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Nature Cell Biology Feb 2021The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP...
The response to poly(ADP-ribose) polymerase inhibitors (PARPi) is dictated by homologous recombination (HR) DNA repair and the abundance of lesions that trap PARP enzymes. It remains unclear, however, if the established role of PARP in promoting chromatin accessibility impacts viability in these settings. Using a CRISPR-based screen, we identified the PAR-binding chromatin remodeller ALC1/CHD1L as a key determinant of PARPi toxicity in HR-deficient cells. ALC1 loss reduced viability of breast cancer gene (BRCA)-mutant cells and enhanced sensitivity to PARPi by up to 250-fold, while overcoming several resistance mechanisms. ALC1 deficiency reduced chromatin accessibility concomitant with a decrease in the association of base damage repair factors. This resulted in an accumulation of replication-associated DNA damage, increased PARP trapping and a reliance on HR. These findings establish PAR-dependent chromatin remodelling as a mechanistically distinct aspect of PARPi responses and therapeutic target in HR-deficient cancers.
Topics: BRCA1 Protein; BRCA2 Protein; CRISPR-Cas Systems; Cell Line, Tumor; Cell Proliferation; Chromatin; Chromatin Assembly and Disassembly; Chromosome Aberrations; DNA Helicases; DNA Repair; DNA-Binding Proteins; Epistasis, Genetic; Genomic Instability; Green Fluorescent Proteins; Homologous Recombination; Humans; Methyl Methanesulfonate; Mutation; Phthalazines; Piperazines; Poly Adenosine Diphosphate Ribose; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Domains
PubMed: 33462394
DOI: 10.1038/s41556-020-00624-3 -
Current Genetics Oct 2020Histone proteins regulate cellular factors' accessibility to DNA, and histone dosage has previously been linked with DNA damage susceptibility and efficiency of DNA... (Review)
Review
Histone proteins regulate cellular factors' accessibility to DNA, and histone dosage has previously been linked with DNA damage susceptibility and efficiency of DNA repair pathways. Surplus histones are known to impede the DNA repair process by interfering with the homologous recombination-mediated DNA repair in Saccharomyces cerevisiae. Here, we discuss the recent finding of association of methyl methanesulfonate (MMS) resistance with the reduced histone H4 gene dosage in the pathogenic yeast Candida glabrata. We have earlier shown that while the low histone H3 gene dosage led to MMS susceptibility, the lack of two H4-encoding ORFs, CgHHF1 and CgHHF2, led to resistance to MMS-induced DNA damage. This resistance was linked with a higher rate of homologous recombination (HR). Taking these findings further, we review the interactome analysis of histones H3 and H4 in C. glabrata. We also report that the arginine residue present at the 95th position in the C-terminal tail of histone H4 protein is required for complementation of the MMS resistance in the Cghhf1Δhhf2Δ mutant, thereby pointing out a probable role of this residue in association with HR factors. Additionally, we present evidence that reduction in H4 protein levels may constitute an important part of varied stress responses in C. glabrata. Altogether, we present an overview of histone H4 dosage, HR-mediated repair of damaged DNA and stress resistance in this opportunistic human fungal pathogen.
Topics: Acetylation; Candida glabrata; DNA Repair; Fungal Proteins; Gene Expression Regulation, Fungal; Genome, Fungal; Histones; Homologous Recombination; Humans
PubMed: 32556547
DOI: 10.1007/s00294-020-01088-6 -
Genetics Aug 2023In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream...
In budding yeast, the transcriptional repressor Opi1 regulates phospholipid biosynthesis by repressing expression of genes containing inositol-sensitive upstream activation sequences. Upon genotoxic stress, cells activate the DNA damage response to coordinate a complex network of signaling pathways aimed at preserving genomic integrity. Here, we reveal that Opi1 is important to modulate transcription in response to genotoxic stress. We find that cells lacking Opi1 exhibit hypersensitivity to genotoxins, along with a delayed G1-to-S-phase transition and decreased gamma-H2A levels. Transcriptome analysis using RNA sequencing reveals that Opi1 plays a central role in modulating essential biological processes during methyl methanesulfonate (MMS)-associated stress, including repression of phospholipid biosynthesis and transduction of mating signaling. Moreover, Opi1 induces sulfate assimilation and amino acid metabolic processes, such as arginine and histidine biosynthesis and glycine catabolism. Furthermore, we observe increased mitochondrial DNA instability in opi1Δ cells upon MMS treatment. Notably, we show that constitutive activation of the transcription factor Ino2-Ino4 is responsible for genotoxin sensitivity in Opi1-deficient cells, and the production of inositol pyrophosphates by Kcs1 counteracts Opi1 function specifically during MMS-induced stress. Overall, our findings highlight Opi1 as a critical sensor of genotoxic stress in budding yeast, orchestrating gene expression to facilitate appropriate stress responses.
Topics: Basic Helix-Loop-Helix Transcription Factors; DNA Damage; Gene Expression Regulation, Fungal; Inositol; Phospholipids; Repressor Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Saccharomycetales; Transcription Factors
PubMed: 37440469
DOI: 10.1093/genetics/iyad130 -
Proceedings of the National Academy of... Feb 2021The ubiquitin E3 ligase Bre1-mediated H2B monoubiquitination (H2Bub) is essential for proper DNA replication and repair in eukaryotes. Deficiency in H2Bub causes genome...
The ubiquitin E3 ligase Bre1-mediated H2B monoubiquitination (H2Bub) is essential for proper DNA replication and repair in eukaryotes. Deficiency in H2Bub causes genome instability and cancer. How the Bre1-H2Bub pathway is evoked in response to DNA replication or repair remains unknown. Here, we identify that the single-stranded DNA (ssDNA) binding factor RPA acts as a key mediator that couples Bre1-mediated H2Bub to DNA replication and repair in yeast. We found that RPA interacts with Bre1 in vitro and in vivo, and this interaction is stimulated by ssDNA. This association ensures the recruitment of Bre1 to replication forks or DNA breaks but does not affect its E3 ligase activity. Disruption of the interaction abolishes the local enrichment of H2Bub, resulting in impaired DNA replication, response to replication stress, and repair by homologous recombination, accompanied by increased genome instability and DNA damage sensitivity. Notably, we found that RNF20, the human homolog of Bre1, interacts with RPA70 in a conserved mode. Thus, RPA functions as a master regulator for the spatial-temporal control of H2Bub chromatin landscape during DNA replication and recombination, extending the versatile roles of RPA in guarding genome stability.
Topics: DNA Repair; DNA Replication; DNA, Single-Stranded; Histones; Homologous Recombination; Methyl Methanesulfonate; Protein Interaction Domains and Motifs; Replication Protein A; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 33602814
DOI: 10.1073/pnas.2017497118 -
ELife Jun 2023The replication checkpoint is essential for accurate DNA replication and repair, and maintenance of genomic integrity when a cell is challenged with genotoxic stress....
The replication checkpoint is essential for accurate DNA replication and repair, and maintenance of genomic integrity when a cell is challenged with genotoxic stress. Several studies have defined the complement of proteins that change subcellular location in the budding yeast following chemically induced DNA replication stress using methyl methanesulfonate (MMS) or hydroxyurea (HU). How these protein movements are regulated remains largely unexplored. We find that the essential checkpoint kinases Mec1 and Rad53 are responsible for regulating the subcellular localization of 159 proteins during MMS-induced replication stress. Unexpectedly, Rad53 regulation of the localization of 52 proteins is independent of its known kinase activator Mec1, and in some scenarios independent of Tel1 or the mediator proteins Rad9 and Mrc1. We demonstrate that Rad53 is phosphorylated and active following MMS exposure in cells lacking Mec1 and Tel1. This noncanonical mode of Rad53 activation depends partly on the retrograde signaling transcription factor Rtg3, which also facilitates proper DNA replication dynamics. We conclude that there are biologically important modes of Rad53 protein kinase activation that respond to replication stress and operate in parallel to Mec1 and Tel1.
Topics: Protein Serine-Threonine Kinases; Cell Cycle Proteins; Saccharomyces cerevisiae Proteins; Intracellular Signaling Peptides and Proteins; Checkpoint Kinase 2; Saccharomyces cerevisiae; Phosphorylation; DNA Damage; Methyl Methanesulfonate; DNA Replication
PubMed: 37278514
DOI: 10.7554/eLife.82483 -
Genes Feb 2022The identification of mutants through forward genetic screens is the backbone of genetics research, yet many mutants identified through these screens have yet to be...
The identification of mutants through forward genetic screens is the backbone of genetics research, yet many mutants identified through these screens have yet to be mapped to the genome. This is especially true of mutants that have been identified as mutagen-sensitive (), but have not yet been mapped to their associated molecular locus. Our study addressed the need for additional gene identification by determining the locus and exploring the function of the -linked mutagen-sensitive gene using three available mutant alleles: , , and . After first confirming that all three alleles were sensitive to methyl methanesulfonate (MMS) using complementation analysis, we used deletion mapping to narrow the candidate genes for Through DNA sequencing, we were able to determine that is the uncharacterized gene which encodes the ortholog of the highly conserved DNA2 protein that is important for DNA replication and repair. We further used the sequence and structure of DNA2 to predict the impact of the allele mutations on the final gene product. Together, these results provide a tool for researchers to further investigate the role of DNA2 in DNA repair processes in
Topics: Animals; DNA Repair; Drosophila; Drosophila melanogaster; Methyl Methanesulfonate; Mutagens
PubMed: 35205357
DOI: 10.3390/genes13020312 -
Plant Direct Jul 2023Phytoplasmas induce diseases in more than 1000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma...
Phytoplasmas induce diseases in more than 1000 plant species and cause substantial ecological damage and economic losses, but the specific pathogenesis of phytoplasma has not yet been clarified. -methyladenosine (mA) is the most common internal modification of the eukaryotic Messenger RNA (mRNA). As one of the species susceptible to phytoplasma infection, the pathogenesis and mechanism of Paulownia has been extensively studied by scholars, but the mA transcriptome map of () has not been reported. Therefore, this study aimed to explore the effect of phytoplasma infection on mA modification of and obtained the whole transcriptome mA map in by mA-seq. The mA-seq results of Paulownia witches' broom (PaWB) disease and healthy samples indicate that PaWB infection increased the degree of mA modification of . The correlation analysis between the RNA-seq and mA-seq data detected that a total of 315 differentially methylated genes were predicted to be significantly differentially expressed at the transcriptome level. Moreover, the functions of PaWB-related genes were predicted by functional enrichment analysis, and two genes related to maintenance of the basic mechanism of stem cells in shoot apical meristem were discovered. One of the genes encodes the receptor protein kinase CLV2 (Paulownia_LG2G000076), and the other gene encodes the homeobox transcription factor STM (Paulownia_LG15G000976). In addition, genes F-box (Paulownia_LG17G000760) and MSH5 (Paulownia_LG8G001160) had exon skipping and mutually exclusive exon types of alternative splicing in PaWB-infected seedling treated with methyl methanesulfonate, and mA modification was found in mA-seq results. Moreover, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) verified that the alternative splicing of these two genes was associated with mA modification. This comprehensive map provides a solid foundation for revealing the potential function of the mRNA mA modification in the process of PaWB. In future studies, we plan to verify genes directly related to PaWB and methylation-related enzymes in Paulownia to elucidate the pathogenic mechanism of PaWB caused by phytoplasma invasion.
PubMed: 37426893
DOI: 10.1002/pld3.508 -
Environmental Science and Pollution... Nov 2023In this study, the toxicity induced by the alkylating agent methyl methanesulfonate (MMS) in Allium cepa L. was investigated. For this aim, bulbs were divided into 4...
In this study, the toxicity induced by the alkylating agent methyl methanesulfonate (MMS) in Allium cepa L. was investigated. For this aim, bulbs were divided into 4 groups as control and application (100, 500 and 4000 µM MMS) and germinated for 72 h at 22-24 °C. At the end of the germination period root tips were collected and made ready for analysis by applying traditional preparation methods. Germination, root elongation, weight, mitotic index (MI) values, micronucleus (MN) and chromosomal abnormality (CAs) numbers, malondialdehyde (MDA) levels, superoxide dismutase (SOD) and catalase (CAT) activities and anatomical structures of bulbs were used as indicators to determine toxicity. Moreover the extent of DNA fragmentation induced by MMS was determined by comet assay. To confirm the DNA fragmentation induced by MMS, the DNA-MMS interaction was examined with molecular docking. Correlation and principal component analyses (PCA) were performed to examine the relationship between all parameters and understand the underlying structure and relationships among these parameters. In the present study, a deep neural network (DNN) with two hidden layers implemented in Matlab has been developed for the comparison of the estimated data with the real data. The effect of MDA levels, SOD and CAT activities at 4 different endpoints resulting from administration of various concentrations of MMS, including MN, MI, CAs and DNA damage, was attempted to be estimated by DNN model. It is assumed that the predicted results are in close agreement with the actual data. The effectiveness of the model was evaluated using 4 different metrics, MAE, MAPE, RMSE and R2, which together show that the model performs commendably. As a result, the highest germination, root elongation, weight gain and MI were measured in the control group. MMS application caused a decrease in all physiological parameters and an increase in cytogenetic (except MI) and biochemical parameters. MMS application caused an increase in antioxidant enzyme levels (SOD and CAT) up to a concentration of 500 µM and a decrease at 4000 µM. MMS application induced different types of CAs and anatomical damages in root meristem cells. The results of the comet assay showed that the severity of DNA fragmentation increased with increasing MMS concentration. Molecular docking analysis showed a strong DNA-MMS interaction. The results of correlation and PCA revealed significant positive and negative interactions between the studied parameters and confirmed the interactions of these parameters with MMS. It has been shown that the DNN model developed in this study is a valuable resource for predicting genotoxicity due to oxidative stress and lipid peroxidation. In addition, this model has the potential to help evaluate the genotoxicity status of various chemical compounds. At the end of the study, it was concluded that MMS strongly supports a versatile toxicity in plant cells and the selected parameters are suitable indicators for determining this toxicity.
Topics: Methyl Methanesulfonate; Molecular Docking Simulation; Antioxidants; Plant Roots; Meristem; Superoxide Dismutase; Chromosome Aberrations; Onions; DNA; DNA Damage
PubMed: 37874518
DOI: 10.1007/s11356-023-30465-0 -
Royal Society Open Science Sep 2021The winged-helix domain of the methyl methanesulfonate and ultraviolet-sensitive 81 (MUS81) is a potential cancer drug target. In this context, marine fungi compounds...
The winged-helix domain of the methyl methanesulfonate and ultraviolet-sensitive 81 (MUS81) is a potential cancer drug target. In this context, marine fungi compounds were indicated to be able to prevent MUS81 structure via atomistic simulations. Eight compounds such as (), (), (), (), (), (), () and () were indicated that they are able to prevent the conformation of MUS81 via forming a strong binding affinity to the enzyme via perturbation approach. The electrostatic interaction is the dominant factor in the binding process of ligands to MUS81. The residues Trp55, Arg59, Leu62, His63 and Arg69 were found to frequently form non-bonded contacts and hydrogen bonds to inhibitors. Moreover, the influence of the ligand , which formed the lowest binding free energy to MUS81, on the structural change of enzyme was investigated using replica exchange molecular dynamics simulations. The obtained results indicated that , which forms a strong binding affinity, can modify the structure of MUS81. Overall, the marine compounds probably inhibit MUS81 due to forming a strong binding affinity to the enzyme as well as altering the enzymic conformation.
PubMed: 34527278
DOI: 10.1098/rsos.210974 -
ChemPlusChem May 2021A series of four V-shaped methylpyrimidinium salts bearing diphenylamino-electron-donating groups appended at various pyrimidine positions were designed. These...
A series of four V-shaped methylpyrimidinium salts bearing diphenylamino-electron-donating groups appended at various pyrimidine positions were designed. These chromophores were obtained by regioselective N(1) monomethylation by methyl methanesulfonate of the pyrimidine core. Linear optical properties were studied experimentally and electronic properties were further completed by (TD)-DFT calculations. The second-order nonlinear optical (NLO) properties were also studied using electric field induced second harmonic generation (EFISH) method in chloroform, and all pyrimidinium salts exhibited μβ >1000×10 esu. The 2,4-disubstituted pyrimidinium core is preferred over 4,6-disubstitution as it enhances the NLO response and increases the dipole moment. (E,E)-2,4-Bis[4-(diphenylamino)styryl]-1-methylpyrimidin-1-ium methanesulfonate appears to be the best NLO-phore in chloroform in the series (μβ =2500×10 esu) and a figure of merit μβ /MW=3.4 10 esu mol g .
PubMed: 33973733
DOI: 10.1002/cplu.202100081