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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 -
Molecular Cell Feb 2021Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to...
Chromatin is a barrier to efficient DNA repair, as it hinders access and processing of certain DNA lesions. ALC1/CHD1L is a nucleosome-remodeling enzyme that responds to DNA damage, but its precise function in DNA repair remains unknown. Here we report that loss of ALC1 confers sensitivity to PARP inhibitors, methyl-methanesulfonate, and uracil misincorporation, which reflects the need to remodel nucleosomes following base excision by DNA glycosylases but prior to handover to APEX1. Using CRISPR screens, we establish that ALC1 loss is synthetic lethal with homologous recombination deficiency (HRD), which we attribute to chromosome instability caused by unrepaired DNA gaps at replication forks. In the absence of ALC1 or APEX1, incomplete processing of BER intermediates results in post-replicative DNA gaps and a critical dependence on HR for repair. Hence, targeting ALC1 alone or as a PARP inhibitor sensitizer could be employed to augment existing therapeutic strategies for HRD cancers.
Topics: Animals; Chromatin Assembly and Disassembly; DNA Helicases; DNA Replication; DNA-(Apurinic or Apyrimidinic Site) Lyase; DNA-Binding Proteins; Homologous Recombination; Mice; Mice, Knockout; Neoplasm Proteins; Neoplasms, Experimental; Nucleosomes; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases
PubMed: 33333017
DOI: 10.1016/j.molcel.2020.12.006 -
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 -
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 -
The EMBO Journal Dec 2023R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we...
R-loops represent a major source of replication stress, but the mechanism by which these structures impede fork progression remains unclear. To address this question, we monitored fork progression, arrest, and restart in Saccharomyces cerevisiae cells lacking RNase H1 and H2, two enzymes responsible for degrading RNA:DNA hybrids. We found that while RNase H-deficient cells could replicate their chromosomes normally under unchallenged growth conditions, their replication was impaired when exposed to hydroxyurea (HU) or methyl methanesulfonate (MMS). Treated cells exhibited increased levels of RNA:DNA hybrids at stalled forks and were unable to generate RPA-coated single-stranded (ssDNA), an important postreplicative intermediate in resuming replication. Similar impairments in nascent DNA resection and ssDNA formation at HU-arrested forks were observed in human cells lacking RNase H2. However, fork resection was fully restored by addition of triptolide, an inhibitor of transcription that induces RNA polymerase degradation. Taken together, these data indicate that RNA:DNA hybrids not only act as barriers to replication forks, but also interfere with postreplicative fork repair mechanisms if not promptly degraded by RNase H.
Topics: Humans; DNA Replication; RNA; Ribonucleases; DNA; Hydroxyurea; Ribonuclease H
PubMed: 37855233
DOI: 10.15252/embj.2022113104 -
Acta Crystallographica. Section E,... Oct 2021Nafamostat dimesylate {systematic name: [amino({6-[(4-{[amino(iminiumyl)methyl]amino}phenyl)carbonyloxy]naphthalen-2-yl})methylidene]azanium bis(methanesulfonate)}, CHNO...
Nafamostat dimesylate {systematic name: [amino({6-[(4-{[amino(iminiumyl)methyl]amino}phenyl)carbonyloxy]naphthalen-2-yl})methylidene]azanium bis(methanesulfonate)}, CHNO ·2CHOS, is a broad-spectrum serine protease inhibitor and has been applied clinically as an anti-coagulant agent during hemodialysis and for treatment of severe acute pancreatitis (SAP). Since nafamostat contains flexible moieties, it is necessary to determine the conformation to understand the structure-activity relationships. The divalent cation has a screw-like motif. The guanidinium group is approximately perpendicular to the naphthyl ring system, subtending a dihedral angle of 84.30 (14)°. In the crystal, the nafamostat mol-ecules form columnar structures surrounded by a hydro-philic region.
PubMed: 34667626
DOI: 10.1107/S2056989021009245 -
Frontiers in Plant Science 2021Protein modification by the small ubiquitin-like modifier (SUMO) plays an important role in multiple plant processes, including growth, development, and the response to... (Review)
Review
Protein modification by the small ubiquitin-like modifier (SUMO) plays an important role in multiple plant processes, including growth, development, and the response to abiotic stresses. Mechanistically, SUMOylation is a sequential multi-enzymatic process where SUMO E3 ligases accelerate SUMO conjugation while also influencing target identity and interactions. This review explores the biological functions of plant SUMO E3 ligases [SAP AND MIZ1 DOMAIN-CONTAINING LIGASE (SIZs), METHYL METHANESULFONATE-SENSITIVITY PROTEIN 21 (MMS21s), and PROTEIN INHIBITOR OF ACTIVATED STAT-LIKE (PIALs)] in relation to their molecular activities and domains. We also explore the sub-cellular localization of SUMO E3 ligases and review evidence suggesting a connection between certain SUMO E3 ligases and DNA that contributes to gene expression regulation.
PubMed: 33897743
DOI: 10.3389/fpls.2021.652170 -
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