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Viruses May 2024Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological...
Equid herpesvirus 4 (EHV-4) is a common respiratory pathogen in horses. It sporadically induces abortion or neonatal death. Although its contribution in neurological disorders is not clearly demonstrated, there is a strong suspicion of its involvement. Despite preventive treatments using vaccines against EHV-1/EHV-4, the resurgence of alpha-EHV infection still constitutes an important threat to the horse industry. Yet very few studies have been conducted on the search for antiviral molecules against EHV-4. A screening of 42 antiviral compounds was performed in vitro on equine fibroblast cells infected with the EHV-4 405/76 reference strain (VR2230). The formation of cytopathic effects was monitored by real-time cell analysis (RTCA), and the viral load was quantified by quantitative PCR. Aciclovir, the most widely used antiviral against alpha-herpesviruses in vivo, does not appear to be effective against EHV-4 in vitro. Potential antiviral activities were confirmed for eight molecules (idoxuridine, vidarabine, pritelivir, cidofovir, valganciclovir, ganciclovir, aphidicolin, and decitabine). Decitabine demonstrates the highest efficacy against EHV-4 in vitro. Transcriptomic analysis revealed the up-regulation of various genes implicated in interferon (IFN) response, suggesting that decitabine triggers the immune antiviral pathway.
Topics: Animals; Antiviral Agents; Horses; Decitabine; Immunity, Innate; Herpesvirus 4, Equid; Fibroblasts; Herpesviridae Infections; Horse Diseases; Viral Load; Cell Line; Virus Replication; Drug Evaluation, Preclinical
PubMed: 38793627
DOI: 10.3390/v16050746 -
Viruses Apr 2024The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including...
The HIV-1 capsid (CA) protein forms the outer shell of the viral core that is released into the cytoplasm upon infection. CA binds various cellular proteins, including CPSF6, that direct HIV-1 integration into speckle-associated domains in host chromatin. Upon HIV-1 infection, CPSF6 forms puncta in the nucleus. Here, we characterised these CPSF6 puncta further in HeLa cells, T-cells and macrophages and confirmed that integration and reverse transcription are not required for puncta formation. Indeed, we found that puncta formed very rapidly after infection, correlating with the time that CA entered the nucleus. In aphidicolin-treated HeLa cells and macrophages, puncta were detected for the length of the experiment, suggesting that puncta are only lost upon cell division. CA still co-localised with CPSF6 puncta at the latest time points, considerably after the peak of reverse transcription and integration. Intriguingly, the number of puncta induced in macrophages did not correlate with the MOI or the total number of nuclear speckles present in each cell, suggesting that CA/CPSF6 is only directed to a few nuclear speckles. Furthermore, we found that CPSF6 already co-localised with nuclear speckles in uninfected T-cells, suggesting that HIV-1 promotes a natural behaviour of CPSF6.
Topics: HIV-1; Humans; mRNA Cleavage and Polyadenylation Factors; T-Lymphocytes; HeLa Cells; Macrophages; Virus Integration; Cell Nucleus; Capsid Proteins; HIV Infections; Capsid
PubMed: 38793552
DOI: 10.3390/v16050670 -
Frontiers in Cell and Developmental... 2024The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome...
The accuracy of replication is one of the most important mechanisms ensuring the stability of the genome. The fork protection complex prevents premature replisome stalling and/or premature disassembly upon stress. Here, we characterize the Timeless-Tipin complex, a component of the fork protection complex. We used microscopy approaches, including colocalization analysis and proximity ligation assay, to investigate the spatial localization of the complex during ongoing replication in human cells. Taking advantage of the replication stress induction and the ensuing polymerase-helicase uncoupling, we characterized the Timeless-Tipin localization within the replisome. Replication stress was induced using hydroxyurea (HU) and aphidicolin (APH). While HU depletes the substrate for DNA synthesis, APH binds directly inside the catalytic pocket of DNA polymerase and inhibits its activity. Our data revealed that the Timeless-Tipin complex, independent of the stress, remains bound on chromatin upon stress induction and progresses together with the replicative helicase. This is accompanied by the spatial dissociation of the complex from the blocked replication machinery. Additionally, after stress induction, Timeless interaction with RPA, which continuously accumulates on ssDNA, was increased. Taken together, the Timeless-Tipin complex acts as a universal guardian of the mammalian replisome in an unperturbed S-phase progression as well as during replication stress.
PubMed: 38487270
DOI: 10.3389/fcell.2024.1346534 -
International Journal of Molecular... Dec 2023The comet assay-based in vitro DNA repair assay has become a common tool for quantifying base excision repair (BER) activity in human lymphocytes or cultured cells....
The comet assay-based in vitro DNA repair assay has become a common tool for quantifying base excision repair (BER) activity in human lymphocytes or cultured cells. Here, we optimized the protocol for studying BER in human placental tissue because the placenta is a non-invasive tissue for biomonitoring of early-life exposures, and it can be used to investigate molecular mechanisms associated with prenatal disorders. The optimal protein concentration of placental protein extracts for optimal damage recognition and incision was 2 mg protein/mL. The addition of aphidicolin did not lead to reduced non-specific incisions and was, therefore, not included in the optimized protocol. The interval between sample collection and analysis did not affect BER activity up to 70 min. Finally, this optimized protocol was tested on pre-eclamptic (PE) placental tissues ( = 11) and significantly lower BER activity in PE placentas compared to controls ( = 9) was observed. This was paralleled by a significant reduction in the expression of BER-related genes and increased DNA oxidation in PE placentas. Our study indicates that BER activity can be determined in placentas, and lower activity is present in PE compared with healthy. These findings should be followed up in prospective clinical investigations to examine BER's role in the advancement of PE.
Topics: Pregnancy; Humans; Female; Pilot Projects; Comet Assay; Prospective Studies; Placenta; DNA Repair; Pre-Eclampsia
PubMed: 38203356
DOI: 10.3390/ijms25010187 -
Journal of Radiation Research Mar 2024Telomere dysfunction induces chromosomal instability, which is a driving force in the development of cancers. To examine X-irradiation's effect on telomere integrity, we...
Telomere dysfunction induces chromosomal instability, which is a driving force in the development of cancers. To examine X-irradiation's effect on telomere integrity, we investigated X-ray-induced abnormalities in telomere signals detected by fluorescence in situ hybridization (telomere FISH) in mouse embryo fibroblast cells. The abnormalities were categorized as either extra telomere signals (ETSs) or loss of telomere signals (LTSs). The results indicated that low doses (0.3-0.5 Gy) of X-rays significantly induced ETS but not LTS and that ETS induction was saturated at doses above 0.5 Gy. In addition, treatment with hydrogen peroxide also induced ETS but not LTS. To clarify the involvement of radicals in inducing ETS, we examined the effect of ascorbic acid (AsA) on telomere FISH signals and found that pre-treatment with AsA (5 mM, 2 h), but not post-treatment, significantly suppressed the induction of ETS by X-irradiation. Importantly, neither pre- nor post-treatment with AsA affected X-ray-induced chromosome aberrations. These results suggest that oxidative DNA damage induced by radicals is involved in the induction of ETS. Furthermore, combined treatment with aphidicolin, a DNA replication inhibitor, elevated the induction of ETS by X-irradiation. This observation suggests that DNA replication stress, potentially triggered by oxidative DNA lesions within telomeres, may contribute to the induction of ETS resulting from X-irradiation. Based on these results, we propose that ETS is a sensitive biological marker of oxidative DNA damage in telomere structures.
Topics: Animals; Mice; In Situ Hybridization, Fluorescence; Telomere; Oxidative Stress; Chromosomal Instability; Chromosome Aberrations; DNA Damage
PubMed: 38171574
DOI: 10.1093/jrr/rrad102 -
Cell Reports Dec 2023Ubiquitination of proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) activates DNA damage tolerance pathways. Currently, we lack a comprehensive...
Ubiquitination of proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) activates DNA damage tolerance pathways. Currently, we lack a comprehensive understanding of how PCNA K164 ubiquitination promotes genome stability. To evaluate this, we generated stable cell lines expressing PCNA from the endogenous PCNA locus. Our data reveal that the inability to ubiquitinate K164 causes perturbations in global DNA replication. Persistent replication stress generates under-replicated regions and is exacerbated by the DNA polymerase inhibitor aphidicolin. We show that these phenotypes are due, in part, to impaired Fanconi anemia group D2 protein (FANCD2)-dependent mitotic DNA synthesis (MiDAS) in PCNA cells. FANCD2 mono-ubiquitination is significantly reduced in PCNA mutants, leading to reduced chromatin association and foci formation, both prerequisites for FANCD2-dependent MiDAS. Furthermore, K164 ubiquitination coordinates direct PCNA/FANCD2 colocalization in mitotic nuclei. Here, we show that PCNA K164 ubiquitination maintains human genome stability by promoting FANCD2-dependent MiDAS to prevent the accumulation of under-replicated DNA.
Topics: Humans; DNA; DNA Damage; DNA Repair; DNA Replication; Fanconi Anemia Complementation Group D2 Protein; Genomic Instability; Proliferating Cell Nuclear Antigen; Ubiquitination
PubMed: 38060446
DOI: 10.1016/j.celrep.2023.113523 -
Nature Communications Nov 2023Keeping replication fork stable is essential for safeguarding genome integrity; hence, its protection is highly regulated. The CTC1-STN1-TEN1 (CST) complex protects...
Keeping replication fork stable is essential for safeguarding genome integrity; hence, its protection is highly regulated. The CTC1-STN1-TEN1 (CST) complex protects stalled forks from aberrant MRE11-mediated nascent strand DNA degradation (NSD). However, the activation mechanism for CST at forks is unknown. Here, we report that STN1 is phosphorylated in its intrinsic disordered region. Loss of STN1 phosphorylation reduces the replication stress-induced STN1 localization to stalled forks, elevates NSD, increases MRE11 access to stalled forks, and decreases RAD51 localization at forks, leading to increased genome instability under perturbed DNA replication condition. STN1 is phosphorylated by both the ATR-CHK1 and the calcium-sensing kinase CaMKK2 in response to hydroxyurea/aphidicolin treatment or elevated cytosolic calcium concentration. Cancer-associated STN1 variants impair STN1 phosphorylation, conferring inability of fork protection. Collectively, our study uncovers that CaMKK2 and ATR-CHK1 target STN1 to enable its fork protective function, and suggests an important role of STN1 phosphorylation in cancer development.
Topics: Humans; Calcium; DNA Replication; Genomic Instability; Hydroxyurea; Neoplasms
PubMed: 38036565
DOI: 10.1038/s41467-023-43685-2 -
ELife Oct 2023Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between...
Chromatin has been shown to undergo diffusional motion, which is affected during gene transcription by RNA polymerase activity. However, the relationship between chromatin mobility and other genomic processes remains unclear. Hence, we set out to label the DNA directly in a sequence unbiased manner and followed labeled chromatin dynamics in interphase human cells expressing GFP-tagged proliferating cell nuclear antigen (PCNA), a cell cycle marker and core component of the DNA replication machinery. We detected decreased chromatin mobility during the S-phase compared to G1 and G2 phases in tumor as well as normal diploid cells using automated particle tracking. To gain insight into the dynamical organization of the genome during DNA replication, we determined labeled chromatin domain sizes and analyzed their motion in replicating cells. By correlating chromatin mobility proximal to the active sites of DNA synthesis, we showed that chromatin motion was locally constrained at the sites of DNA replication. Furthermore, inhibiting DNA synthesis led to increased loading of DNA polymerases. This was accompanied by accumulation of the single-stranded DNA binding protein on the chromatin and activation of DNA helicases further restricting local chromatin motion. We, therefore, propose that it is the loading of replisomes but not their catalytic activity that reduces the dynamics of replicating chromatin segments in the S-phase as well as their accessibility and probability of interactions with other genomic regions.
Topics: Humans; Chromatin; DNA Replication; S Phase; Cell Cycle; DNA Helicases
PubMed: 37906089
DOI: 10.7554/eLife.87572 -
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