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Journal of Fungi (Basel, Switzerland) May 2024DNA damage checkpoints are essential for coordinating cell cycle arrest and gene transcription during DNA damage response. Exploring the targets of checkpoint kinases in...
DNA damage checkpoints are essential for coordinating cell cycle arrest and gene transcription during DNA damage response. Exploring the targets of checkpoint kinases in and other fungi has expanded our comprehension of the downstream pathways involved in DNA damage response. While the function of checkpoint kinases, specifically Rad53, is well documented in the fungal pathogen , their targets remain poorly understood. In this study, we explored the impact of deleting on the global transcription profiles and observed alterations in genes associated with ribosome biogenesis, DNA replication, and cell cycle. However, the deletion of only affected a limited number of known DNA damage-responsive genes, including and . Unlike , the downregulation of transcription in under the influence of Methyl Methanesulfonate (MMS) did not depend on Dun1 but still relied on Rad53 and Rad9. In addition, the transcription factor Mcm1 was identified as a regulator of transcription, with evidence of dynamic binding to its promoter region; however, this dynamic binding was interrupted following the deletion of . Furthermore, Rad53 was observed to directly interact with the promoter region of , thus suggesting a potential role in governing its transcription. Overall, checkpoints regulate global gene transcription in and show species-specific regulation on ; these discoveries improve our understanding of the signaling pathway related to checkpoints in this pathogen.
PubMed: 38921373
DOI: 10.3390/jof10060387 -
DNA Repair Jun 2024To identify new molecular components of the Brh2-governed homologous recombination (HR)-network in the highly radiation-resistant fungus Ustilago maydis, we undertook a...
To identify new molecular components of the Brh2-governed homologous recombination (HR)-network in the highly radiation-resistant fungus Ustilago maydis, we undertook a genetic screen for suppressors of blm- hydroxyurea (HU)-sensitivity. Twenty DNA-damage sensitive mutants were obtained, three of which showing slow-growth phenotypes. Focusing on the "normally" growing candidates we identified five mutations, two in previously well-defined genes (Rec2 and Rad51) and the remaining three in completely uncharacterized genes (named Rec3, Bls9 and Zdr1). A common feature among these novel factors is their prominent role in DNA repair. Rec3 contains the P-loop NTPase domain which is most similar to that found in U. maydis Rec2 protein, and like Rec2, Rec3 plays critical roles in induced allelic recombination, is crucial for completion of meiosis, and with regard to DNA repair Δrec3 and Δrec2 are epistatic to one another. Importantly, overexpression of Brh2 in Δrec3 can effectively restore DNA-damage resistance, indicating a close functional connection between Brh2 and Rec3. The Bls9 does not seem to have any convincing domains that would give a clue as to its function. Nevertheless, we present evidence that, besides being involved in DNA-repair, Bls9 is also necessary for HR between chromosome homologs. Moreover, Δbls9 showed epistasis with Δbrh2 with respect to killing by DNA-damaging agents. Both, Rec3 and Bls9, play an important role in protecting the genome from mutations. Zdr1 is Cys2-His2 zinc finger (C2H2-ZF) protein, whose loss does not cause a detectable change in HR. Also, the functions of both Bls9 and Zdr1 genes are dispensable in meiosis and sporulation. However, Zdr1 appears to have overlapping activities with Blm and Mus81 in protecting the organism from methyl methanesulfonate- and diepoxybutane-induced DNA-damage. Finally, while deletion of Rec3 and Zdr1 can suppress HU-sensitivity of blm-, Δgen1, and Δmus81 mutants, interestingly loss of Bls9 does not rescue HU-sensitivity of Δgen1.
PubMed: 38861762
DOI: 10.1016/j.dnarep.2024.103709 -
Mutation Research. Genetic Toxicology... 2024Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety...
Currently, there is no test system, whether in vitro or in vivo, capable of examining all endpoints required for genotoxicity evaluation used in pre-clinical drug safety assessment. The objective of this study was to develop a model which could assess all the required endpoints and possesses robust human metabolic activity, that could be used in a streamlined, animal-free manner. Liver-on-chip (LOC) models have intrinsic human metabolic activity that mimics the in vivo environment, making it a preferred test system. For our assay, the LOC was assembled using primary human hepatocytes or HepaRG cells, in a MPS-T12 plate, maintained under microfluidic flow conditions using the PhysioMimix® Microphysiological System (MPS), and co-cultured with human lymphoblastoid (TK6) cells in transwells. This system allows for interaction between two compartments and for the analysis of three different genotoxic endpoints, i.e. DNA strand breaks (comet assay) in hepatocytes, chromosome loss or damage (micronucleus assay) and mutation (Duplex Sequencing) in TK6 cells. Both compartments were treated at 0, 24 and 45 h with two direct genotoxicants: methyl methanesulfonate (MMS) and ethyl methanesulfonate (EMS), and two genotoxicants requiring metabolic activation: benzo[a]pyrene (B[a]P) and cyclophosphamide (CP). Assessment of cytochrome activity, RNA expression, albumin, urea and lactate dehydrogenase production, demonstrated functional metabolic capacities. Genotoxicity responses were observed for all endpoints with MMS and EMS. Increases in the micronucleus and mutations (MF) frequencies were also observed with CP, and %Tail DNA with B[a]P, indicating the metabolic competency of the test system. CP did not exhibit an increase in the %Tail DNA, which is in line with in vivo data. However, B[a]P did not exhibit an increase in the % micronucleus and MF, which might require an optimization of the test system. In conclusion, this proof-of-principle experiment suggests that LOC-MPS technology is a promising tool for in vitro hazard identification genotoxicants.
Topics: Humans; Hepatocytes; Mutagens; Micronucleus Tests; Mutagenicity Tests; Liver; Lab-On-A-Chip Devices; DNA Damage; Comet Assay; Cyclophosphamide; Methyl Methanesulfonate; Cell Line; Benzo(a)pyrene; Coculture Techniques; Ethyl Methanesulfonate; Mutation
PubMed: 38821675
DOI: 10.1016/j.mrgentox.2024.503762 -
Dose-response : a Publication of... 2024We have been conducting a collaborative study on the thresholds of mutagens. In our previous examinations of cell activity and cell proliferation as endpoints, both...
BACKGROUND
We have been conducting a collaborative study on the thresholds of mutagens. In our previous examinations of cell activity and cell proliferation as endpoints, both displayed hormesis. This time, we conducted experiments to determine thresholds using the micronucleus test as an endpoint.
METHODS
The micronucleus test was conducted using Chinese hamster CHL/IU cells and mouse lymphoid L5178Y cells. Additionally, we conducted preliminary investigations into the gene expression using human TK6 cells.
RESULTS
When adhesive CHL/IU cells were treated with mitomycin C (MMC), and the hormetic response was examined, hormesis was not observed clearly. When L5178Y cells were treated with methyl methanesulfonate (EMS), AF-2, MMC, and colchicine, all of them exhibited an adaptive response. Additionally, cross-adaptive responses using AF-2 and MMC or EMS and MMC were conducted, both combinations showed a cross-adaptive response. When the gene expression patterns of six genes were investigated by RT-PCR after treatment with MMC, EMS, and HO using TK6 cells, two genes, and , were induced in a dose- and time-dependent manner.
CONCLUSION
Adaptive responses arise from preconditioning. As hormesis is inherently linked to preconditioning, adaptive responses observed in this study strongly suggest that hormesis was induced, hence existence of thresholds.
PubMed: 38715588
DOI: 10.1177/15593258241252040 -
International Journal of Biological... May 2024Genotoxic DNA damaging agents are the choice of chemicals for studying DNA repair pathways and the associated genome instability. One such preferred laboratory chemical...
Characterization of structural, genotoxic, and immunological effects of methyl methanesulfonate (MMS) induced DNA modifications: Implications for inflammation-driven carcinogenesis.
Genotoxic DNA damaging agents are the choice of chemicals for studying DNA repair pathways and the associated genome instability. One such preferred laboratory chemical is methyl methanesulfonate (MMS). MMS, an SN2-type alkylating agent known for its ability to alkylate adenine and guanine bases, causes strand breakage. Exploring the outcomes of MMS interaction with DNA and the associated cytotoxicity will pave the way to decipher how the cell confronts methylation-associated stress. This study focuses on an in-depth understanding of the structural instability, induced antigenicity on the DNA molecule, cross-reactive anti-DNA antibodies, and cytotoxic potential of MMS in peripheral lymphocytes and cancer cell lines. The findings are decisive in identifying the hazardous nature of MMS to alter the intricacies of DNA and morphology of the cell. Structural alterations were assessed through UV-Vis, fluorescence, liquid chromatography, and mass spectroscopy (LCMS). The thermal instability of DNA was analyzed using duplex melting temperature profiles. Scanning and transmission electron microscopy revealed gross topographical and morphological changes. MMS-modified DNA exhibited increased antigenicity in animal subjects. MMS was quite toxic for the cancer cell lines (HCT116, A549, and HeLa). This research will offer insights into the potential role of MMS in inflammatory carcinogenesis and its progression.
Topics: Methyl Methanesulfonate; Humans; DNA; DNA Damage; Inflammation; Animals; Carcinogenesis; HeLa Cells; A549 Cells; Lymphocytes; HCT116 Cells
PubMed: 38653426
DOI: 10.1016/j.ijbiomac.2024.131743 -
Chemical Research in Toxicology May 2024The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion , but it is not believed to have a large direct physiological...
The major product of DNA-methylating agents, N7-methyl-2'-deoxyguanosine (MdG), is a persistent lesion , but it is not believed to have a large direct physiological impact. However, MdG reacts with histone proteins to form reversible DNA-protein cross-links (DPC), a family of DNA lesions that can significantly threaten cell survival. In this paper, we developed a tandem mass spectrometry method for quantifying the amounts of MdG and DPC in nuclear DNA by taking advantage of their chemical lability and the concurrent release of N7-methylguanine. Using this method, we determined that DPC is formed in less than 1% yield based upon the levels of MdG in methyl methanesulfonate (MMS)-treated HeLa cells. Despite its low chemical yield, DPC contributes to MMS cytotoxicity. Consequently, cells that lack efficient DPC repair by the DPC protease SPRTN are hypersensitive to MMS. This investigation shows that the downstream chemical and biochemical effects of initially formed DNA damage can have significant biological consequences. With respect to MdG formation, the initial DNA lesion is only the beginning.
Topics: Humans; HeLa Cells; DNA; Deoxyguanosine; Methyl Methanesulfonate; Tandem Mass Spectrometry; Cell Survival; DNA Damage; Cross-Linking Reagents; DNA-Binding Proteins
PubMed: 38652696
DOI: 10.1021/acs.chemrestox.4c00076 -
Non-coding RNA Research Sep 2024In recent years, various long non-coding RNAs (lncRNAs) involved in DNA damage response (DDR) have been identified and studied to deepen our understanding. However,...
In recent years, various long non-coding RNAs (lncRNAs) involved in DNA damage response (DDR) have been identified and studied to deepen our understanding. However, there are rare reports on the association between lncRNAs and base excision repair (BER). Our designed DNA microarray identified dozens of functionally unknown lncRNAs, and their transcription levels significantly increased upon exposure to DNA damage inducers. One of them, named (Long noncoding RNA Interacts with PARP-1), exhibited a significant alteration in transcription in response to methyl methanesulfonate (MMS) and temozolomide (TMZ) treatments. knockdown or knockout cell lines are sensitive to MMS and TMZ, indicating that plays a crucial role in DDR. The loss or insufficiency of significantly influences the efficiency of BER in human cells, and it suggests that participates in the BER pathway. The interaction between and a key factor in BER, poly (ADP-ribose) polymerase 1 (PARP-1), has been confirmed. We identified and characterized , a lncRNA, which is involved in DDR, significantly influences BER efficiency, and interacts with the BER key factor PARP-1. This advances our understanding of the connection between lncRNAs and BER, presenting the potential for the discovery of new drug targets.
PubMed: 38577022
DOI: 10.1016/j.ncrna.2024.03.010 -
MSphere Apr 2024Histone lysine acetyltransferase MYST-associated NuA4 complex is conserved from yeast to humans and plays key roles in cell cycle regulation, gene transcription, and DNA...
Histone lysine acetyltransferase MYST-associated NuA4 complex is conserved from yeast to humans and plays key roles in cell cycle regulation, gene transcription, and DNA replication/repair. Here, we identified a MYST-associated complex, PfNuA4, which contains 11 of the 13 conserved NuA4 subunits. Reciprocal pulldowns using PfEAF2, a shared component between the NuA4 and SWR1 complexes, not only confirmed the PfNuA4 complex but also identified the PfSWR1 complex, a histone remodeling complex, although their identities are low compared to the homologs in yeast or humans. Notably, both H2A.Z/H2B.Z were associated with the PfSWR1 complex, indicating that this complex is involved in the deposition of H2A.Z/H2B.Z, the variant histone pair that is enriched in the activated promoters. Overexpression of PfMYST resulted in earlier expression of genes involved in cell cycle regulation, DNA replication, and merozoite invasion, and upregulation of the genes related to antigenic variation and DNA repair. Consistently, PfMYST overexpression led to high basal phosphorylated PfH2A (γ-PfH2A), the mark of DNA double-strand breaks, and conferred protection against genotoxic agent methyl methanesulfonate (MMS), X-rays, and artemisinin, the first-line antimalarial drug. In contrast, the knockdown of PfMYST caused a delayed parasite recovery upon MMS treatment. MMS induced the gradual disappearance of PfMYST in the cytoplasm and concomitant accumulation of PfMYST in the nucleus, suggesting cytoplasm-nucleus shuttling of PfMYST. Meanwhile, PfMYST colocalized with the γ-PfH2A, indicating PfMYST was recruited to the DNA damage sites. Collectively, PfMYST plays critical roles in cell cycle regulation, gene transcription, and DNA replication/DNA repair in this low-branching parasitic protist.IMPORTANCEUnderstanding gene regulation and DNA repair in malaria parasites is critical for identifying targets for antimalarials. This study found PfNuA4, a PfMYST-associated, histone modifier complex, and PfSWR1, a chromatin remodeling complex in malaria parasite . These complexes are divergent due to the low identities compared to their homologs from yeast and humans. Furthermore, overexpression of PfMYST resulted in substantial transcriptomic changes, indicating that PfMYST is involved in regulating the cell cycle, antigenic variation, and DNA replication/repair. Consistently, PfMYST was found to protect against DNA damage caused by the genotoxic agent methyl methanesulfonate, X-rays, and artemisinin, the first-line antimalarial drug. Additionally, DNA damage led to the relocation of cytoplasmic PfMYST to the nucleus and colocalization of PfMYST with γ-PfH2A, the mark of DNA damage. In summary, this study demonstrated that the PfMYST complex has critical functions in regulating cell cycle, antigenic variation, and DNA replication/DNA repair in .
Topics: Plasmodium falciparum; DNA Repair; Protozoan Proteins; Histone Acetyltransferases; Humans; DNA Replication; Histones; Gene Expression Regulation
PubMed: 38564734
DOI: 10.1128/msphere.00140-24 -
International Journal of Molecular... Feb 2024DNA lesions trigger DNA damage checkpoint (DDC) signaling which arrests cell cycle progression and promotes DNA damage repair. In , phosphorylation of histone H2A...
DNA lesions trigger DNA damage checkpoint (DDC) signaling which arrests cell cycle progression and promotes DNA damage repair. In , phosphorylation of histone H2A (γH2A, equivalent to γH2AX in mammals) is an early chromatin mark induced by DNA damage that is recognized by a group of DDC and DNA repair factors. We find that γH2A negatively regulates the G2/M checkpoint in response to the genotoxin camptothecin, which is a DNA topoisomerase I poison. γH2A also suppresses DDC signaling induced by the DNA alkylating agent methyl methanesulfonate. These results differ from prior findings, which demonstrate positive or no roles of γH2A in DDC in response to other DNA damaging agents such as phleomycin and ionizing radiation, which suggest that γH2A has DNA damage-specific effects on DDC signaling. We also find evidence supporting the notion that γH2A regulates DDC signaling by mediating the competitive recruitment of the DDC mediator Rad9 and the DNA repair factor Rtt107 to DNA lesions. We propose that γH2A/γH2AX serves to create a dynamic balance between DDC and DNA repair that is influenced by the nature of DNA damage.
Topics: Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; DNA Damage; Histones; DNA
PubMed: 38473708
DOI: 10.3390/ijms25052462 -
Mutation Research. Genetic Toxicology... 2024Preclinical and clinical studies have shown that molecular hydrogen (H) has anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Safety data are available in...
Preclinical and clinical studies have shown that molecular hydrogen (H) has anti-oxidant, anti-inflammatory, and anti-apoptotic properties. Safety data are available in the literature and acute toxicity has been tested in isolated cells and laboratory animals. We have evaluates the genotoxicity of H in vivo in rats after 72 h exposure, following the International Council for Harmonization guidelines ICH S2 (R1). The study was conducted on three groups of male Wistar rats: a negative control group, a positive control group receiving methyl methanesulfonate, and a H-treated group receiving a 3.1% H gas mixture for 72 h. Alkaline comet, formamidopyrimidine DNA glycosylase (Fpg)-modified comet and bone marrow micronucleus assays were performed. H exposure increased neither comet-tail DNA intensity (DNA damage) nor frequency of "hedgehogs" in blood, liver, lungs, or bronchoalveolar lavage fluid. No increase in Fpg-sensitive sites in lungs, no induction of micronucleus formation, and no imbalance of immature erythrocyte to total erythrocyte ratio (IME%) was observed in rats exposed to H. The ICH S2 (R1) test-battery revealed no in vivo genotoxicity in Wistar rats after 72 h inhalation of a mixture containing 3.1% H.
Topics: Male; Rats; Animals; Hydrogen; Rats, Wistar; DNA Damage; Comet Assay; Antioxidants; DNA-Formamidopyrimidine Glycosylase
PubMed: 38432775
DOI: 10.1016/j.mrgentox.2024.503736