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Nature Aug 2021The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination. The BRCA1-BARD1 complex...
The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks. We further show that RING domains in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.
Topics: BRCA1 Protein; Cryoelectron Microscopy; DNA Repair; Histones; Homologous Recombination; Humans; Models, Molecular; Mutation; Neoplasms; Nucleosomes; Protein Domains; Tumor Suppressor Proteins; Tumor Suppressor p53-Binding Protein 1; Ubiquitin; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 34321665
DOI: 10.1038/s41586-021-03716-8 -
Nature Nov 2021Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different...
Extrachromosomal circular DNA elements (eccDNAs) have been described in the literature for several decades, and are known for their broad existence across different species. However, their biogenesis and functions are largely unknown. By developing a new circular DNA enrichment method, here we purified and sequenced full-length eccDNAs with Nanopore sequencing. We found that eccDNAs map across the entire genome in a close to random manner, suggesting a biogenesis mechanism of random ligation of genomic DNA fragments. Consistent with this idea, we found that apoptosis inducers can increase eccDNA generation, which is dependent on apoptotic DNA fragmentation followed by ligation by DNA ligase 3. Importantly, we demonstrated that eccDNAs can function as potent innate immunostimulants in a manner that is independent of eccDNA sequence but dependent on eccDNA circularity and the cytosolic DNA sensor Sting. Collectively, our study not only revealed the origin, biogenesis and immunostimulant function of eccDNAs but also uncovered their sensing pathway and potential clinical implications in immune response.
Topics: Animals; Apoptosis; Cells, Cultured; Chromosome Mapping; DNA Fragmentation; DNA Ligase ATP; DNA, Circular; Endodeoxyribonucleases; Gene Expression Regulation; Genome; Immunity, Innate; Male; Membrane Proteins; Mice; Poly-ADP-Ribose Binding Proteins
PubMed: 34671165
DOI: 10.1038/s41586-021-04009-w -
Cell Feb 2021Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing...
Mutations in DNA damage response (DDR) genes endanger genome integrity and predispose to cancer and genetic disorders. Here, using CRISPR-dependent cytosine base editing screens, we identify > 2,000 sgRNAs that generate nucleotide variants in 86 DDR genes, resulting in altered cellular fitness upon DNA damage. Among those variants, we discover loss- and gain-of-function mutants in the Tudor domain of the DDR regulator 53BP1 that define a non-canonical surface required for binding the deubiquitinase USP28. Moreover, we characterize variants of the TRAIP ubiquitin ligase that define a domain, whose loss renders cells resistant to topoisomerase I inhibition. Finally, we identify mutations in the ATM kinase with opposing genome stability phenotypes and loss-of-function mutations in the CHK2 kinase previously categorized as variants of uncertain significance for breast cancer. We anticipate that this resource will enable the discovery of additional DDR gene functions and expedite studies of DDR variants in human disease.
Topics: Amino Acid Sequence; Ataxia Telangiectasia Mutated Proteins; Base Sequence; CRISPR-Cas Systems; Camptothecin; Cell Line; DNA Damage; DNA Repair; Female; Gene Editing; Genetic Testing; Humans; Mutation; Phenotype; Protein Binding; Protein Domains; RNA, Guide, CRISPR-Cas Systems; Topoisomerase Inhibitors; Tumor Suppressor p53-Binding Protein 1; Ubiquitin Thiolesterase; Ubiquitin-Protein Ligases
PubMed: 33606978
DOI: 10.1016/j.cell.2021.01.041 -
Cell Death and Differentiation Nov 2019Ferroptosis is a recently identified form of regulated cell death defined by the iron-dependent accumulation of lipid reactive oxygen species. Ferroptosis has been...
Ferroptosis is a recently identified form of regulated cell death defined by the iron-dependent accumulation of lipid reactive oxygen species. Ferroptosis has been studied in various diseases such as cancer, Parkinson's disease, and stroke. However, the exact function and mechanism of ferroptosis in ischemia/reperfusion (I/R) injury, especially in the intestine, remains unknown. Considering the unique conditions required for ferroptosis, we hypothesize that ischemia promotes ferroptosis immediately after intestinal reperfusion. In contrast to conventional strategies employed in I/R studies, we focused on the ischemic phase. Here we verified ferroptosis by assessing proferroptotic changes after ischemia along with protein and lipid peroxidation levels during reperfusion. The inhibition of ferroptosis by liproxstatin-1 ameliorated I/R-induced intestinal injury. Acyl-CoA synthetase long-chain family member 4 (ACSL4), which is a key enzyme that regulates lipid composition, has been shown to contribute to the execution of ferroptosis, but its role in I/R needs clarification. In the present study, we used rosiglitazone (ROSI) and siRNA to inhibit ischemia/hypoxia-induced ACSL4 in vivo and in vitro. The results demonstrated that ACSL4 inhibition before reperfusion protected against ferroptosis and cell death. Further investigation revealed that special protein 1 (Sp1) was a crucial transcription factor that increased ACSL4 transcription by binding to the ACSL4 promoter region. Collectively, this study demonstrates that ferroptosis is closely associated with intestinal I/R injury, and that ACSL4 has a critical role in this lethal process. Sp1 is an important factor in promoting ACSL4 expression. These results suggest a unique and effective mechanistic approach for intestinal I/R injury prevention and treatment.
Topics: Animals; Caco-2 Cells; Cell Line, Tumor; Coenzyme A Ligases; DNA-Binding Proteins; Ferroptosis; Humans; Intestines; Lipid Peroxidation; Mice; Mice, Inbred C57BL; Models, Animal; Promoter Regions, Genetic; Quinoxalines; Reactive Oxygen Species; Reperfusion Injury; Rosiglitazone; Sp1 Transcription Factor; Spiro Compounds
PubMed: 30737476
DOI: 10.1038/s41418-019-0299-4 -
Molecular Cell Jul 2021Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect...
Mammalian DNA base excision repair (BER) is accelerated by poly(ADP-ribose) polymerases (PARPs) and the scaffold protein XRCC1. PARPs are sensors that detect single-strand break intermediates, but the critical role of XRCC1 during BER is unknown. Here, we show that protein complexes containing DNA polymerase β and DNA ligase III that are assembled by XRCC1 prevent excessive engagement and activity of PARP1 during BER. As a result, PARP1 becomes "trapped" on BER intermediates in XRCC1-deficient cells in a manner similar to that induced by PARP inhibitors, including in patient fibroblasts from XRCC1-mutated disease. This excessive PARP1 engagement and trapping renders BER intermediates inaccessible to enzymes such as DNA polymerase β and impedes their repair. Consequently, PARP1 deletion rescues BER and resistance to base damage in XRCC1 cells. These data reveal excessive PARP1 engagement during BER as a threat to genome integrity and identify XRCC1 as an "anti-trapper" that prevents toxic PARP1 activity.
Topics: Animals; Cell Line; DNA; DNA Breaks, Single-Stranded; DNA Damage; DNA Ligase ATP; DNA Polymerase beta; DNA Repair; DNA-Binding Proteins; Fibroblasts; Humans; Poly (ADP-Ribose) Polymerase-1; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases; Protein Binding; X-ray Repair Cross Complementing Protein 1
PubMed: 34102106
DOI: 10.1016/j.molcel.2021.05.009 -
Nature Communications Jan 2022Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC), and approximately 20% of patients experience treatment failure due to tumour...
Radiotherapy is the primary treatment for patients with nasopharyngeal carcinoma (NPC), and approximately 20% of patients experience treatment failure due to tumour radioresistance. However, the exact regulatory mechanism remains poorly understood. Here, we show that the deubiquitinase USP44 is hypermethylated in NPC, which results in its downregulation. USP44 enhances the sensitivity of NPC cells to radiotherapy in vitro and in vivo. USP44 recruits and stabilizes the E3 ubiquitin ligase TRIM25 by removing its K48-linked polyubiquitin chains at Lys439, which further facilitates the degradation of Ku80 and inhibits its recruitment to DNA double-strand breaks (DSBs), thus enhancing DNA damage and inhibiting DNA repair via non-homologous end joining (NHEJ). Knockout of TRIM25 reverses the radiotherapy sensitization effect of USP44. Clinically, low expression of USP44 indicates a poor prognosis and facilitates tumour relapse in NPC patients. This study suggests the USP44-TRIM25-Ku80 axis provides potential therapeutic targets for NPC patients.
Topics: Apoptosis; Carcinogenesis; Cell Line; Cell Line, Tumor; DNA Breaks, Double-Stranded; DNA Methylation; DNA Repair; G2 Phase Cell Cycle Checkpoints; Gene Expression Regulation, Neoplastic; HEK293 Cells; Humans; Ku Autoantigen; Nasopharyngeal Carcinoma; Nasopharyngeal Neoplasms; Promoter Regions, Genetic; Radiation Tolerance; Survival Analysis; Transcription Factors; Tripartite Motif Proteins; Ubiquitin Thiolesterase; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 35079021
DOI: 10.1038/s41467-022-28158-2 -
Nucleic Acids Research Sep 2023Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA...
Histone deacetylase 6 (HDAC6) mediates DNA damage signaling by regulating the mismatch repair and nucleotide excision repair pathways. Whether HDAC6 also mediates DNA double-strand break (DSB) repair is unclear. Here, we report that HDAC6 negatively regulates DSB repair in an enzyme activity-independent manner. In unstressed cells, HDAC6 interacts with H2A/H2A.X to prevent its interaction with the E3 ligase RNF168. Upon sensing DSBs, RNF168 rapidly ubiquitinates HDAC6 at lysine 116, leading to HDAC6 proteasomal degradation and a restored interaction between RNF168 and H2A/H2A.X. H2A/H2A.X is ubiquitinated by RNF168, precipitating the recruitment of DSB repair factors (including 53BP1 and BRCA1) to chromatin and subsequent DNA repair. These findings reveal novel regulatory machinery based on an HDAC6-RNF168 axis that regulates the H2A/H2A.X ubiquitination status. Interfering with this axis might be leveraged to disrupt a key mechanism of cancer cell resistance to genotoxic damage and form a potential therapeutic strategy for cancer.
Topics: Humans; Cell Line, Tumor; DNA Damage; DNA Repair; Histone Deacetylase 6; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 37503842
DOI: 10.1093/nar/gkad631 -
American Journal of Physiology.... Jul 2020Mitochondrial injury in granulosa cells is associated with the pathogenesis of polycystic ovary syndrome (PCOS). However, the protective effects of melatonin against...
Mitochondrial injury in granulosa cells is associated with the pathogenesis of polycystic ovary syndrome (PCOS). However, the protective effects of melatonin against mitochondrial injury in the granulosa cells of PCOS remain unclear. In this study, decreased mitochondrial membrane potential and mtDNA content, increased number of autophagosomes were found in the granulosa cells of PCOS patients and the dihydrotestosterone (DHT)-treated KGN cells, with decreased protein level of the autophagy substrate p62 and increased levels of the cellular autophagy markers Beclin 1 and LC3B-II, while the protein levels of PTEN-induced kinase-1 (PINK1) and Parkin were increased and the level of sirtuin 1 (SIRT1) was decreased. DHT-induced PCOS-like mice also showed enhanced mitophagy and decreased mRNA expression. Melatonin treatment significantly increased the protein level of SIRT1 and decreased the levels of PINK1/Parkin, whereas it ameliorated the mitochondrial dysfunction and PCOS phenotype in vitro and in vivo. However, when the KGN cells were treated with siRNA to knock down SIRT1 expression, melatonin treatment failed to repress the excessive mitophagy. In conclusion, melatonin protects against mitochondrial injury in granulosa cells of PCOS by enhancing SIRT1 expression to inhibit excessive PINK1/Parkin-mediated mitophagy.
Topics: Adult; Animals; Antioxidants; Autophagosomes; Autophagy; Beclin-1; Case-Control Studies; Cell Line; DNA, Mitochondrial; Dihydrotestosterone; Female; Granulosa Cells; Humans; Melatonin; Membrane Potential, Mitochondrial; Mice; Microtubule-Associated Proteins; Mitophagy; Polycystic Ovary Syndrome; Protein Kinases; Sirtuin 1; Ubiquitin-Protein Ligases
PubMed: 32343612
DOI: 10.1152/ajpendo.00006.2020 -
Molecular Cell Oct 2021PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach...
PRIMPOL repriming allows DNA replication to skip DNA lesions, leading to ssDNA gaps. These gaps must be filled to preserve genome stability. Using a DNA fiber approach to directly monitor gap filling, we studied the post-replicative mechanisms that fill the ssDNA gaps generated in cisplatin-treated cells upon increased PRIMPOL expression or when replication fork reversal is defective because of SMARCAL1 inactivation or PARP inhibition. We found that a mechanism dependent on the E3 ubiquitin ligase RAD18, PCNA monoubiquitination, and the REV1 and POLζ translesion synthesis polymerases promotes gap filling in G2. The E2-conjugating enzyme UBC13, the RAD51 recombinase, and REV1-POLζ are instead responsible for gap filling in S, suggesting that temporally distinct pathways of gap filling operate throughout the cell cycle. Furthermore, we found that BRCA1 and BRCA2 promote gap filling by limiting MRE11 activity and that simultaneously targeting fork reversal and gap filling enhances chemosensitivity in BRCA-deficient cells.
Topics: Antineoplastic Agents; BRCA1 Protein; BRCA2 Protein; Cell Line, Tumor; DNA Breaks, Single-Stranded; DNA Helicases; DNA Primase; DNA Repair; DNA Replication; DNA, Neoplasm; DNA-Binding Proteins; DNA-Directed DNA Polymerase; G2 Phase; Genomic Instability; HEK293 Cells; Humans; MRE11 Homologue Protein; Multifunctional Enzymes; Neoplasms; Nucleotidyltransferases; Proliferating Cell Nuclear Antigen; S Phase; Time Factors; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 34624216
DOI: 10.1016/j.molcel.2021.09.013 -
Cell Death and Differentiation Jan 2023The precise regulation of STING homeostasis is essential for its antiviral function. Post-translational modification, especially ubiquitination, is important for the...
The precise regulation of STING homeostasis is essential for its antiviral function. Post-translational modification, especially ubiquitination, is important for the regulation of STING homeostasis. Previous studies have focused on how STING is degraded, but little is known about its maintenance. Here, we show that UFM1 specific ligase UFL1 promotes innate immune response by maintaining STING expression independent of UFMylation. Mechanistically, UFL1 inhibits TRIM29 to interact with STING, thereby reducing its ubiquitination at K338/K347/K370 and subsequent proteasomal degradation. DNA virus infection reduces the UFL1 expression, which may promote STING degradation and facilitate viral expansion. Our study identifies UFL1 as a crucial regulator for the maintenance of STING stability and antiviral function, and provides novel insights into the mechanistic explanation for the immunological escape of DNA virus.
Topics: Ubiquitin-Protein Ligases; Antiviral Agents; Ubiquitination; Protein Processing, Post-Translational; Immunity; Immunity, Innate
PubMed: 35871231
DOI: 10.1038/s41418-022-01041-9