Atypical mitotic DNA synthesis sequencing (MiDAS-seq) in U2OS cells depleted for RAD51
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA804143 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR17914843.fastq.gz | Fastqsanger.gz |
Items per page: 5 1 - 1 of 1 |
Similar Datasets
Project description:DNA replication stress is an established driver of cancer-associated chromosomal rearrangements. Replication stress perturbs the duplication of late-replicating loci and activates a mitotic DNA repair pathway (termed MiDAS) for completion of replication. We here investigated RAD51-independent MiDAS.
2022-06-27 | GSE196278 | GEO
Project description:The nascent transcriptome during G1, Early-s, Late-S, and G2/M cell cycle phases and atypical MiDAS-seq in RAD51 knockdown U2OS cells
| PRJNA804140 | ENA
Project description:Multiple replication abnormalities cause cells lacking BRCA2 to enter mitosis with under-replicated DNA and to activate mitotic DNA synthesis (MiDAS). However, the precise position of these MiDAS sites, as well as their origin, remains unknown. Here we labelled mitotic nascent DNA and performed high-throughput sequencing to identify at high-resolution the sites where MiDAS occurs in the absence of BRCA2. This approach revealed 150 genomic loci affected by MiDAS, which map within regions replicating during early S-phase and are therefore distinct from the aphidicolin-induced common fragile sites. Moreover, these sites largely localise near early firing origins and within genes transcribed in early S, suggesting that they stem from transcription-replication conflicts (TCRs). Inhibiting transcription with 5,6-dichloro-1-β-D-ribofuranosylbenzimidazole (DRB) during early S-phase abrogates MiDAS. Strikingly, MiDAS sites co-localise with genomic loci where R-loops form in unchallenged conditions, suggesting that R-loop accumulation caused by BRCA2 inactivation leads to DNA lesion which are repaired by MiDAS. RAD52 is required in this process, as its abrogation in BRCA2-deficient cells reduces the rate of MiDAS and causes DNA damage accumulation in G1. Furthermore, MiDAS sites triggered by BRCA2 inactivation are hotspots for genomic rearrangement in BRCA2-mutated breast tumours. These results indicate that BRCA2 acts in early S-phase to protect TRC- and R-loop-induced DNA lesions, thereby preventing them from becoming a source of genomic instability and tumorigenesis.
2022-08-01 | GSE196163 | GEO
Project description:Deregulation of origin firing and licensing, shortage of deoxyribonucleotides in the cell and interference between transcription and replication are among the causes of DNA replication stress in cancer cells. This leads in various ways (notably, through common fragile sites expression and uncomplete replication of late-replicating regions of the genome during S phase) to the necessity of finishing DNA replication in mitosis by a mechanisms called Mitotic DNA synthesis (MiDAS), which is related to Break-Induced Replication (BIR). Even if it is of primary importance for cancer cells, the molecular mechanism of MiDAS, and generally of BIR, is not yet well understood. Recently, the third subunit of the eukaryotic DNA polymerase delta (POLD3) has been recognized as a key player of BIR, though its role is not yet clear. In this work, using a protocol established in our group to map at high resolution newly replicated DNA at MiDAS sites, we provide new insights into the molecular role of POLD3 in this repair mechanism. In particular, by analyzing MiDAS in mutant HeLa clones lacking the PCNA-interacting domain of POLD3, we demonstrate that the interaction between POLD3 and PCNA is required for coordinating leading and lagging strand synthesis in MiDAS. This work represents an important step forward towards the comprehension of the molecular mechansisms of Mitotic DNA synthesis, the full understanding of which is of primary importance for the possible development of novel cancer therapies targeting BIR-related pathways.
2025-10-01 | GSE244399 | GEO
Project description:DNA replication stress promotes cancer development and is associated with DNA synthesis beyond S-phase. Replication stress-induced DNA lesions and under-replicated DNA can persist into G2 and mitosis, where they undergo mitotic DNA synthesis (MiDAS). After cell division, inherited DNA lesions are protected by genome caretaker proteins and show signs of post-mitotic DNA synthesis (post-MiDAS) in G1. The genomic regions that undergo post-MiDAS, the cellular factors involved, and the implications for genome integrity remain poorly understood. Here, we provide evidence for post-MiDAS activity at centromeres and telomeres in cancer cells employing alternative lengthening of telomeres (ALT) and present data suggesting that a subset of these fragile genomic regions can cluster during post-MiDAS in G1. We further show that post-MiDAS is promoted by local SUMOylation and BLM recruitment into ALT-associated PML bodies (APBs) and involves DNA damage tolerance factors and proteins associated with ALT and MiDAS. Consistent with G1-specific regulation, complementary quantitative proteomics and targeted siRNA screening reveal that APC/C, together with its G1 co-activator CDH1 but independent of its mitotic co-activator CDC20, restrains excessive post-MiDAS in G1. Finally, we show that CDH1 depletion selectively impairs the survival of ALT-positive cancer cell lines, indicating that post-MiDAS might represent a potential ALT-specific vulnerability. This dataset contains TurboID-RPA32 proximity labeling mass spectrometry data generated from FACS-sorted G1-phase U-2 OS FUCCI TurboID-RPA32-HA cells to identify proteins in proximity to RPA32.
2026-08-26 | PXD069839 | Pride
Project description:DNA replication stress promotes cancer development and is associated with DNA synthesis beyond S-phase. Replication stress-induced DNA lesions and under-replicated DNA can persist into G2 and mitosis, where they undergo mitotic DNA synthesis (MiDAS). After cell division, inherited DNA lesions are protected by genome caretaker proteins and show signs of post-mitotic DNA synthesis (post-MiDAS) in G1. The genomic regions that undergo post-MiDAS, the cellular factors involved, and the implications for genome integrity remain poorly understood. Here, we provide evidence for post-MiDAS activity at centromeres and telomeres in cancer cells employing alternative lengthening of telomeres (ALT) and present data suggesting that a subset of these fragile genomic regions can cluster during post-MiDAS in G1. We further show that post-MiDAS is promoted by local SUMOylation and BLM recruitment into ALT-associated PML bodies (APBs) and involves DNA damage tolerance factors and proteins associated with ALT and MiDAS. Consistent with G1-specific regulation, complementary quantitative proteomics and targeted siRNA screening reveal that APC/C, together with its G1 co-activator CDH1 but independent of its mitotic co-activator CDC20, restrains excessive post-MiDAS in G1. Finally, we show that CDH1 depletion selectively impairs the survival of ALT-positive cancer cell lines, indicating that post-MiDAS might represent a potential ALT-specific vulnerability. This dataset contains RPA70 immunoprecipitation mass spectrometry data generated from chromatin-bound protein extracts of FACS-sorted G1-phase U-2 OS FUCCI cells to identify the interactome of RPA70 in G1.
2026-08-26 | PXD069592 | Pride
Project description:DNA replication stress promotes cancer development and is associated with DNA synthesis beyond S-phase. Replication stress-induced DNA lesions and under-replicated DNA can persist into G2 and mitosis, where they undergo mitotic DNA synthesis (MiDAS). After cell division, inherited DNA lesions are protected by genome caretaker proteins and show signs of post-mitotic DNA synthesis (post-MiDAS) in G1. The genomic regions that undergo post-MiDAS, the cellular factors involved, and the implications for genome integrity remain poorly understood. Here, we provide evidence for post-MiDAS activity at centromeres and telomeres in cancer cells employing alternative lengthening of telomeres (ALT) and present data suggesting that a subset of these fragile genomic regions can cluster during post-MiDAS in G1. We further show that post-MiDAS is promoted by local SUMOylation and BLM recruitment into ALT-associated PML bodies (APBs) and involves DNA damage tolerance factors and proteins associated with ALT and MiDAS. Consistent with G1-specific regulation, complementary quantitative proteomics and targeted siRNA screening reveal that APC/C, together with its G1 co-activator CDH1 but independent of its mitotic co-activator CDC20, restrains excessive post-MiDAS in G1. Finally, we show that CDH1 depletion selectively impairs the survival of ALT-positive cancer cell lines, indicating that post-MiDAS might represent a potential ALT-specific vulnerability. This dataset contains RPA70 immunoprecipitation mass spectrometry data generated from whole-cell protein extracts of FACS-sorted G1-phase U-2 OS FUCCI cells to identify the interactome of RPA70 in G1.
2026-08-26 | PXD069739 | Pride
Project description:Human U2OS cells were stimulated with selective agonist SDF-1 of atypical chemokine receptor 3 (ACKR3) for 24 hours. Extracellular vesicles were isolated from U2OS cell culture media. Total RNAs were purified from extracellular vesicle samples. We used TaqMan Low Density Arrays to quantitate miRNA expression in extracellular vesicles secreted by U2OS cells after GPCR activation.
2025-07-30 | GSE270077 | GEO
Project description:DNA double-strand breaks (DSBs) accumulate in mitosis in response to DNA replication stress or deficiency in breast cancer susceptibility type 1 and 2 (BRCA1/2). This is a critical threat to genome stability as the classical DSB repair pathways non-homologous end joining (NHEJ) and homologous recombination (HR) are repressed(1-7). Instead, mitotic repair relies on the processes of microhomology-mediated end joining (MMEJ) or mitotic DNA synthesis (MiDAS), but how these pathways are coordinated and regulated remains unclear. Here, we reveal that the CIP2A-TOPBP1 axis orchestrates DSB repair in mitosis by promoting CDK1-driven phosphorylation of SLX4 at Thr1260, recruiting the SMX complex to promote MiDAS. In addition, the CIP2A-TOPBP1 axis regulates mitotic Polθ recruitment and MMEJ. This dual mechanism ensures coordinated DSBs repair by integrating spatial control of TOPBP1-CIP2A chromatin recruitment with the temporal regulation provided by mitotic kinases, thereby preserving genome stability. These findings provide new insights into DSB repair dynamics in mitosis and offer a mechanistic rationale for the synthetic lethality of CIP2A in BRCA1/2-deficient tumors through the simultaneous impairment of break induced replication (BIR)-like MiDAS and MMEJ, which are genetically redundant pathways to HR.
2025-09-19 | PXD068371 | Pride