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Nature Jan 2024Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression. However, the precise mechanism...
Oncogene-induced replication stress generates endogenous DNA damage that activates cGAS-STING-mediated signalling and tumour suppression. However, the precise mechanism of cGAS activation by endogenous DNA damage remains enigmatic, particularly given that high-affinity histone acidic patch (AP) binding constitutively inhibits cGAS by sterically hindering its activation by double-stranded DNA (dsDNA). Here we report that the DNA double-strand break sensor MRE11 suppresses mammary tumorigenesis through a pivotal role in regulating cGAS activation. We demonstrate that binding of the MRE11-RAD50-NBN complex to nucleosome fragments is necessary to displace cGAS from acidic-patch-mediated sequestration, which enables its mobilization and activation by dsDNA. MRE11 is therefore essential for cGAS activation in response to oncogenic stress, cytosolic dsDNA and ionizing radiation. Furthermore, MRE11-dependent cGAS activation promotes ZBP1-RIPK3-MLKL-mediated necroptosis, which is essential to suppress oncogenic proliferation and breast tumorigenesis. Notably, downregulation of ZBP1 in human triple-negative breast cancer is associated with increased genome instability, immune suppression and poor patient prognosis. These findings establish MRE11 as a crucial mediator that links DNA damage and cGAS activation, resulting in tumour suppression through ZBP1-dependent necroptosis.
Topics: Humans; Cell Proliferation; Cell Transformation, Neoplastic; DNA Damage; MRE11 Homologue Protein; Necroptosis; Nucleosomes; Nucleotidyltransferases; Radiation, Ionizing; Triple Negative Breast Neoplasms; Genomic Instability
PubMed: 38200309
DOI: 10.1038/s41586-023-06889-6 -
Nature Jul 2023Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres. This...
Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments (PICh) to capture the telomeric DNA damage response proteome during BITS. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.
Topics: Animals; Cell Cycle Proteins; Chromatin; DNA Breaks, Double-Stranded; DNA Polymerase III; DNA Repair; DNA Replication; DNA-Binding Proteins; Exodeoxyribonucleases; Homologous Recombination; Mammals; Proliferating Cell Nuclear Antigen; Proteomics; Rad51 Recombinase; Telomere; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination; Templates, Genetic
PubMed: 37316655
DOI: 10.1038/s41586-023-06177-3 -
Molecular Cell Feb 2024Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and...
Inactivating mutations in the BRCA1 and BRCA2 genes impair DNA double-strand break (DSB) repair by homologous recombination (HR), leading to chromosomal instability and cancer. Importantly, BRCA1/2 deficiency also causes therapeutically targetable vulnerabilities. Here, we identify the dependency on the end resection factor EXO1 as a key vulnerability of BRCA1-deficient cells. EXO1 deficiency generates poly(ADP-ribose)-decorated DNA lesions during S phase that associate with unresolved DSBs and genomic instability in BRCA1-deficient but not in wild-type or BRCA2-deficient cells. Our data indicate that BRCA1/EXO1 double-deficient cells accumulate DSBs due to impaired repair by single-strand annealing (SSA) on top of their HR defect. In contrast, BRCA2-deficient cells retain SSA activity in the absence of EXO1 and hence tolerate EXO1 loss. Consistent with a dependency on EXO1-mediated SSA, we find that BRCA1-mutated tumors show elevated EXO1 expression and increased SSA-associated genomic scars compared with BRCA1-proficient tumors. Overall, our findings uncover EXO1 as a promising therapeutic target for BRCA1-deficient tumors.
Topics: Humans; BRCA1 Protein; BRCA2 Protein; DNA Damage; DNA Repair; DNA Repair Enzymes; Exodeoxyribonucleases; Homologous Recombination; Neoplasms
PubMed: 38266640
DOI: 10.1016/j.molcel.2023.12.039 -
The Journal of Experimental Medicine Jun 2023DNASE1 (D1) and DNASE1L3 (D1L3) synergistically reduce the severity of systemic infections caused by Staphylococcus aureus. In this issue of JEM, Lacey et al. (2023. J....
DNASE1 (D1) and DNASE1L3 (D1L3) synergistically reduce the severity of systemic infections caused by Staphylococcus aureus. In this issue of JEM, Lacey et al. (2023. J. Exp. Med.https://doi.org/10.1084/jem.20221086) develop D1-/-, D1L3-/-, and D1-/-D1L3-/- mice to show that exogenous addition of the DNase formulation Dornase alfa can facilitate removal of biofilms.
Topics: Mice; Animals; Endodeoxyribonucleases
PubMed: 37129875
DOI: 10.1084/jem.20230421 -
Biochemistry Jul 2023Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in...
Werner syndrome protein (WRN) is a multifunctional enzyme with helicase, ATPase, and exonuclease activities that are necessary for numerous DNA-related transactions in the human cell. Recent studies identified WRN as a synthetic lethal target in cancers characterized by genomic microsatellite instability resulting from defects in DNA mismatch repair pathways. WRN's helicase activity is essential for the viability of these high microsatellite instability (MSI-H) cancers and thus presents a therapeutic opportunity. To this end, we developed a multiplexed high-throughput screening assay that monitors exonuclease, ATPase, and helicase activities of full-length WRN. This screening campaign led to the discovery of 2-sulfonyl/sulfonamide pyrimidine derivatives as novel covalent inhibitors of WRN helicase activity. The compounds are specific for WRN versus other human RecQ family members and show competitive behavior with ATP. Examination of these novel chemical probes established the sulfonamide NH group as a key driver of compound potency. One of the leading compounds, H3B-960, showed consistent activities in a range of assays (IC = 22 nM, = 40 nM, = 32 nM), and the most potent compound identified, H3B-968, has inhibitory activity IC ∼ 10 nM. These kinetic properties trend toward other known covalent druglike molecules. Our work provides a new avenue for screening WRN for inhibitors that may be adaptable to different therapeutic modalities such as targeted protein degradation, as well as a proof of concept for the inhibition of WRN helicase activity by covalent molecules.
Topics: Humans; Werner Syndrome; Exodeoxyribonucleases; RecQ Helicases; High-Throughput Screening Assays; Microsatellite Instability; Werner Syndrome Helicase; Neoplasms
PubMed: 37403936
DOI: 10.1021/acs.biochem.2c00599 -
Nature Biotechnology Mar 2024A number of mitochondrial diseases in humans are caused by point mutations that could be corrected by base editors, but delivery of CRISPR guide RNAs into the...
A number of mitochondrial diseases in humans are caused by point mutations that could be corrected by base editors, but delivery of CRISPR guide RNAs into the mitochondria is difficult. In this study, we present mitochondrial DNA base editors (mitoBEs), which combine a transcription activator-like effector (TALE)-fused nickase and a deaminase for precise base editing in mitochondrial DNA. Combining mitochondria-localized, programmable TALE binding proteins with the nickase MutH or Nt.BspD6I(C) and either the single-stranded DNA-specific adenine deaminase TadA8e or the cytosine deaminase ABOBEC1 and UGI, we achieve A-to-G or C-to-T base editing with up to 77% efficiency and high specificity. We find that mitoBEs are DNA strand-selective mitochondrial base editors, with editing results more likely to be retained on the nonnicked DNA strand. Furthermore, we correct pathogenic mitochondrial DNA mutations in patient-derived cells by delivering mitoBEs encoded in circular RNAs. mitoBEs offer a precise, efficient DNA editing tool with broad applicability for therapy in mitochondrial genetic diseases.
Topics: Humans; Gene Editing; DNA, Mitochondrial; CRISPR-Cas Systems; RNA, Guide, CRISPR-Cas Systems; Mitochondria; Mitochondrial Diseases; Deoxyribonuclease I; Cytosine
PubMed: 37217751
DOI: 10.1038/s41587-023-01791-y -
Science Advances Sep 2023Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic...
Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.
Topics: Humans; Eukaryota; Deoxyribonuclease I; RNA; Deoxyribonucleases; Viruses
PubMed: 37756409
DOI: 10.1126/sciadv.adk0171 -
Nature Jan 2024Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage...
Bacteria encode hundreds of diverse defence systems that protect them from viral infection and inhibit phage propagation. Gabija is one of the most prevalent anti-phage defence systems, occurring in more than 15% of all sequenced bacterial and archaeal genomes, but the molecular basis of how Gabija defends cells from viral infection remains poorly understood. Here we use X-ray crystallography and cryo-electron microscopy (cryo-EM) to define how Gabija proteins assemble into a supramolecular complex of around 500 kDa that degrades phage DNA. Gabija protein A (GajA) is a DNA endonuclease that tetramerizes to form the core of the anti-phage defence complex. Two sets of Gabija protein B (GajB) dimers dock at opposite sides of the complex and create a 4:4 GajA-GajB assembly (hereafter, GajAB) that is essential for phage resistance in vivo. We show that a phage-encoded protein, Gabija anti-defence 1 (Gad1), directly binds to the Gabija GajAB complex and inactivates defence. A cryo-EM structure of the virally inhibited state shows that Gad1 forms an octameric web that encases the GajAB complex and inhibits DNA recognition and cleavage. Our results reveal the structural basis of assembly of the Gabija anti-phage defence complex and define a unique mechanism of viral immune evasion.
Topics: Bacteria; Bacterial Proteins; Bacteriophages; Cryoelectron Microscopy; Crystallography, X-Ray; Deoxyribonucleases; DNA, Viral; Immune Evasion; Protein Multimerization
PubMed: 37992757
DOI: 10.1038/s41586-023-06855-2 -
Frontiers in Immunology 2023Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a serious autoimmune disease that is characterized by vascular necrosis. The pathogenesis... (Review)
Review
Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) is a serious autoimmune disease that is characterized by vascular necrosis. The pathogenesis of AAV includes ANCA-mediated neutrophil activation, subsequent release of inflammatory cytokines and reactive oxygen species (ROS), and formation of neutrophil extracellular traps (NETs). Excessive NETs could participate not only in ANCA-mediated vascular injury but also in the production of ANCAs per se as autoantigens. Thus, a vicious cycle of NET formation and ANCA production is critical for AAV pathogenesis. Elucidating the molecular signaling pathways in aberrant neutrophil activation and NETs clearance systems will allow specific therapeutics to regulate these pathways. Currently, standard therapy with high doses of glucocorticoids and immunosuppressants has improved outcomes in patients with AAV. However, AAV frequently develops in elderly people, and adverse effects such as severe infections in the standard regimens might contribute to the mortality. Mechanistically, cytokines or complement factors activate and prime neutrophils for ANCA-binding; thus, C5a receptor blocker has garnered attention as potential replacement for glucocorticoids in clinical settings. Recent studies have demonstrated that receptor-interacting protein kinases (RIPK3) and cyclophilin D (CypD), which regulate cell necrosis, may be involved in ANCA-induced NETs formation. Meanwhile, targeting NETs clearance, including the addition of deoxyribonuclease I (DNase I) and macrophage engulfment, may improve vasculitis. In this review, we focus on the pathogenesis of NETs and discuss potential targeted therapies for AAV based on recent experimental evidence.
Topics: Humans; Aged; Extracellular Traps; Antibodies, Antineutrophil Cytoplasmic; Neutrophils; Necrosis; Anti-Neutrophil Cytoplasmic Antibody-Associated Vasculitis
PubMed: 37781373
DOI: 10.3389/fimmu.2023.1261151 -
Nucleic Acids Research Jun 2023DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair...
DNA double-strand break (DSB) repair via homologous recombination is initiated by end resection. The extent of DNA end resection determines the choice of the DSB repair pathway. Nucleases for end resection have been extensively studied. However, it is still unclear how the potential DNA structures generated by the initial short resection by MRE11-RAD50-NBS1 are recognized and recruit proteins, such as EXO1, to DSB sites to facilitate long-range resection. We found that the MSH2-MSH3 mismatch repair complex is recruited to DSB sites through interaction with the chromatin remodeling protein SMARCAD1. MSH2-MSH3 facilitates the recruitment of EXO1 for long-range resection and enhances its enzymatic activity. MSH2-MSH3 also inhibits access of POLθ, which promotes polymerase theta-mediated end-joining (TMEJ). Collectively, we present a direct role of MSH2-MSH3 in the initial stages of DSB repair by promoting end resection and influencing the DSB repair pathway by favoring homologous recombination over TMEJ.
Topics: DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Repair; Exodeoxyribonucleases; Homologous Recombination; MutS Homolog 2 Protein; Humans; Cell Line; DNA Helicases; MutS Homolog 3 Protein
PubMed: 37140056
DOI: 10.1093/nar/gkad308