human cells, we proteolytically cleaved the cohesin complex from interphase chromatin and examined changes in chromosomal organization as well as transcriptome. We observed a general loss of local chromosomal interactions upon disruption of cohesin complex, but the topological domains remain intact. However, we found that depletion of CTCF by RNA interference in these cells not only reduced intra-domain interactions but also increased inter-domain interactions. Further more, distinct groups of genes become mis-regulated upon depletion of cohesin and CTCF. Taken together, these observations suggest that CTCF and cohesin contribute in different ways to chromatin organization and gene regulation. Hi-C and mRNA-seq experiments in Cohesin and CTCF depleted HEK293 cells

2013-12-16 | E-GEOD-44267 | biostudies-arrayexpress

Comprehensive characterization of Minichromosome Maintenance Complex (MCM) protein interactions using affinity and proximity purifications coupled to mass spectrometry.

| MSV000080198 | MassIVE