A unified mechanism for PARP inhibitor-induced PARP1 chromatin retention at DNA damage sites in living cells

Cell Reports, Journal Year: 2024, Volume and Issue: 43(5), P. 114234 - 114234

Published: May 1, 2024

Poly(ADP-ribose) polymerase (PARP) inhibitors (PARPis) not only suppress PARP1 catalytic activity but also prolong its association to damaged chromatin. Here, through live-cell imaging, we quantify the alterations in dynamics and elicited by seven PARPis over a wide range of concentrations deliver unified mechanism PARPi-induced chromatin retention. We find that gross retention at DNA damage sites is jointly governed inhibition allosteric trapping, albeit strictly independent manner—catalytic causes multiple unproductive binding-dissociation cycles PARP1, while trapping prolongs lesion-bound state greatly increase overall Importantly, stronger produces greater temporal shifts downstream repair events superior cytotoxicity, highlighting retention, complex precisely quantifiable characteristic PARPis, as valuable biomarker for PARPi efficacy. Our approach can be promptly repurposed interrogating properties DNA-repair-targeting compounds beyond PARPis.

Language: Английский

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VCP/p97-associated proteins are binders and debranching enzymes of K48–K63-branched ubiquitin chains

Nature Structural & Molecular Biology, Journal Year: 2024, Volume and Issue: unknown

Published: July 8, 2024

Abstract Branched ubiquitin (Ub) chains constitute a sizable fraction of Ub polymers in human cells. Despite their abundance, our understanding branched function cell signaling has been stunted by the absence accessible methods and tools. Here we identify cellular branched-chain-specific binding proteins devise approaches to probe K48–K63-branched function. We establish method monitor cleavage linkages within complex unveil ATXN3 MINDY as debranching enzymes. engineer K48–K63 branch-specific nanobody reveal molecular basis its specificity crystal structures nanobody-branched chain complexes. Using this nanobody, detect increased K48–K63-Ub branching following valosin-containing protein (VCP)/p97 inhibition after DNA damage. Together with discovery that multiple VCP/p97-associated bind or debranch K48–K63-linked Ub, these results suggest for VCP/p97-related processes.

Language: Английский

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In and out of Replication Stress: PCNA/RPA1-Based Dynamics of Fork Stalling and Restart in the Same Cell

International Journal of Molecular Sciences, Journal Year: 2025, Volume and Issue: 26(2), P. 667 - 667

Published: Jan. 14, 2025

Replication forks encounter various impediments, which induce fork stalling and threaten genome stability, yet the precise dynamics of restart at single-cell level remain elusive. Herein, we devise a live-cell microscopy-based approach to follow hydroxyurea-induced subsequent 30 s resolution. We measure two distinct processes during stalling. One is rapid PCNA removal, reflects drop in DNA synthesis. The other gradual RPA1 accumulation up 2400 nt ssDNA per despite an active intra-S checkpoint. Restoring nucleotide pool enables prompt without post-replicative smooth cell cycle progression. ATR, but not ATM inhibition, accelerates nine-fold, leading exhaustion within 20 min. Fork under ATR inhibition led persistence ~600 after S-phase, reached 2500 ATR/ATM co-inhibition, with both scenarios mitotic catastrophe. MRE11 had no effect on PCNA/RPA1 regardless activity. E3 ligase RAD18 was recruited stalled replication parallel removal. Our results shed light depletion provide valuable tool for interrogating effects stress-inducing anti-cancer agents.

Language: Английский

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Insights into the SARS-CoV-2 ORF6 Mechanism of Action

International Journal of Molecular Sciences, Journal Year: 2023, Volume and Issue: 24(14), P. 11589 - 11589

Published: July 18, 2023

ORF6 is responsible for suppressing the immune response of cells infected by SARS-CoV-2 virus. It also most toxic protein SARS-CoV-2, and its actions are associated with viral pathogenicity. Here, we study in silico vitro structure protein, interaction RAE1 mechanism action behind high toxicity. We show both computationally experimentally that ORF6, embedded cytoplasmic membranes, binds to sequesters it cytoplasm, thus depleting availability nucleus impairing nucleocytoplasmic mRNA transport. This negatively affects cellular genome stability compromising cell cycle progression into S-phase promoting accumulation RNA–DNA hybrids. Understanding multiple ways which DNA replication may have important implications elucidating pathogenicity developing therapeutic strategies mitigate deleterious effects on host cells.

Language: Английский

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Overexpression of the NMig1 Gene Encoding a NudC Domain Protein Enhances Root Growth and Abiotic Stress Tolerance in Arabidopsis thaliana

Frontiers in Plant Science, Journal Year: 2020, Volume and Issue: 11

Published: June 11, 2020

The family of NudC proteins has representatives in all eukaryotes and plays essential evolutionarily conserved roles many aspects organismal development stress response, including nuclear migration, cell division, folding stabilisation other proteins. This study investigates an undescribed Arabidopsis homolog the Aspergillus nidulans gene, named NMig1 (for Nuclear Migration 1), which shares high sequence similarity to plant mammalian NudC-like genes. Expression was highly upregulated response several abiotic factors, such as heat shock, drought salinity. Constitutive overexpression led enhanced root growth lateral under optimal conditions. Exposure resulted relatively weaker inhibition length branching NMig1-overexpressing plants, compared wild-type Col-0. expression level antioxidant enzyme-encoding genes stress-associated considerably induced transgenic plants. increased major enzymes greater potential correlated well with lower levels reactive oxygen species (ROS) lipid peroxidation. In addition, associated strong upregulation encoding shock Therefore, our data demonstrate that could be considered a potentially important target gene for further use, breeding more resilient crops improved architecture stress.

Language: Английский

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Diffusion of activated ATM explains γH2AX and MDC1 spread beyond the DNA damage site

iScience, Journal Year: 2024, Volume and Issue: 27(9), P. 110826 - 110826

Published: Aug. 26, 2024

During DNA repair, ATM-induced H2AX histone phosphorylation and MDC1 recruitment spread megabases beyond the damage site. While loop extrusion has been suggested to drive this spread, underlying mechanism remains unclear. Herein, we provide two lines of evidence that is not only driver damage-induced γH2AX spread. First, cohesin loader NIPBL subunit RAD21 accumulate considerably later than at micro-IR-induced damage. Second, auxin-induced depletion does affect γH2AX/MDC1 following micro-irradiation or DSB induction by zeocin. To determine if diffusion activated ATM could account for observed behavior, measured exchange rate constants within damaged unperturbed chromatin. Using these measurements, introduced a quantitative model in which freely diffusing phosphorylates H2AX. This faithfully describes dynamics subsequent complex lesions.

Language: Английский

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DNArepairK: An Interactive Database for Exploring the Impact of Anticancer Drugs onto the Dynamics of DNA Repair Proteins

Biomedicines, Journal Year: 2021, Volume and Issue: 9(9), P. 1238 - 1238

Published: Sept. 16, 2021

Cells are constantly exposed to numerous mutagens that produce diverse types of DNA lesions. Eukaryotic cells have evolved an impressive array repair mechanisms able detect and these lesions, thus preventing genomic instability. The process is subjected precise spatiotemporal coordination, proteins recruited lesions in orderly fashion, depending on their function. Here, we present DNArepairK, a unique open-access database contains the kinetics recruitment removal 70 fluorescently tagged complex damage sites living HeLa Kyoto cells. An interactive graphical representation data complemented with live cell imaging movies facilitates straightforward comparisons between dynamics contributing different pathways. Notably, most included DNArepairK represented by both nontreated PARP1/2 inhibitor-treated (talazoparib) cells, thereby providing unprecedented overview effects anticancer drugs regular response. We believe exclusive dataset available will be value scientists exploring response but, also, inform guide development evaluation novel repair-targeting drugs.

Language: Английский

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Interferon-γ as a Potential Inhibitor of SARS-CoV-2 ORF6 Accessory Protein

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(4), P. 2155 - 2155

Published: Feb. 10, 2024

The ORF6 protein of the SARS-CoV-2 virus plays a crucial role in blocking innate immune response infected cells by inhibiting interferon pathways. Additionally, it binds to and immobilises RAE1 on cytoplasmic membranes, thereby mRNA transport from nucleus cytoplasm. In all these cases, host cell proteins are tethered flexible C-terminus ORF6. A possible strategy inhibit biological activity is bind its with suitable ligands. Our silico experiments suggest that hIFNγ high affinity, thus impairing interactions and, consequently, viral invasion. vitro studies reported here reveal shift localisation overexpressing upon treatment predominantly mainly nuclear, resulting restoration export nucleus. We also explored expression GFP transfected-with-ORF6 means fluorescence microscopy qRT-PCR, finding unblocks trafficking reinstates level. ability cytokine block reflected minimising negative effects DNA replication reducing accumulated RNA-DNA hybrids. results, therefore, as promising inhibitor most toxic protein.

Language: Английский

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Dynamics of Replication-Associated Protein Levels through the Cell Cycle

International Journal of Molecular Sciences, Journal Year: 2024, Volume and Issue: 25(15), P. 8230 - 8230

Published: July 28, 2024

The measurement of dynamic changes in protein level and localization throughout the cell cycle is major relevance to studies cellular processes tightly coordinated with cycle, such as replication, transcription, DNA repair, checkpoint control. Currently available methods include biochemical assays cells bulk following synchronization, which determine levels poor temporal no spatial resolution. Taking advantage genetic engineering live-cell microscopy, we performed time-lapse imaging expressing fluorescently tagged proteins under control their endogenous regulatory elements order follow cycle. We effectively discern between phases S subphases based on fluorescence intensity distribution co-expressed proliferating nuclear antigen (PCNA)-mCherry. This allowed us precisely compare multiple replication-associated factors, including Rap1-interacting factor 1 (RIF1), minichromosome maintenance complex component 6 (MCM6), origin recognition subunit (ORC1, Claspin, high spatiotemporal resolution HeLa Kyoto cells. Combining these data mass spectrometry-based measurements concentrations reveals concentration Our approach provides a practical basis for detailed interrogation dynamics context

Language: Английский

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The Effect of Dia2 Protein Deficiency on the Cell Cycle, Cell Size, and Recruitment of Ctf4 Protein in Saccharomyces cerevisiae

Molecules, Journal Year: 2021, Volume and Issue: 27(1), P. 97 - 97

Published: Dec. 24, 2021

Cells have evolved elaborate mechanisms to regulate DNA replication machinery and cell cycles in response damage stress order prevent genomic instability cancer. The E3 ubiquitin ligase SCF

Language: Английский

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