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Nature Microbiology May 2020Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular...
Chronic hepatitis B virus (HBV) infections result in 887,000 deaths annually. The central challenge in curing HBV is eradication of the stable covalently closed circular DNA (cccDNA) form of the viral genome, which is formed by the repair of lesion-bearing HBV relaxed circular DNA delivered by the virions to hepatocytes. The complete and minimal set of host factors involved in cccDNA formation is unknown, largely due to the lack of a biochemical system that fully reconstitutes cccDNA formation. Here, we have developed experimental systems where various HBV relaxed-circular-DNA substrates are repaired to form cccDNA by both cell extracts and purified human proteins. Using yeast- and human-extract screenings, we identified five core components of lagging-strand synthesis as essential for cccDNA formation: proliferating cell nuclear antigen, the replication factor C complex, DNA polymerase δ, flap endonuclease 1 and DNA ligase 1. We reconstituted cccDNA formation with purified human homologues, establishing these as a minimal set of factors for cccDNA formation. We further demonstrated that treatment with the DNA-polymerase inhibitor aphidicolin diminishes cccDNA formation both in biochemical assays and in HBV-infected human cells. Together, our findings define key components in HBV cccDNA formation.
Topics: Cell Line; DNA Ligase ATP; DNA Replication; DNA, Circular; DNA, Viral; Flap Endonucleases; Genome, Viral; Hepatitis B virus; Hepatocytes; Humans; Proliferating Cell Nuclear Antigen; Replication Protein C; Virion; Virus Replication
PubMed: 32152586
DOI: 10.1038/s41564-020-0678-0 -
Antiviral Research Nov 2020Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection...
Equid herpesvirus-1 infections cause respiratory, neurological and reproductive syndromes. Despite preventive treatments with vaccines, resurgence of EHV-1 infection still constitutes a major threat to equine industry. However, no antiviral compound is available to treat infected horses. In this study, 2891 compounds were screened against EHV-1 using impedance measurement. 22 compounds have been found to be effective in vitro against EHV-1. Valganciclovir, ganciclovir, decitabine, aphidicolin, idoxuridine and pritelivir (BAY 57-1293) are the most effective compounds identified, and their antiviral potency was further assessed on E. Derm, RK13 and EEK cells and against 3 different field strains of EHV-1 (ORF30 2254 A/G/C). We also provide evidences of synergistic interactions between valganciclovir and decitabine in our in vitro antiviral assay as determined by MacSynergy II, isobologramm and Chou-Talalay methods. Finally, we showed that deoxycytidine reverts the antiviral effect of decitabine, thus supporting some competition at the level of nucleoside phosphorylation by deoxycytidine kinase and/or DNA synthesis. Deoxycitidine analogues, like decitabine, is a family of compounds identified for the first time with promising antiviral efficacy against herpesviruses.
Topics: Animals; Antiviral Agents; Cell Line; Decitabine; Drug Combinations; Drug Discovery; Drug Synergism; Ganciclovir; Herpesviridae Infections; Herpesvirus 1, Equid; High-Throughput Screening Assays; Horses; Rabbits; Valganciclovir
PubMed: 32926887
DOI: 10.1016/j.antiviral.2020.104931 -
Cell Reports Oct 2019Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance...
Signaling by the ubiquitin-related SUMO pathway relies on coordinated conjugation and deconjugation events. SUMO-specific deconjugating enzymes counterbalance SUMOylation, but comprehensive insight into their substrate specificity and regulation is missing. By characterizing SENP6, we define an N-terminal multi-SIM domain as a critical determinant in targeting SENP6 to SUMO chains. Proteomic profiling reveals a network of SENP6 functions at the crossroads of chromatin organization and DNA damage response (DDR). SENP6 acts as a SUMO eraser at telomeric and centromeric chromatin domains and determines the SUMOylation status and chromatin association of the cohesin complex. Importantly, SENP6 is part of the hPSO4/PRP19 complex that drives ATR-Chk1 activation. SENP6 deficiency impairs chromatin association of the ATR cofactor ATRIP, thereby compromising the activation of Chk1 signaling in response to aphidicolin-induced replicative stress and sensitizing cells to DNA damage. We propose a general role of SENP6 in orchestrating chromatin dynamics and genome stability networks by balancing chromatin residency of protein complexes.
Topics: Amino Acid Motifs; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; Checkpoint Kinase 1; Chromatin; Chromosomal Proteins, Non-Histone; Chromosomes, Human; Cysteine Endopeptidases; Genome, Human; Genomic Instability; HEK293 Cells; HeLa Cells; Humans; Nuclear Proteins; Protein Binding; Small Ubiquitin-Related Modifier Proteins; Sumoylation; Transcription Factors; Cohesins
PubMed: 31597105
DOI: 10.1016/j.celrep.2019.08.106 -
Mutagenesis Apr 2021In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal...
In vitro genotoxicity assays utilising human skin models are becoming important tools for the safety assessment of chemicals whose primary exposure is via the dermal route. In order to explore metabolic competency and inducibility of CYP450 activating enzymes, 3D reconstructed human skin tissues were topically treated with 2-acetylaminofluorene (2-AAF) and its genotoxic metabolites, N-hydroxy-2-acetylaminofluorene (N-OH-2-AAF) and N-hydroxy-2-aminofluorene (N-OH-2-AF), which primarily cause DNA damage by forming DNA adducts. 2-AAF did not increase DNA damage measured in the reconstructed skin micronucleus (RSMN) assay when administered in multiple applications at 24 h intervals but was detected in the skin comet assay in the presence of the DNA polymerase inhibitor aphidicolin (APC). Similarly, no increase was found with N-OH-2-AAF in the RSMN assay after multiple treatments whereas a single 3 h exposure to N-OH-2-AAF caused a large dose-related increase in the skin comet assay. A significant increase in the RSMN assay was only obtained with the highly reactive N-OH-2-AF metabolite after multiple treatments over 72 h, whereas N-OH-2-AF caused a strong increase after a single 3 h exposure in the skin comet assay. In support of these results, DNA adduct formation, measured by the 32P-postlabelling assay, was examined. Adduct levels after 2-AAF treatment for 3 h were minimal but increased >10-fold after multiple exposures over 48 h, suggesting that enzyme(s) that metabolise 2-AAF are induced in the skin models. As expected, a single 3 h exposure to N-OH-2-AAF and N-OH-2-AF resulted in adduct levels that were at least 10-fold greater than those after multiple exposures to 2-AAF despite ~100-fold lower tested concentrations. Our results demonstrate that DNA damage caused by 2-AAF metabolites is more efficiently detected in the skin comet assay than the RSMN assay and after multiple exposures and enzyme induction, 2-AAF-induced DNA damage can be detected in the APC-modified comet assay.
Topics: 2-Acetylaminofluorene; Carcinogens; DNA Adducts; DNA Damage; Fluorenes; Humans; Hydroxyacetylaminofluorene; Micronucleus Tests; Mutagens; Skin
PubMed: 31816077
DOI: 10.1093/mutage/gez044 -
Developmental Biology Jan 2024Sea cucumbers have an extraordinary regenerative capability. Under stressful conditions, Holothuria glaberrima can eviscerate their internal organs, including the...
Sea cucumbers have an extraordinary regenerative capability. Under stressful conditions, Holothuria glaberrima can eviscerate their internal organs, including the digestive tract. From the mesentery, a rudiment grows and gives rise to a new intestine within a few weeks. In the last decades, the cellular events that occur during intestinal regeneration have been characterized, including apoptosis, cell proliferation, and muscle cell dedifferentiation. Nevertheless, their contribution to the formation and early growth of the rudiment is still unknown. Furthermore, these cellular events' relationship and potential interdependence remain a mystery. Using modulators to inhibit apoptosis and cell proliferation, we tested whether rudiment growth or other regenerative cellular events like muscle cell dedifferentiation were affected. We found that inhibition of apoptosis by zVAD and cell proliferation by aphidicolin and mitomycin did not affect the overall size of the rudiment seven days post-evisceration (7-dpe). Interestingly, animals treated with aphidicolin showed higher levels of muscle cell dedifferentiation in the distal mesentery, which could act as a compensatory mechanism. On the other hand, inhibition of apoptosis led to a decrease in cell proliferation in the rudiment and a delay in the spatiotemporal progression of muscle cell dedifferentiation throughout the rudiment-mesentery structure. Our findings suggest that neither apoptosis nor cell proliferation significantly contributes to early rudiment growth during intestinal regeneration in the sea cucumber. Nevertheless, apoptosis may play an essential role in modulating cell proliferation in the rudiment (a process known as apoptosis-induced proliferation) and the timing for the progression of muscle cell dedifferentiation. These findings provide new insights into the role and relationship of cellular events during intestinal regeneration in an emerging regeneration model.
Topics: Animals; Sea Cucumbers; Aphidicolin; Intestines; Cell Proliferation; Apoptosis; Cell Dedifferentiation
PubMed: 37925124
DOI: 10.1016/j.ydbio.2023.11.001 -
Journal of B.U.ON. : Official Journal... 2021Retraction of: 'Inhibitory effect of Aphidicolin - a tetracyclic diterpene - on the proliferation and apoptotic induction in human cervical cancer (HeLa) cells', by...
Retraction of: 'Inhibitory effect of Aphidicolin - a tetracyclic diterpene - on the proliferation and apoptotic induction in human cervical cancer (HeLa) cells', by En-Yan Yu, Rui-Yan Zhao, Dong-Sheng Wang, JBUON 2015;20(6):1480-1486; PMID:26854444. Following the publication of the above article, readers drew to our attention that part of the data was unreliable. The authors were requested to provide the raw data to prove the originality, but were unable to do so. After an investigation, the Editors of JBUON decided to retract this article. We thank the readers for bringing this matter to our attention. We apologize for any inconvenience it may cause.
PubMed: 33721476
DOI: No ID Found -
Protoplasma Sep 2020Although intermediate filaments (IFs) are biochemically and immunologically suggested to exist in plant cells, there are few molecular genetic studies related to the...
Although intermediate filaments (IFs) are biochemically and immunologically suggested to exist in plant cells, there are few molecular genetic studies related to the proteins that form these structures. In this study, Arabidopsis AT3G05270 was selected as a candidate gene for a protein constituting IF in plant cells. The protein encoded by AT3G05270 has a large α-helix as well as the IF protein motif indispensable for maintaining the structures of IF. Moreover, fluorescence signals of this protein fused with GFP exhibited cytoskeleton-like filamentous structures in plant cells. Thus, we named the protein encoded by AT3G05270 as Intermediate Filament Motif Protein 1 (IFMoP1). The structures composed of IFMoP1 and their localizations were examined in IFMoP1-GFP-expressing tobacco BY-2 cells whose cell cycle was synchronized using aphidicolin, a DNA synthesis inhibitor, and propyzamide, a microtubule-disrupting agent. The IFMoP1-GFP signals were present at the spindles and phragmoplasts in the mitotic phase. In addition, the frequency of cells with cytoskeleton-like filamentous structures composed of IFMoP1-GFP increased with the increase in cells that completed cell division, and then decreased after several hours. In terms of the relationship in intracellular localization between IFMoP1 and microtubules, the filamentous structures composed of IFMoP1 were present independently of microtubules during interphase. In living cells, these filamentous structures moved along with the nucleus. IFMoP1 co-localized with spindle and phragmoplast microtubules during mitosis, as well as with a part of the cortical microtubules in interphase.
Topics: Cell Cycle; Intermediate Filaments; Microtubules; Plant Cells
PubMed: 32488394
DOI: 10.1007/s00709-020-01512-1 -
Emerging Topics in Life Sciences Dec 2023Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and...
Common fragile sites (CFS) are specific genomic regions prone to chromosomal instability under conditions of DNA replication stress. CFSs manifest as breaks, gaps, and constrictions on metaphase chromosomes under mild replication stress. These replication-sensitive CFS regions are preferentially unstable during cancer development, as reflected by their association with copy number variants (CNVs) frequently arise in most tumor types. Over the years, it became clear that a combination of different characteristics underlies the enhanced sensitivity of CFSs to replication stress. As of today, there is a strong evidence that the core fragility regions along CFSs overlap with actively transcribed large genes with delayed replication timing upon replication stress. Recently, the mechanistic basis for CFS instability was further extended to regions which span topologically associated domain (TAD) boundaries, generating a fragility signature composed of replication, transcription and genome organization. The presence of difficult-to-replicate AT-rich repeats was one of the early features suggested to characterize a subgroup of CFSs. These long stretches of AT-dinucleotide have the potential to fold into stable secondary structures which may impede replication fork progression, leaving the region under-replicated. Here, we focus on the molecular mechanisms underlying repeat instability at CFSs and on the proteins involved in the resolution of secondary structure impediments arising along repetitive sequence elements which are essential for the maintenance of genome stability.
Topics: Humans; DNA Replication; Chromosome Fragile Sites; DNA Replication Timing; Chromosomal Instability; DNA
PubMed: 37876349
DOI: 10.1042/ETLS20230023 -
Plant Cell Reports Aug 2020Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments...
Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S-M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (HO), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.
Topics: Apoptosis; Cell Nucleus; DNA Damage; DNA Fragmentation; DNA Replication; Gene Expression Regulation, Plant; Glutathione; Hydrogen Peroxide; Interphase; Meristem; Mitosis; Onions; Protein Biosynthesis; Reactive Oxygen Species; Seedlings; Transcription, Genetic; Uracil
PubMed: 32328702
DOI: 10.1007/s00299-020-02545-9 -
Cell Research Nov 2020DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA...
DNA replication stress, a feature of human cancers, often leads to instability at specific genomic loci, such as the common fragile sites (CFSs). Cells experiencing DNA replication stress may also exhibit mitotic DNA synthesis (MiDAS). To understand the physiological function of MiDAS and its relationship to CFSs, we mapped, at high resolution, the genomic sites of MiDAS in cells treated with the DNA polymerase inhibitor aphidicolin. Sites of MiDAS were evident as well-defined peaks that were largely conserved between cell lines and encompassed all known CFSs. The MiDAS peaks mapped within large, transcribed, origin-poor genomic regions. In cells that had been treated with aphidicolin, these regions remained unreplicated even in late S phase; MiDAS then served to complete their replication after the cells entered mitosis. Interestingly, leading and lagging strand synthesis were uncoupled in MiDAS, consistent with MiDAS being a form of break-induced replication, a repair mechanism for collapsed DNA replication forks. Our results provide a better understanding of the mechanisms leading to genomic instability at CFSs and in cancer cells.
Topics: Cell Line, Tumor; Chromosome Breakage; Chromosome Fragile Sites; DNA; DNA Replication Timing; Genome, Human; Genomic Instability; Humans; Mitosis; Molecular Sequence Annotation; Neoplasms; Replication Origin; Sequence Analysis, DNA
PubMed: 32561860
DOI: 10.1038/s41422-020-0358-x