Resveratrol induces H3 and H4K16 deacetylation and H2A.X phosphorylation in Toxoplasma gondii

Abstract Objective Resveratrol (RSV) is a multitarget drug that has demonstrated activity against Toxoplasma gondii in macrophage and human foreskin fibroblast (HFF) cell line infection models. However, the mechanism of action of RSV has not yet been determined. Thus, with the aim of identifying a possible mechanism of the anti-T. gondii activity of this compound, we analyzed the effects of RSV on histones H3 and H4 lysine 16 acetylation (H4K16). We also analyzed RSV-induced DNA damage to intracellular tachyzoites by using the DNA damage marker phosphorylated histone H2A.X (γH2AX). Results RSV inhibited intracellular T. gondii tachyzoite growth at concentrations below the toxic threshold for host cells. The IC50 value after 24 h of treatment was 53 μM. RSV induced a reduction in H4K16 acetylation (H4K16ac), a marker associated with transcription, DNA replication and homologous recombination repair. A similar deacetylation effect was observed on histone H3. RSV also increased T. gondii H2A.X phosphorylation at the SQE motif (termed γH2A.X), which is a DNA damage-associated posttranslational modification. Our findings suggest a possible link between RSV and DNA damage or repair processes that is possibly associated with DNA replication stress.

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Resveratrol induces H3 and H4K16 de-acetylation and H2A.X phosphorylation in Toxoplasma gondii

10.21203/rs.3.rs-94395/v1 ◽

2020 ◽

Author(s):

Susana Marisol Contreras ◽

Agustina Ganuza ◽

Maria Martha Corvi ◽

Sergio Angel

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Toxoplasma Gondii ◽

Repair Process ◽

Host Cells ◽

Target Drug ◽

Infection Models ◽

Recombination Repair ◽

Dna Replication Stress ◽

H4k16 Acetylation

Abstract Objective: Objective: Resveratrol (RSV) is a multi-target drug that has demonstrated activity against Toxoplasma gondii in macrophage and HFF cell line infection models. However, its mechanism of action has not been determined yet. With the aim of determining a possible mechanism of anti-T. gondii action, we analyzed the effect of RSV on histones H3 and H4 lysine 16 acetylation (H4K16). RSV-induced DNA damage of intracellular tachyzoites was assessed as well by using the DNA damage marker phosphorylated histone H2A.X (gH2AX).Results: RSV inhibited intracellular T. gondii tachyzoite growth at concentrations below the toxic effect on host cells. The IC50 value in a 24-hours treatment was 53 mM. RSV induced a reduction in H4K16 acetylation (H4K16ac), a mark associated to transcription, DNA replication and homologous recombination repair. The same deacetylating effect was observed on histone H3. RSV also enhanced the SQE motif phosphorylation on T. gondii H2A.X (termed γH2A.X), a DNA damage associated PTM. Our findings suggest a possible link between RSV and DNA damage or DNA repair process maybe associated to DNA replication stress.

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Resveratrol induces H2A.X phosphorylation and H4K16 de-acetylation in Toxoplasma gondii

10.1101/2020.08.25.266031 ◽

2020 ◽

Author(s):

Susana M. Contreras ◽

Agustina Ganuza ◽

María M. Corvi ◽

Sergio O. Angel

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Toxoplasma Gondii ◽

Redox Homeostasis ◽

Repair Process ◽

Host Cells ◽

Target Drug ◽

Toxic Concentration ◽

Dna Replication Stress ◽

H3 Acetylation

AbstractResveratrol (RSV) is a multi-target drug that demonstrated activity against Toxoplasma gondii in macrophage and HFF cell line infection models. In addition to modulate redox homeostasis, RSV is also an activator of Sir2, a type III HDAC. RSV inhibited intracellular T. gondii tachyzoite growth at concentrations below the toxic effect on host cells. The IC50 value in a 24-hours treatment was 53 μM. After 96 hours of treatment the maximum non-toxic concentration for host cell, 20 μM, only inhibited T. gondii growth a 50%. RSV induced a reduction in H4K16 acetylation (H4K16ac), a mark associated to transcription, DNA replication and homologous recombination repair, without any effect on H3 acetylation (H3ac). RSV also enhanced the SQE motif phosphorylation on T. gondii H2A.X (termed γH2A.X), a DNA damage associated PTM. Sirtinol, a specific Sir2 inhibitor also inhibited T. gondii but did not altered the acetylation status of H3 and H4K16 as well as H2A.X phosphorylation. Our findings suggest a possible link between RSV and DNA damage or DNA repair process maybe due to DNA replication stress and/or another undetermined mechanism.

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Protective Mechanisms Against DNA Replication Stress in the Nervous System

Genes ◽

10.3390/genes11070730 ◽

2020 ◽

Vol 11 (7) ◽

pp. 730

Author(s):

Clara Forrer Charlier ◽

Rodrigo A. P. Martins

Keyword(s):

Dna Damage ◽

Nervous System ◽

Dna Replication ◽

Neurological Diseases ◽

Replication Stress ◽

Nervous System Development ◽

Stress Generation ◽

Genome Replication ◽

Cns Development ◽

Dna Replication Stress

The precise replication of DNA and the successful segregation of chromosomes are essential for the faithful transmission of genetic information during the cell cycle. Alterations in the dynamics of genome replication, also referred to as DNA replication stress, may lead to DNA damage and, consequently, mutations and chromosomal rearrangements. Extensive research has revealed that DNA replication stress drives genome instability during tumorigenesis. Over decades, genetic studies of inherited syndromes have established a connection between the mutations in genes required for proper DNA repair/DNA damage responses and neurological diseases. It is becoming clear that both the prevention and the responses to replication stress are particularly important for nervous system development and function. The accurate regulation of cell proliferation is key for the expansion of progenitor pools during central nervous system (CNS) development, adult neurogenesis, and regeneration. Moreover, DNA replication stress in glial cells regulates CNS tumorigenesis and plays a role in neurodegenerative diseases such as ataxia telangiectasia (A-T). Here, we review how replication stress generation and replication stress response (RSR) contribute to the CNS development, homeostasis, and disease. Both cell-autonomous mechanisms, as well as the evidence of RSR-mediated alterations of the cellular microenvironment in the nervous system, were discussed.

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DNA double-strand breaks in the Toxoplasma gondii-infected cells by the action of reactive oxygen species

Parasites & Vectors ◽

10.1186/s13071-020-04324-7 ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Haohan Zhuang ◽

Chaoqun Yao ◽

Xianfeng Zhao ◽

Xueqiu Chen ◽

Yimin Yang ◽

...

Keyword(s):

Dna Damage ◽

Toxoplasma Gondii ◽

Double Strand Breaks ◽

Host Cells ◽

Oxygen Species ◽

Dna Double Strand Breaks ◽

Strand Breaks ◽

Damage Response ◽

Infected Cells

Abstract Background Toxoplasma gondii is an obligate parasite of all warm-blooded animals around the globe. Once infecting a cell, it manipulates the host’s DNA damage response that is yet to be elucidated. The objectives of the present study were three-fold: (i) to assess DNA damages in T. gondii-infected cells in vitro; (ii) to ascertain causes of DNA damage in T. gondii-infected cells; and (iii) to investigate activation of DNA damage responses during T. gondii infection. Methods HeLa, Vero and HEK293 cells were infected with T. gondii at a multiplicity of infection (MOI) of 10:1. Infected cells were analyzed for a biomarker of DNA double-strand breaks (DSBs) γH2AX at 10 h, 20 h or 30 h post-infection using both western blot and immunofluorescence assay. Reactive oxygen species (ROS) levels were measured using 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA), and ROS-induced DNA damage was inhibited by a ROS inhibitor N-acetylcysteine (NAC). Lastly, DNA damage responses were evaluated by detecting the active form of ataxia telangiectasia mutated/checkpoint kinase 2 (ATM/CHK2) by western blot. Results γH2AX levels in the infected HeLa cells were significantly increased over time during T. gondii infection compared to uninfected cells. NAC treatment greatly reduced ROS and concomitantly diminished γH2AX in host cells. The phosphorylated ATM/CHK2 were elevated in T. gondii-infected cells. Conclusions Toxoplasma gondii infection triggered DNA DSBs with ROS as a major player in host cells in vitro. It also activated DNA damage response pathway ATM/CHK2. Toxoplasma gondii manages to keep a balance between survival and apoptosis of its host cells for the benefit of its own survival.

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The deubiquitinase USP36 Regulates DNA replication stress and confers therapeutic resistance through PrimPol stabilization

Nucleic Acids Research ◽

10.1093/nar/gkaa1090 ◽

2020 ◽

Vol 48 (22) ◽

pp. 12711-12726

Author(s):

Yuanliang Yan ◽

Zhijie Xu ◽

Jinzhou Huang ◽

Guijie Guo ◽

Ming Gao ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Stress Response ◽

Critical Role ◽

Replication Stress ◽

Regulatory Mechanisms ◽

Therapeutic Resistance ◽

Chemotherapy Response ◽

Dna Replication Stress ◽

Damage Tolerant

Abstract PrimPol has been recently identified as a DNA damage tolerant polymerase that plays an important role in replication stress response. However, the regulatory mechanisms of PrimPol are not well defined. In this study, we identify that the deubiquitinase USP36 interferes with degradation of PrimPol to regulate the replication stress response. Mechanistically, USP36 is deubiquitinated following DNA replication stress, which in turn facilitates its upregulation and interaction with PrimPol. USP36 deubiquitinates K29-linked polyubiquitination of PrimPol and increases its protein stability. Depletion of USP36 results in replication stress-related defects and elevates cell sensitivity to DNA-damage agents, such as cisplatin and olaparib. Moreover, USP36 expression positively correlates with the level of PrimPol protein and poor prognosis in patient samples. These findings indicate that the regulation of PrimPol K29-linked ubiquitination by USP36 plays a critical role in DNA replication stress and chemotherapy response.

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Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

Molecular Biology of the Cell ◽

10.1091/mbc.e08-06-0639 ◽

2009 ◽

Vol 20 (3) ◽

pp. 983-994 ◽

Cited By ~ 13

Author(s):

Xia Yi ◽

Hilda I. de Vries ◽

Katarzyna Siudeja ◽

Anil Rana ◽

Willy Lemstra ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Synthesis ◽

Replication Stress ◽

Epigenetic Mechanism ◽

Dna Integrity ◽

Silent Chromatin ◽

Chromatin Compaction ◽

Cycle Arrest ◽

Dna Replication Stress

Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall (stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 (two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress.

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Dissecting DNA damage response pathways by analysing protein localization and abundance changes during DNA replication stress

Nature Cell Biology ◽

10.1038/ncb2549 ◽

2012 ◽

Vol 14 (9) ◽

pp. 966-976 ◽

Cited By ~ 291

Author(s):

Johnny M. Tkach ◽

Askar Yimit ◽

Anna Y. Lee ◽

Michael Riffle ◽

Michael Costanzo ◽

...

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Dna Damage Response ◽

Protein Localization ◽

Replication Stress ◽

Damage Response ◽

Dna Replication Stress

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In vitro Effect of Harmine Alkaloid and Its N-Methyl Derivatives Against Toxoplasma gondii

Frontiers in Microbiology ◽

10.3389/fmicb.2021.716534 ◽

2021 ◽

Vol 12 ◽

Author(s):

Maria L. Alomar ◽

Juan G. Yañuk ◽

Sergio O. Angel ◽

M. Micaela Gonzalez ◽

Franco M. Cabrerizo

Keyword(s):

Dna Damage ◽

Toxoplasma Gondii ◽

Dna Damage Response ◽

Biological Activities ◽

Host Cells ◽

Dependent Manner ◽

Damage Response ◽

Methyl Derivatives ◽

Vitro Effect

Toxoplasmosis is one of the most prevalent and neglected zoonotic global diseases caused by Toxoplasma gondii. The current pharmacological treatments show clinical limitations, and therefore, the search for new drugs is an urgent need in order to eradicate this infection. Due to their intrinsic biological activities, β-carboline (βC) alkaloids might represent a good alternative that deserves further investigations. In this context, the in vitro anti-T. gondii activity of three βCs, harmine (1), 2-methyl-harminium (2), and 9-methyl-harmine (3), was evaluated herein. Briefly, the three alkaloids exerted direct effects on the parasite invasion and/or replication capability. Replication rates of intracellular treated tachyzoites were also affected in a dose-dependent manner, at noncytotoxic concentrations for host cells. Additionally, cell cycle analysis revealed that both methyl-derivatives 2 and 3 induce parasite arrest in S/M phases. Compound 3 showed the highest irreversible parasite growth inhibition, with a half maximal inhibitory concentration (IC50) value of 1.8 ± 0.2 μM and a selectivity index (SI) of 17.2 at 4 days post infection. Due to high replication rates, tachyzoites are frequently subjected to DNA double-strand breaks (DSBs). This highly toxic lesion triggers a series of DNA damage response reactions, starting with a kinase cascade that phosphorylates a large number of substrates, including the histone H2A.X to lead the early DSB marker γH2A.X. Western blot studies showed that basal expression of γH2A.X was reduced in the presence of 3. Interestingly, the typical increase in γH2A.X levels produced by camptothecin (CPT), a drug that generates DSB, was not observed when CPT was co-administered with 3. These findings suggest that 3 might disrupt Toxoplasma DNA damage response.

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The activation loop residue serine 173 of S.pombe Chk1 kinase is critical for the response to DNA replication stress

10.1101/164244 ◽

2017 ◽

Author(s):

Naomi Coulton ◽

Thomas Caspari

Keyword(s):

Dna Damage ◽

Dna Replication ◽

Replication Stress ◽

Genetic Alterations ◽

Activation Loop ◽

Replication Forks ◽

Dna Polymerase Delta ◽

Dna Replication Stress ◽

Summary Statement ◽

Chk1 Kinase

AbstractWhy the DNA damage checkpoint kinase Chk1 protects the genome of lower and higher eukaryotic cells differentially is still unclear. Mammalian Chk1 regulates replication origins, safeguards DNA replication forks and promotes fork progression. Conversely, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A) in the activation loop of fission yeast Chk1 abolishes the G1-M and S-M checkpoints without affecting the G2-M arrest. Although Chk1-S173A is fully phosphorylated at serine 345 by the DNA damage sensor Rad3 (ATR) when DNA replication forks break, cells fail to stop the cell cycle. Mutant cells are uniquely sensitive to the DNA alkylation agent methyl- methanesulfate (MMS). This MMS sensitivity is genetically linked with the lagging strand DNA polymerase delta. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 is impaired. Serine 173 is equivalent to lysine 166 in human Chk1, an amino acid important for substrate specificity. We conclude that the removal of serine 173 impairs the phosphorylation of a Chk1 target that is important to protect cells from DNA replication stress.Summary statementMutation of serine-173 in the activation loop of Chk1 kinase may promote cancer as it abolishes the response to genetic alterations that arise while chromosomes are being copied.

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