The role of PCNA ubiquitylation during unperturbed genome replication in S.pombe
Ontology highlight
ABSTRACT:
ORGANISM(S): Schizosaccharomyces pombe
PROVIDER: GSE70033 | GEO | 2017/05/12
SECONDARY ACCESSION(S): PRJNA287522
REPOSITORIES: GEO
Similar Datasets
Project description:To establish if PCNA ubiquitylation affects the DNA replication kinetics of specific loci, we compared rates of DNA replication across the genome between rad18+ and rad18-delta cells using the BrdU-IP sequencing method. We mapped BrdU-incorporation at 300-bp resolution and subsequent informatics analysis was performed to generate datasets of replication kinetics.
2017-05-12 | E-GEOD-70033 | biostudies-arrayexpress
Project description:Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway, and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial and most efforts aimed to identify BRCA1/BARD1 ubiquitination substrates rely on indirect evidence. Here, we observed that the BRCA1/BARD1 ubiquitin E3 activity was not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identified substrates for BRCA1/BARD1. We found that PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18. PCNA ubiquitination by BRCA1/BARD1 avoids the formation of ssDNA gaps during DNA replication and promotes continuous DNA synthesis. These results address the controversy about the function of BRCA1/BARD1 E3 activity in Homologous Recombination.
2024-05-07 | PXD050319 | Pride
Project description:Deficiencies in the BRCA1 tumor suppressor gene are the main cause of hereditary breast and ovarian cancer. BRCA1 is involved in the Homologous Recombination DNA repair pathway, and, together with BARD1, forms a heterodimer with ubiquitin E3 activity. The relevance of the BRCA1/BARD1 ubiquitin E3 activity for tumor suppression and DNA repair remains controversial and most efforts aimed to identify BRCA1/BARD1 ubiquitination substrates rely on indirect evidence. Here, we observed that the BRCA1/BARD1 ubiquitin E3 activity was not required for Homologous Recombination or resistance to Olaparib. Using TULIP2 methodology, which enables the direct identification of E3-specific ubiquitination substrates, we identified substrates for BRCA1/BARD1. PCNA is ubiquitinated by BRCA1/BARD1 in unperturbed conditions independently of RAD18, avoiding the formation of ssDNA gaps during DNA replication and promoting replication fork stability upon replication stress, solving the controversy about the function of BRCA1/BARD1 E3 activity in Homologous Recombination.
2024-05-07 | PXD039167 | Pride
Project description:Mechanistic model of the Post-Replication Repair (PRR), the pathway involved in the bypass
of DNA lesions induced by sunlight exposure and UV radiation. PRR acts through two different mechanisms,
activated by mono- and poly-ubiquitylation of the DNA sliding clamp, called Proliferating Cell Nuclear Antigen (PCNA).
This model has been defined according to the stochastic formulation of chemical kinetics [Gillespie DT, J Phys Chem 1977, 81(25):2340-2361],
which requires to specify the set of molecular species occurring in the pathway and their respective interactions,
formally described as a set of biochemical reactions.
The volume considered for this system is 1.666667e-17L; this value can be used to convert the model into the deterministic formulation.
2013-09-06 | BIOMD0000000475 | BioModels
Project description:CRISPR Cas9-based functional genomics screening is a powerful approach for identifying and characterizing novel oncology drug targets. Here, we elucidate the synthetic lethal mechanism of deubiquitinating enzyme USP1 in cancers with underlying DNA damage vulnerabilities, specifically BRCA1/2 mutant tumors and a subset of BRCA1/2 wild-type (WT) tumors. In sensitive cells, pharmacological inhibition of USP1 leads to decreased DNA synthesis concomitant with the induction of S-phase-specific DNA damage. Genome-wide CRISPR-Cas9 screens identified RAD18 and UBE2K, which promote PCNA mono- and polyubiquitination respectively, as downstream mediators of USP1 dependency. The accumulation of mono- and polyubiquitinated PCNA following USP1 inhibition was associated with a reduction in total PCNA protein levels. Ectopic expression of WT and ubiquitin-dead K164R PCNA reversed USP1 inhibitor sensitivity. Our results demonstrate, for the first time, that USP1 dependency hinges on the aberrant processing of mono- and polyubiquitinated PCNA. Moreover, this mechanism of USP1 dependency extends beyond BRCA1/2 mutant tumors to a novel subset of BRCA1/2 WT cancer enriched in ovarian and lung lineages. We further show PARP and USP1 inhibition are strongly synergistic in BRCA1/2 mutant cell lines and xenograft models. We postulate USP1 dependency unveils a previously uncharacterized vulnerability linked to post-translational modifications of PCNA. Taken together, USP1 inhibition may represent a unique therapeutic strategy for BRCA1/2 mutant tumors and a subset of BRCA1/2 WT tumors.
2022-09-27 | GSE214121 | GEO
Project description: Not available
| S-EPMC5440044 | biostudies-literature
Project description:The influence of mono-ubiquitylation of histone H2B (H2Bub) on transcription via nucleosome reassembly has been widely documented. Recently, it has also been shown that H2Bub promotes recovery from replication stress; however, the underling molecular mechanism remains unclear. Here, we show that H2B ubiquitylation coordinates activation of the intra-S replication checkpoint and chromatin re-assembly, in order to limit fork progression and DNA damage in the presence of replication stress. In particular, we show that the absence of H2Bub affects replication dynamics (enhanced fork progression and reduced origin firing), leading to γH2A accumulation and increased hydroxyurea sensitivity. Further genetic analysis indicates a role for H2Bub in transducing Rad53 phosphorylation. Concomitantly, we found that a change in replication dynamics is not due to a change in dNTP level, but is mediated by reduced Rad53 activation and destabilization of the RecQ helicase Sgs1 at the fork. Furthermore, we demonstrate that H2Bub facilitates the dissociation of the histone chaperone Asf1 from Rad53, and nucleosome reassembly behind the fork is compromised in cells lacking H2Bub. Taken together, these results indicate that the regulation of H2B ubiquitylation is a key event in the maintenance of genome stability, through coordination of intra-S checkpoint activation, chromatin assembly and replication fork progression. S.cerevisiae oligonucleotide microarrays were provided by Affymetrix (S.cerevisiae Tiling 1.0R, P/N 900645). BrdU and proteins ChIP-chip analyses were carried out as described (Fachinetti et al., M Cell, 2010).
2014-09-12 | E-GEOD-61030 | biostudies-arrayexpress
Project description:Microarray analyses were performed to compare the gene expression profiles of wild-type and several mutant strains of the ciliated protozoan Tetrahymena thermophila. Elimination of H3K4 methylation (hht2-K4Q) and knockout of either of the ubiquitylation enzymes (delta-UBC2 and delta-BRE1) affects a broader spectrum of genes than elimination of H2B ubiquitylation (htb1-K115R).
2009-10-27 | GSE18743 | GEO
Project description:Microarray analyses were performed to compare the gene expression profiles of wild-type and several mutant strains of the ciliated protozoan Tetrahymena thermophila. Elimination of H3K4 methylation (hht2-K4Q) and knockout of either of the ubiquitylation enzymes (delta-UBC2 and delta-BRE1) affects a broader spectrum of genes than elimination of H2B ubiquitylation (htb1-K115R). Cells at mid-logarithmic growing phase (cell density of 200,000 cells/ml) were collected. Then total RNAs were extracted and hybridized.
2010-06-20 | E-GEOD-18743 | biostudies-arrayexpress
Project description:Targeting transcription replication conflicts, a major source of endogenous DNA double stranded breaks and genomic instability, could have important therapeutic implications. When transcription and DNA replication machineries encounter each other on chromosomes, one way to resolve the conflict is temporarily dislodging RNA polymerase II from the conflict sites to enable the replication fork to proceed. Proliferating cell nuclear antigen (PCNA), an essential component of replisomes, plays a critical role in this process. We discovered a small molecule ligand (AOH1996) of PCNA, which targets a binding pocket adjacent to the outer surface region of PCNA known to interact with PIP box and APIM motif proteins. Orally administrable, AOH1996 suppresses tumor growth but causes no discernable side effects. In addition, we found that AOH1996 treatment can stabilize interaction between PCNA and RPB1, the largest subunit of RNA polymerase II. To determine whether the effect of AOH1996 is mediated through affecting PCNA interaction with RPB1, we mutated Y418 within the APIM motif of RPB1 to an alanine in the SK-N-AS cell line by CRISPR and compared changes in protein expression profiles caused by AOH1996 in the parent and mutant SK-N-AS cells.
2023-08-03 | PXD027782 | Pride