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PLoS Biology Mar 2024Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA)...
Impediments in replication fork progression cause genomic instability, mutagenesis, and severe pathologies. At stalled forks, RPA-coated single-stranded DNA (ssDNA) activates the ATR kinase and directs fork remodeling, 2 key early events of the replication stress response. RFWD3, a recently described Fanconi anemia (FA) ubiquitin ligase, associates with RPA and promotes its ubiquitylation, facilitating late steps of homologous recombination (HR). Intriguingly, RFWD3 also regulates fork progression, restart and stability via poorly understood mechanisms. Here, we used proteomics to identify putative RFWD3 substrates during replication stress in human cells. We show that RFWD3 interacts with and ubiquitylates the SMARCAL1 DNA translocase directly in vitro and following DNA damage in vivo. SMARCAL1 ubiquitylation does not trigger its subsequent proteasomal degradation but instead disengages it from RPA thereby regulating its function at replication forks. Proper regulation of SMARCAL1 by RFWD3 at stalled forks protects them from excessive MUS81-mediated cleavage in response to UV irradiation, thereby limiting DNA replication stress. Collectively, our results identify RFWD3-mediated SMARCAL1 ubiquitylation as a novel mechanism that modulates fork remodeling to avoid genome instability triggered by aberrant fork processing.
Topics: Humans; DNA, Single-Stranded; DNA Replication; Replication Protein A; Protein Binding; Ubiquitination; DNA Damage; Genomic Instability; DNA Helicases; Ubiquitin-Protein Ligases
PubMed: 38502677
DOI: 10.1371/journal.pbio.3002552 -
The Journal of Clinical Investigation Jan 2024As the leading cause of disability worldwide, low back pain (LBP) is recognized as a pivotal socioeconomic challenge to the aging population and is largely attributed to...
As the leading cause of disability worldwide, low back pain (LBP) is recognized as a pivotal socioeconomic challenge to the aging population and is largely attributed to intervertebral disc degeneration (IVDD). Elastic nucleus pulposus (NP) tissue is essential for the maintenance of IVD structural and functional integrity. The accumulation of senescent NP cells with an inflammatory hypersecretory phenotype due to aging and other damaging factors is a distinctive hallmark of IVDD initiation and progression. In this study, we reveal a mechanism of IVDD progression in which aberrant genomic DNA damage promoted NP cell inflammatory senescence via activation of the cyclic GMP-AMP synthase/stimulator of IFN genes (cGAS/STING) axis but not of absent in melanoma 2 (AIM2) inflammasome assembly. Ataxia-telangiectasia-mutated and Rad3-related protein (ATR) deficiency destroyed genomic integrity and led to cytosolic mislocalization of genomic DNA, which acted as a powerful driver of cGAS/STING axis-dependent inflammatory phenotype acquisition during NP cell senescence. Mechanistically, disassembly of the ATR-tripartite motif-containing 56 (ATR-TRIM56) complex with the enzymatic liberation of ubiquitin-specific peptidase 5 (USP5) and TRIM25 drove changes in ATR ubiquitination, with ATR switching from K63- to K48-linked modification, c thereby promoting ubiquitin-proteasome-dependent dynamic instability of ATR protein during NP cell senescence progression. Importantly, an engineered extracellular vesicle-based strategy for delivering ATR-overexpressing plasmid cargo efficiently diminished DNA damage-associated NP cell senescence and substantially mitigated IVDD progression, indicating promising targets and effective approaches to ameliorate the chronic pain and disabling effects of IVDD.
Topics: Humans; Aged; Intervertebral Disc Degeneration; Nucleus Pulposus; Aging; Cellular Senescence; Nucleotidyltransferases; Intervertebral Disc; Tripartite Motif Proteins; Ubiquitin-Protein Ligases; Ataxia Telangiectasia Mutated Proteins
PubMed: 38488012
DOI: 10.1172/JCI165140 -
Journal of the American Chemical Society Mar 2024Given the prevalent advancements in DNA- and RNA-based PROTACs, there remains a significant need for the exploration and expansion of more specific DNA-based tools, thus...
Given the prevalent advancements in DNA- and RNA-based PROTACs, there remains a significant need for the exploration and expansion of more specific DNA-based tools, thus broadening the scope and repertoire of DNA-based PROTACs. Unlike conventional A- or B-form DNA, Z-form DNA is a configuration that exclusively manifests itself under specific stress conditions and with specific target sequences, which can be recognized by specific reader proteins, such as ADAR1 or ZBP1, to exert downstream biological functions. The core of our innovation lies in the strategic engagement of Z-form DNA with ADAR1 and its degradation is achieved by leveraging a VHL ligand conjugated to Z-form DNA to recruit the E3 ligase. This ingenious construct engendered a series of Z-PROTACs, which we utilized to selectively degrade the Z-DNA-binding protein ADAR1, a molecule that is frequently overexpressed in cancer cells. This meticulously orchestrated approach triggers a cascade of PANoptotic events, notably encompassing apoptosis and necroptosis, by mitigating the blocking effect of ADAR1 on ZBP1, particularly in cancer cells compared with normal cells. Moreover, the Z-PROTAC design exhibits a pronounced predilection for ADAR1, as opposed to other Z-DNA readers, such as ZBP1. As such, Z-PROTAC likely elicits a positive immunological response, subsequently leading to a synergistic augmentation of cancer cell death. In summary, the Z-DNA-based PROTAC (Z-PROTAC) approach introduces a modality generated by the conformational change from B- to Z-form DNA, which harnesses the structural specificity intrinsic to potentiate a selective degradation strategy. This methodology is an inspiring conduit for the advancement of PROTAC-based therapeutic modalities, underscoring its potential for selectivity within the therapeutic landscape of PROTACs to target undruggable proteins.
Topics: DNA, Z-Form; Proteolysis Targeting Chimera; Proteolysis; Adenosine Deaminase; RNA; Ubiquitin-Protein Ligases; DNA-Binding Proteins
PubMed: 38469801
DOI: 10.1021/jacs.3c13646 -
Nature Communications Mar 2024This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of...
This study establishes the physiological role of Fused in Sarcoma (FUS) in mitochondrial DNA (mtDNA) repair and highlights its implications to the pathogenesis of FUS-associated neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS). Endogenous FUS interacts with and recruits mtDNA Ligase IIIα (mtLig3) to DNA damage sites within mitochondria, a relationship essential for maintaining mtDNA repair and integrity in healthy cells. Using ALS patient-derived FUS mutant cell lines, a transgenic mouse model, and human autopsy samples, we discovered that compromised FUS functionality hinders mtLig3's repair role, resulting in increased mtDNA damage and mutations. These alterations cause various manifestations of mitochondrial dysfunction, particularly under stress conditions relevant to disease pathology. Importantly, rectifying FUS mutations in patient-derived induced pluripotent cells (iPSCs) preserves mtDNA integrity. Similarly, targeted introduction of human DNA Ligase 1 restores repair mechanisms and mitochondrial activity in FUS mutant cells, suggesting a potential therapeutic approach. Our findings unveil FUS's critical role in mitochondrial health and mtDNA repair, offering valuable insights into the mechanisms underlying mitochondrial dysfunction in FUS-associated motor neuron disease.
Topics: Animals; Humans; Mice; Amyotrophic Lateral Sclerosis; DNA, Mitochondrial; Ligases; Mice, Transgenic; Mitochondrial Diseases; Motor Neuron Disease; Mutation; RNA-Binding Protein FUS; DNA Ligase ATP
PubMed: 38461154
DOI: 10.1038/s41467-024-45978-6 -
Molecular Cell Apr 2024Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept...
Although mismatch repair (MMR) is essential for correcting DNA replication errors, it can also recognize other lesions, such as oxidized bases. In G0 and G1, MMR is kept in check through unknown mechanisms as it is error-prone during these cell cycle phases. We show that in mammalian cells, D-type cyclins are recruited to sites of oxidative DNA damage in a PCNA- and p21-dependent manner. D-type cyclins inhibit the proteasomal degradation of p21, which competes with MMR proteins for binding to PCNA, thereby inhibiting MMR. The ability of D-type cyclins to limit MMR is CDK4- and CDK6-independent and is conserved in G0 and G1. At the G1/S transition, the timely, cullin-RING ubiquitin ligase (CRL)-dependent degradation of D-type cyclins and p21 enables MMR activity to efficiently repair DNA replication errors. Persistent expression of D-type cyclins during S-phase inhibits the binding of MMR proteins to PCNA, increases the mutational burden, and promotes microsatellite instability.
Topics: Animals; Cyclins; Proliferating Cell Nuclear Antigen; DNA Mismatch Repair; Cyclin-Dependent Kinase Inhibitor p21; Interphase; Mammals
PubMed: 38458201
DOI: 10.1016/j.molcel.2024.02.010 -
Indian Journal of Ophthalmology Jul 2024Retinoblastoma (RB) is the most common intraocular tumor in pediatric age group. The role of genetics has been explored in predicting survival prognosis, but its role in...
PURPOSE
Retinoblastoma (RB) is the most common intraocular tumor in pediatric age group. The role of genetics has been explored in predicting survival prognosis, but its role in predicting globe salvage remains largely unexplored. We hereby aim to isolate cell-free DNA (cfDNA) from aqueous humor (AH) in RB eyes and validate its use for genetic studies.
METHODS
AH was obtained from 26 eyes undergoing enucleation (arm A) or intravitreal chemotherapy (arm B). Isolation of cfDNA was done using QIAamp ® Circulating Nucleic Acid kit, and the cfDNA was utilized for targeted sequencing of RB1 gene.
RESULTS
We could isolate cfDNA in all eyes (72% unilateral and 28% bilateral) with a distribution peak between 140 and 160 bp and a mean concentration of 27.75 ng/µl for arm A and 14 ng/µl for arm B. Targeted sequencing done on four samples showed RB1 gene mutations, namely, inframe deletion (c. 78-80del, p.Pro29del), start-loss mutation (c.1A>T, p.Met1?), nonsense mutations (c.2236G>T, p.Glu746Ter), (c.1659T>A, p.Cys553Ter), and (c.2065C>T, p.Gln689Ter), and novel missense mutations (c.672C>A, p.Asp224Glu) and c.692C>T (p.Pro231Leu). Genetic profile of cfDNA extracted from AH and genomic DNA from the tumor tissue was comparable.
CONCLUSION
Our study supports the previous reports that AH may be used as a source of tumor-derived cfDNA. This is the first report from South Asia on isolation and genetic analysis of cfDNA from AH of RB eyes and, therefore, a big step forward in paving the role of tumor genetics in RB. Further studies are required to elucidate concordance between the tumor and AH genetic profile.
Topics: Humans; Retinoblastoma; Retinal Neoplasms; Aqueous Humor; Male; Female; Child, Preschool; Infant; DNA, Neoplasm; Mutation; Eye Enucleation; Child; Biomarkers, Tumor; India; Retinoblastoma Binding Proteins; Asia, Southern; Ubiquitin-Protein Ligases
PubMed: 38454873
DOI: 10.4103/IJO.IJO_234_23 -
Genes & Development Mar 2024Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo...
A maternal-effect variant causes nuclear and cytoplasmic abnormalities in oocytes, as well as failure of epigenetic reprogramming and zygotic genome activation in embryos.
Maternal inactivation of genes encoding components of the subcortical maternal complex (SCMC) and its associated member, PADI6, generally results in early embryo lethality. In humans, SCMC gene variants were found in the healthy mothers of children affected by multilocus imprinting disturbances (MLID). However, how the SCMC controls the DNA methylation required to regulate imprinting remains poorly defined. We generated a mouse line carrying a missense variant that was identified in a family with Beckwith-Wiedemann syndrome and MLID. If homozygous in female mice, this variant resulted in interruption of embryo development at the two-cell stage. Single-cell multiomic analyses demonstrated defective maturation of mutant oocytes and incomplete DNA demethylation, down-regulation of zygotic genome activation (ZGA) genes, up-regulation of maternal decay genes, and developmental delay in two-cell embryos developing from mutant oocytes but little effect on genomic imprinting. Western blotting and immunofluorescence analyses showed reduced levels of UHRF1 in oocytes and abnormal localization of DNMT1 and UHRF1 in both oocytes and zygotes. Treatment with 5-azacytidine reverted DNA hypermethylation but did not rescue the developmental arrest of mutant embryos. Taken together, this study demonstrates that PADI6 controls both nuclear and cytoplasmic oocyte processes that are necessary for preimplantation epigenetic reprogramming and ZGA.
Topics: Animals; Child; Female; Humans; Mice; CCAAT-Enhancer-Binding Proteins; Cytoplasm; DNA Methylation; Embryonic Development; Genomic Imprinting; Oocytes; Ubiquitin-Protein Ligases; Zygote
PubMed: 38453481
DOI: 10.1101/gad.351238.123 -
Frontiers in Immunology 2024African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly mortal and hemorrhagic infectious disease in pigs. Previous studies have indicated that...
African swine fever (ASF) caused by African swine fever virus (ASFV) is a highly mortal and hemorrhagic infectious disease in pigs. Previous studies have indicated that ASFV modulates interferon (IFN) production. In this study, we demonstrated that ASFV pA151R negatively regulated type I IFN production. Ectopic expression of pA151R dramatically inhibited K63-linked polyubiquitination and Ser172 phosphorylation of TANK-binding kinase 1 (TBK1). Mechanically, we demonstrated that E3 ligase TNF receptor-associated factor 6 (TRAF6) participated in the ubiquitination of TBK1 in cGAS-STING signaling pathway. We showed that pA151R interacted with TRAF6 and degraded it through apoptosis pathway, leading to the disruption of TBK1 and TRAF6 interaction. Moreover, we clarified that the amino acids H102, C109, C132, and C135 in pA151R were crucial for pA151R to inhibit type I interferon production. In addition, we verified that overexpression of pA151R facilitated DNA virus Herpes simplex virus 1 (HSV-1) replication by inhibiting IFN-β production. Importantly, knockdown of pA151R inhibited ASFV replication and enhanced IFN-β production in porcine alveolar macrophages (PAMs). Our findings will help understand how ASFV escapes host antiviral immune responses and develop effective ASFV vaccines.
Topics: Animals; Swine; Ubiquitin-Protein Ligases; African Swine Fever Virus; TNF Receptor-Associated Factor 6; African Swine Fever; Ubiquitination
PubMed: 38449860
DOI: 10.3389/fimmu.2024.1339510 -
The EMBO Journal Apr 2024MAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase...
MAGEA4 is a cancer-testis antigen primarily expressed in the testes but aberrantly overexpressed in several cancers. MAGEA4 interacts with the RING ubiquitin ligase RAD18 and activates trans-lesion DNA synthesis (TLS), potentially favouring tumour evolution. Here, we employed NMR and AlphaFold2 (AF) to elucidate the interaction mode between RAD18 and MAGEA4, and reveal that the RAD6-binding domain (R6BD) of RAD18 occupies a groove in the C-terminal winged-helix subdomain of MAGEA4. We found that MAGEA4 partially displaces RAD6 from the RAD18 R6BD and inhibits degradative RAD18 autoubiquitination, which could be countered by a competing peptide of the RAD18 R6BD. AlphaFold2 and cross-linking mass spectrometry (XL-MS) also revealed an evolutionary invariant intramolecular interaction between the catalytic RING and the DNA-binding SAP domains of RAD18, which is essential for PCNA mono-ubiquitination. Using interaction proteomics, we found that another Type-I MAGE, MAGE-C2, interacts with the RING ubiquitin ligase TRIM28 in a manner similar to the MAGEA4/RAD18 complex, suggesting that the MAGEA4 peptide-binding groove also serves as a ligase-binding cleft in other type-I MAGEs. Our data provide new insights into the mechanism and regulation of RAD18-mediated PCNA mono-ubiquitination.
Topics: Proliferating Cell Nuclear Antigen; Ubiquitin-Conjugating Enzymes; Ubiquitin-Protein Ligases; Ubiquitination; Ubiquitin; Peptides; DNA Damage
PubMed: 38448672
DOI: 10.1038/s44318-024-00058-9 -
Cell Reports Mar 2024The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 for DNA methylation maintenance during DNA replication. Here,...
The multi-domain protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains, 1) recruits DNMT1 for DNA methylation maintenance during DNA replication. Here, we show that MOF (males absent on the first) acetylates UHRF1 at K670 in the pre-RING linker region, whereas HDAC1 deacetylates UHRF1 at the same site. We also identify that K667 and K668 can also be acetylated by MOF when K670 is mutated. The MOF/HDAC1-mediated acetylation in UHRF1 is cell-cycle regulated and peaks at G1/S phase, in line with the function of UHRF1 in recruiting DNMT1 to maintain DNA methylation. In addition, UHRF1 acetylation significantly enhances its E3 ligase activity. Abolishing UHRF1 acetylation at these sites attenuates UHRF1-mediated H3 ubiquitination, which in turn impairs DNMT1 recruitment and DNA methylation. Taken together, these findings identify MOF as an acetyltransferase for UHRF1 and define a mechanism underlying the regulation of DNA methylation maintenance through MOF-mediated UHRF1 acetylation.
Topics: Male; Humans; DNA Methylation; Histones; Acetylation; Ubiquitin-Protein Ligases; CCAAT-Enhancer-Binding Proteins; Ubiquitination; DNA (Cytosine-5-)-Methyltransferase 1
PubMed: 38446667
DOI: 10.1016/j.celrep.2024.113908