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Nature Communications Oct 2022The pathways involved in suppressing DNA replication stress and the associated DNA damage are critical to maintaining genome integrity. The Mre11 complex is unique among...
The pathways involved in suppressing DNA replication stress and the associated DNA damage are critical to maintaining genome integrity. The Mre11 complex is unique among double strand break (DSB) repair proteins for its association with the DNA replication fork. Here we show that Mre11 complex inactivation causes DNA replication stress and changes in the abundance of proteins associated with nascent DNA. One of the most highly enriched proteins at the DNA replication fork upon Mre11 complex inactivation was the ubiquitin like protein ISG15. Mre11 complex deficiency and drug induced replication stress both led to the accumulation of cytoplasmic DNA and the subsequent activation of innate immune signaling via cGAS-STING-Tbk1. This led to ISG15 induction and protein ISGylation, including constituents of the replication fork. ISG15 plays a direct role in preventing replication stress. Deletion of ISG15 was associated with replication fork stalling, tonic ATR activation, genomic aberrations, and sensitivity to aphidicolin. These data reveal a previously unrecognized role for ISG15 in mitigating DNA replication stress and promoting genomic stability.
Topics: Aphidicolin; DNA; DNA Damage; DNA Repair; DNA Replication; Nucleotidyltransferases; Ubiquitins
PubMed: 36216822
DOI: 10.1038/s41467-022-33535-y -
Communications Biology Sep 2022Topoisomerase I (TOP1) controls the topological state of DNA during DNA replication, and its dysfunction due to treatment with an inhibitor, such as camptothecin (CPT),...
Topoisomerase I (TOP1) controls the topological state of DNA during DNA replication, and its dysfunction due to treatment with an inhibitor, such as camptothecin (CPT), causes replication arrest and cell death. Although CPT has excellent cytotoxicity, it has the disadvantage of instability under physiological conditions. Therefore, new types of TOP1 inhibitor have attracted particular attention. Here, we characterised the effect of a non-camptothecin inhibitor, Genz-644282 (Genz). First, we found that treatment with Genz showed cytotoxicity by introducing double-strand breaks (DSBs), which was suppressed by co-treatment with aphidicolin. Genz-induced DSB formation required the functions of TOP1. Next, we explored the advantages of Genz over CPT and found it was effective against CPT-resistant TOP1 carrying either N722S or N722A mutation. The effect of Genz was also confirmed at the cellular level using a CPT-resistant cell line carrying N722S mutation in the TOP1 gene. Moreover, we found arginine residue 364 plays a crucial role for the binding of Genz. Because tyrosine residue 723 is the active centre for DNA cleavage and re-ligation by TOP1, asparagine residue 722 plays crucial roles in the accessibility of the drug. Here, we discuss the mechanism of action of Genz on TOP1 inhibition.
Topics: Aphidicolin; Arginine; Asparagine; Camptothecin; DNA; DNA Topoisomerases, Type I; Naphthyridines; Tyrosine
PubMed: 36114357
DOI: 10.1038/s42003-022-03920-w -
Toxins Aug 2022Studies on microorganism response spaceflight date back to 1960. However, nothing conclusive is known concerning the effects of spaceflight on virulence and...
Studies on microorganism response spaceflight date back to 1960. However, nothing conclusive is known concerning the effects of spaceflight on virulence and environmental tolerance of entomopathogenic fungi; thus, this area of research remains open to further exploration. In this study, the entomopathogenic fungus (strain SB010) was exposed to spaceflight (ChangZheng 5 space shuttle during 5 May 2020 to 8 May 2020) as a part of the Key Research and Development Program of Guangdong Province, China, in collaboration with the China Space Program. The study revealed significant differences between the secondary metabolite profiles of the wild isolate (SB010) and the spaceflight-exposed isolate (BHT021, BH030, BHT098) of . Some of the secondary metabolites/toxins, including enniatin A2, brevianamide F, macrosporin, aphidicolin, and diacetoxyscirpenol, were only produced by the spaceflight-exposed isolate (BHT021, BHT030). The study revealed increased insecticidal activities for of crude protein extracts of spaceflight mutants (BHT021 and BH030, respectively) against 5 days post application when compared crude protein extracts of the wild isolate (SB010). The data obtained support the idea of using space mutation as a tool for development/screening of fungal strains producing higher quantities of secondary metabolites, ultimately leading to increased toxicity/virulence against the target insect host.
Topics: Animals; Beauveria; China; Insecta; Space Flight; Virulence
PubMed: 36006216
DOI: 10.3390/toxins14080555 -
Molecular Cell Sep 2022Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS)....
Aberrant replication causes cells lacking BRCA2 to enter mitosis with under-replicated DNA, which activates a repair mechanism known as mitotic DNA synthesis (MiDAS). Here, we identify genome-wide the sites where MiDAS reactions occur when BRCA2 is abrogated. High-resolution profiling revealed that these sites are different from MiDAS at aphidicolin-induced common fragile sites in that they map to genomic regions replicating in the early S-phase, which are close to early-firing replication origins, are highly transcribed, and display R-loop-forming potential. Both transcription inhibition in early S-phase and RNaseH1 overexpression reduced MiDAS in BRCA2-deficient cells, indicating that transcription-replication conflicts (TRCs) and R-loops are the source of MiDAS. Importantly, the MiDAS sites identified in BRCA2-deficient cells also represent hotspots for genomic rearrangements in BRCA2-mutated breast tumors. Thus, our work provides a mechanism for how tumor-predisposing BRCA2 inactivation links transcription-induced DNA damage with mitotic DNA repair to fuel the genomic instability characteristic of cancer cells.
Topics: Aphidicolin; BRCA2 Protein; Chromosome Fragile Sites; DNA; DNA Damage; DNA Replication; Genomic Instability; Humans; Mitosis
PubMed: 36002001
DOI: 10.1016/j.molcel.2022.07.011 -
Scientific Reports Aug 2022Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal...
Recent studies revealed classes of recurrent DNA double-strand breaks (DSBs) in neural stem/progenitor cells, including transcription-associated, promoter-proximal breaks and recurrent DSB clusters in late-replicating, long neural genes that may give rise to somatic brain mosaicism. The mechanistic factors promoting these different classes of DSBs in neural stem/progenitor cells are not understood. Here, we elucidated the genome-wide landscape of RNA:DNA hybrid structures called "R-loops" in primary neural stem/progenitor cells undergoing aphidicolin-induced, mild replication stress to assess the potential contribution of R-loops to the different, recurrent classes of DNA break "hotspots". We find that R-loops in neural stem/progenitor cells undergoing mild replication stress are present primarily in early-replicating, transcribed regions and in genes with promoter GC skew that are associated with cell lineage-specific processes. Surprisingly, most long, neural genes that form recurrent DSB clusters do not show R-loop formation under conditions of mild replication stress. Our findings are consistent with a role of R-loop-associated processes in promoter-proximal DNA break formation in highly transcribed, early replicating regions but suggest that R-loops do not drive replication stress-induced, recurrent DSB cluster formation in most long, neural genes.
Topics: DNA; DNA Breaks, Double-Stranded; DNA Repair; Neural Stem Cells; R-Loop Structures
PubMed: 35927309
DOI: 10.1038/s41598-022-17452-0 -
Cell Reports Apr 2022Mitotic DNA synthesis (MiDAS) has been proposed to restart DNA synthesis during mitosis because of replication fork stalling in late interphase caused by mild...
Mitotic DNA synthesis (MiDAS) has been proposed to restart DNA synthesis during mitosis because of replication fork stalling in late interphase caused by mild replication stress (RS). Contrary to this proposal, we find that cells exposed to mild RS in fact maintain continued DNA replication throughout G2 and during G2-M transition in RAD51- and RAD52-dependent manners. Persistent DNA synthesis is necessary to resolve replication intermediates accumulated in G2 and disengage an ATR-imposed block to mitotic entry. Because of its continual nature, DNA synthesis at very late replication sites can overlap with chromosome condensation, generating the phenomenon of mitotic DNA synthesis. Unexpectedly, we find that the commonly used CDK1 inhibitor RO3306 interferes with replication to preclude detection of G2 DNA synthesis, leading to the impression of a mitosis-driven response. Our study reveals the importance of persistent DNA replication and checkpoint control to lessen the risk for severe genome under-replication under mild RS.
Topics: DNA; DNA Replication; Mitosis
PubMed: 35443178
DOI: 10.1016/j.celrep.2022.110701 -
Frontiers in Toxicology 2021Studies in rodent models have been the accepted approach by regulatory agencies to evaluate potential developmental neurotoxicity (DNT) of chemicals for decades. These...
Studies in rodent models have been the accepted approach by regulatory agencies to evaluate potential developmental neurotoxicity (DNT) of chemicals for decades. These studies, however, are inefficient and cannot meet the demand for the thousands of chemicals that need to be assessed for DNT hazard. As such, several new approach methods (NAMs) have been developed to circumvent limitations of these traditional studies. The DNT NAMs, some of which utilize human-derived cell models, are intended to be employed in a testing battery approach, each focused on a specific neurodevelopmental process. The need for multiple assays, however, to evaluate each process can prolong testing and prioritization of chemicals for more in depth assessments. Therefore, a multi-endpoint higher-throughput approach to assess DNT hazard potential would be of value. Accordingly, we have adapted a high-throughput phenotypic profiling (HTPP) approach for use with human-derived neural progenitor (hNP1) cells. HTPP is a fluorescence-based assay that quantitatively measures alterations in cellular morphology. This approach, however, required optimization of several laboratory procedures prior to chemical screening. First, we had to determine an appropriate cell plating density in 384-well plates. We then had to identify the minimum laminin concentration required for optimal cell growth and attachment. And finally, we had to evaluate whether addition of antibiotics to the culture medium would alter cellular morphology. We selected 6,000 cells/well as an appropriate plating density, 20 µg/ml laminin for optimal cell growth and attachment, and antibiotic addition in the culture medium. After optimizing hNP1 cell culture conditions for HTPP, it was then necessary to select appropriate in-plate assay controls from a reference chemical set. These reference chemicals were previously demonstrated to elicit unique phenotypic profiles in various other cell types. Aphidicolin, bafilomycin A1, berberine chloride, and cucurbitacin I induced robust phenotypic profiles as compared to dimethyl sulfoxide vehicle control in the hNP1 cells, and thus can be employed as in-plate assay controls for subsequent chemical screens. We have optimized HTPP for hNP1 cells, and consequently this approach can now be assessed as a potential NAM for DNT hazard evaluation and results compared to previously developed DNT assays.
PubMed: 35295155
DOI: 10.3389/ftox.2021.803987 -
International Journal of Molecular... Feb 2022Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA...
Mosaicism is the most important limitation for one-step gene editing in embryos by CRISPR/Cas9 because cuts and repairs sometimes take place after the first DNA replication of the zygote. To try to minimize the risk of mosaicism, in this study a reversible DNA replication inhibitor was used after the release of CRISPR/Cas9 in the cell. There is no previous information on the use of aphidicolin in porcine embryos, so the reversible inhibition of DNA replication and the effect on embryo development of different concentrations of this drug was first evaluated. The effect of incubation with aphidicolin was tested with CRISPR/Cas9 at different concentrations and different delivery methodologies. As a result, the reversible inhibition of DNA replication was observed, and it was concentration dependent. An optimal concentration of 0.5 μM was established and used for subsequent experiments. Following the use of this drug with CRISPR/Cas9, a halving of mosaicism was observed together with a detrimental effect on embryo development. In conclusion, the use of reversible inhibition of DNA replication offers a way to reduce mosaicism. Nevertheless, due to the reduction in embryo development, it would be necessary to reach a balance for its use to be feasible.
Topics: Animals; Animals, Genetically Modified; Aphidicolin; CRISPR-Cas Systems; Cell Nucleus; DNA Replication; Embryo, Mammalian; Embryonic Development; Eukaryota; Gene Editing; Mosaicism; Swine; Zygote
PubMed: 35216252
DOI: 10.3390/ijms23042135 -
Frontiers in Genetics 2021Glioblastoma multiforme (GBM) is a malignant tumor of the central nervous system (CNS). The poor prognosis of GBM due to resistance to therapy has been associated with...
Glioblastoma multiforme (GBM) is a malignant tumor of the central nervous system (CNS). The poor prognosis of GBM due to resistance to therapy has been associated with high chromosomal instability (CIN). Replication stress is a major cause of CIN that manifests as chromosome rearrangements, fragility, and breaks, including those cytologically expressed within specific chromosome regions named common fragile sites (CFSs). In this work, we characterized the expression of human CFSs in the glioblastoma U-251 MG cell line upon treatment with the inhibitor of DNA polymerase alpha aphidicolin (APH). We observed 52 gaps/breaks located within previously characterized CFSs. We found 17 to be CFSs in GBM cells upon treatment with APH, showing a frequency equal to at least 1% of the total gaps/breaks. We report that two CFSs localized to regions FRA2E (2p13/p12) and FRA2F (2q22) were only found in U-251 MG cells, but not lymphocytes or fibroblasts, after APH treatment. Notably, these glioblastoma-specific CFSs had a relatively high expression compared to the other CFSs with breakage frequency between ∼7 and 9%. Presence of long genes, incomplete replication, and delayed DNA synthesis during mitosis (MiDAS) after APH treatment suggest that an impaired replication process may contribute to this loci-specific fragility in U-251 MG cells. Altogether, our work offers a characterization of common fragile site expression in glioblastoma U-251 MG cells that may be further exploited for cytogenetic and clinical studies to advance our understanding of this incurable cancer.
PubMed: 35154254
DOI: 10.3389/fgene.2021.810793 -
International Journal of Molecular... Dec 2021The expression of PD-L1 by tumor cells is mainly associated with its immunosuppressive effect. In fact, PD-1/PD-L1 immune checkpoint inhibitors demonstrated remarkable...
The expression of PD-L1 by tumor cells is mainly associated with its immunosuppressive effect. In fact, PD-1/PD-L1 immune checkpoint inhibitors demonstrated remarkable effects in advanced cancer patients including HNSCC. In this context, irradiation is currently being investigated as a synergistic treatment modality to immunotherapy. However, the majority of HNSCC patients still show little improvement or even hyperprogression. Interestingly, there is increasing evidence for additional cell-intrinsic functions of PD-L1 in tumor cells. In previous studies, we showed that PD-L1 has a strong influence on proliferation, migration, invasion, and survival after irradiation. We demonstrated that cellular expression and localization of PD-L1 differed depending on sensitivity to irradiation. Here, we show that PD-L1 is also differentially expressed during cell cycle progression of HNSCC. Furthermore, cellular localization of PD-L1 also changes depending on a particular cell cycle phase. Moreover, distinct observations occurred depending on the general differentiation status. Overall, the function of PD-L1 cannot be generalized. Rather, it depends on the differentiation status and microenvironment. PD-L1 expression and localization are variable, depending on different factors. These findings may provide insight into why differential response to PD-1/PD-L1 antibody therapy can occur. Detailed understanding of cell-intrinsic PD-L1 functions will further allow antibody-based immunotherapy to be optimized.
Topics: B7-H1 Antigen; Cell Cycle; Gene Expression Regulation, Neoplastic; Humans; Protein Transport; Squamous Cell Carcinoma of Head and Neck
PubMed: 34884892
DOI: 10.3390/ijms222313087