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The FEBS Journal May 2022The chromatin-based DNA damage response pathway is tightly orchestrated by histone post-translational modifications, including histone H2A ubiquitination. Ubiquitination... (Review)
Review
The chromatin-based DNA damage response pathway is tightly orchestrated by histone post-translational modifications, including histone H2A ubiquitination. Ubiquitination plays an integral role in regulating cellular processes including DNA damage signaling and repair. The ubiquitin E3 ligase RNF168 is essential in assembling a cohort of DNA repair proteins at the damaged chromatin via its enzymatic activity. RNF168 ubiquitinates histone H2A(X) at the N terminus and generates a specific docking scaffold for ubiquitin-binding motif-containing proteins. The regulation of RNF168 at damaged chromatin and the mechanistic implication in the recruitment of DNA repair proteins to the damaged sites remain an area of active investigation. Here, we review the function and regulation of RNF168 in the context of ubiquitin-mediated DNA damage signaling and repair. We will also discuss the unanswered questions that require further investigation and how understanding RNF168 targeting specificity could benefit the therapeutic development for cancer treatment.
Topics: Chromatin; DNA Damage; Histones; Humans; Ubiquitin; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 33797206
DOI: 10.1111/febs.15857 -
Cell Reports Dec 2020The present study demonstrates that topoisomerase 3B (TOP3B) forms both RNA and DNA cleavage complexes (TOP3Bccs) in vivo and reveals a pathway for repairing TOP3Bccs....
The present study demonstrates that topoisomerase 3B (TOP3B) forms both RNA and DNA cleavage complexes (TOP3Bccs) in vivo and reveals a pathway for repairing TOP3Bccs. For inducing and detecting cellular TOP3Bccs, we engineer a "self-trapping" mutant of TOP3B (R338W-TOP3B). Transfection with R338W-TOP3B induces R-loops, genomic damage, and growth defect, which highlights the importance of TOP3Bcc repair mechanisms. To determine how cells repair TOP3Bccs, we deplete tyrosyl-DNA phosphodiesterases (TDP1 and TDP2). TDP2-deficient cells show elevated TOP3Bccs both in DNA and RNA. Conversely, overexpression of TDP2 lowers cellular TOP3Bccs. Using recombinant human TDP2, we demonstrate that TDP2 can process both denatured and proteolyzed TOP3Bccs. We also show that cellular TOP3Bccs are ubiquitinated by the E3 ligase TRIM41 before undergoing proteasomal processing and excision by TDP2.
Topics: Amino Acid Substitution; DNA; DNA Cleavage; DNA Repair; DNA Topoisomerases, Type I; DNA-Binding Proteins; Gene Knockout Techniques; HCT116 Cells; HEK293 Cells; Humans; Phosphoric Diester Hydrolases; Proteolysis; R-Loop Structures; RNA; RNA Cleavage; Recombinant Proteins; Ubiquitin-Protein Ligases; Ubiquitination
PubMed: 33378676
DOI: 10.1016/j.celrep.2020.108569 -
Structure (London, England : 1993) Mar 2022DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there...
DNA ligases act in the final step of many DNA repair pathways and are commonly regulated by the DNA sliding clamp proliferating cell nuclear antigen (PCNA), but there are limited insights into the physical basis for this regulation. Here, we use single-particle cryoelectron microscopy (cryo-EM) to analyze an archaeal DNA ligase and heterotrimeric PCNA in complex with a single-strand DNA break. The cryo-EM structures highlight a continuous DNA-binding surface formed between DNA ligase and PCNA that supports the distorted conformation of the DNA break undergoing repair and contributes to PCNA stimulation of DNA ligation. DNA ligase is conformationally flexible within the complex, with its domains fully ordered only when encircling the repaired DNA to form a stacked ring structure with PCNA. The structures highlight DNA ligase structural transitions while docked on PCNA, changes in DNA conformation during ligation, and the potential for DNA ligase domains to regulate PCNA accessibility to other repair factors.
Topics: Cryoelectron Microscopy; DNA; DNA Ligase ATP; DNA Ligases; DNA Replication; Nucleic Acid Conformation; Proliferating Cell Nuclear Antigen; Protein Binding
PubMed: 34838188
DOI: 10.1016/j.str.2021.11.002 -
Structure (London, England : 1993) Mar 2022In this issue of Structure, Sverzhinsky et al. (2022) report structures of archaeal DNA ligase bound to the proliferating cell nuclear antigen (PCNA) sliding clamp and...
In this issue of Structure, Sverzhinsky et al. (2022) report structures of archaeal DNA ligase bound to the proliferating cell nuclear antigen (PCNA) sliding clamp and a nicked DNA substrate. The structures provide snapshots of ligation intermediates, which reveal a dynamic nature of the complex and explain how PCNA stimulates the DNA ligase activity.
Topics: Cryoelectron Microscopy; DNA; DNA Ligase ATP; DNA Ligases; Proliferating Cell Nuclear Antigen; Protein Binding
PubMed: 35245433
DOI: 10.1016/j.str.2022.02.008 -
The Journal of Cell Biology May 2023Replication fork reversal is an important mechanism to protect the stability of stalled forks and thereby preserve genomic integrity. While multiple enzymes have been...
Replication fork reversal is an important mechanism to protect the stability of stalled forks and thereby preserve genomic integrity. While multiple enzymes have been identified that can remodel forks, their regulation remains poorly understood. Here, we demonstrate that the ubiquitin ligase RFWD3, whose mutation causes Fanconi Anemia, promotes recruitment of the DNA translocase ZRANB3 to stalled replication forks and ubiquitinated sites of DNA damage. Using electron microscopy, we show that RFWD3 stimulates fork remodeling in a ZRANB3-epistatic manner. Fork reversal is known to promote nascent DNA degradation in BRCA2-deficient cells. Consistent with a role for RFWD3 in fork reversal, inactivation of RFWD3 in these cells rescues fork degradation and collapse, analogous to ZRANB3 inactivation. RFWD3 loss impairs ZRANB3 localization to spontaneous nuclear foci induced by inhibition of the PCNA deubiquitinase USP1. We demonstrate that RFWD3 promotes PCNA ubiquitination and interaction with ZRANB3, providing a mechanism for RFWD3-dependent recruitment of ZRANB3. Together, these results uncover a new role for RFWD3 in regulating ZRANB3-dependent fork remodeling.
Topics: DNA; DNA Damage; DNA Replication; DNA-Binding Proteins; Proliferating Cell Nuclear Antigen; Humans; Ubiquitin-Protein Ligases; DNA Helicases; Ubiquitination
PubMed: 37036693
DOI: 10.1083/jcb.202106022 -
PLoS Genetics Dec 2022DNA replication is a vulnerable time for genome stability maintenance. Intrinsic stressors, as well as oncogenic stress, can challenge replication by fostering conflicts...
DNA replication is a vulnerable time for genome stability maintenance. Intrinsic stressors, as well as oncogenic stress, can challenge replication by fostering conflicts with transcription and stabilizing DNA:RNA hybrids. RAD18 is an E3 ubiquitin ligase for PCNA that is involved in coordinating DNA damage tolerance pathways to preserve genome stability during replication. In this study, we show that RAD18 deficient cells have higher levels of transcription-replication conflicts and accumulate DNA:RNA hybrids that induce DNA double strand breaks and replication stress. We find that these effects are driven in part by failure to recruit the Fanconi Anemia protein FANCD2 at difficult to replicate and R-loop prone genomic sites. FANCD2 activation caused by splicing inhibition or aphidicolin treatment is critically dependent on RAD18 activity. Thus, we highlight a RAD18-dependent pathway promoting FANCD2-mediated suppression of R-loops and transcription-replication conflicts.
Topics: Humans; DNA Repair; Fanconi Anemia; Ubiquitin-Protein Ligases; Fanconi Anemia Complementation Group D2 Protein; DNA; DNA Damage; DNA Replication; RNA; Genomic Instability; DNA-Binding Proteins
PubMed: 36480547
DOI: 10.1371/journal.pgen.1010309 -
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 Jan 2023Human cells license tens of thousands of origins of replication in G1 and then must stop all licensing before DNA synthesis in S phase to prevent re-replication and...
Human cells license tens of thousands of origins of replication in G1 and then must stop all licensing before DNA synthesis in S phase to prevent re-replication and genome instability that ensue when an origin is licensed on replicated DNA. However, the E3 ubiquitin ligase CRL4 only starts to degrade the licensing factor CDT1 after origin firing, raising the question of how cells prevent re-replication before CDT1 is fully degraded. Here, using quantitative microscopy and in-vitro-reconstituted human DNA replication, we show that CDT1 inhibits DNA synthesis during an overlap period when CDT1 is still present after origin firing. CDT1 inhibits DNA synthesis by suppressing CMG helicase at replication forks, and DNA synthesis commences once CDT1 is degraded. Thus, in contrast to the prevailing model that human cells prevent re-replication by strictly separating licensing from firing, licensing and firing overlap, and cells instead separate licensing from DNA synthesis.
Topics: Humans; S Phase; Cell Cycle Proteins; DNA Replication; Ubiquitin-Protein Ligases; DNA; DNA Helicases
PubMed: 36608667
DOI: 10.1016/j.molcel.2022.12.004 -
DNA Repair Oct 2021The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to...
The nonhomologous DNA end joining pathway is required for repair of most double-strand breaks in the mammalian genome. Here we use a purified biochemical NHEJ system to compare the joining of free DNA with recombinant mononucleosomal and dinucleosomal substrates to investigate ligation and local DNA end resection. We find that the nucleosomal state permits ligation in a manner dependent on the presence of free DNA flanking the nucleosome core particle. Local resection at DNA ends by the Artemis:DNA-PKcs nuclease complex is completely suppressed in all mononucleosome substrates regardless of flanking DNA up to a length of 14 bp. Like mononucleosomes, dinucleosomes lacking flanking free DNA are not joined. Therefore, the nucleosomal state imposes severe constraints on NHEJ nuclease and ligase activities.
Topics: Animals; Cell Line; DNA; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligases; DNA-Activated Protein Kinase; HeLa Cells; Humans; Nucleosomes; Spodoptera; Xenopus
PubMed: 34339948
DOI: 10.1016/j.dnarep.2021.103193 -
The Journal of Biological Chemistry Jun 2024APE2 plays important roles in the maintenance of genomic and epigenomic stability including DNA repair and DNA damage response. Accumulating evidence has suggested that...
APE2 plays important roles in the maintenance of genomic and epigenomic stability including DNA repair and DNA damage response. Accumulating evidence has suggested that APE2 is upregulated in multiple cancers at the protein and mRNA levels and that APE2 upregulation is correlative with higher and lower overall survival of cancer patients depending on tumor type. However, it remains unknown how APE2 protein abundance is maintained and regulated in cells. Here, we provide the first evidence of APE2 regulation via the posttranslational modification ubiquitin. APE2 is poly-ubiquitinated via K48-linked chains and degraded via the ubiquitin-proteasome system where K371 is the key residue within APE2 responsible for its ubiquitination and degradation. We further characterize MKRN3 as the E3 ubiquitin ligase for APE2 ubiquitination in cells and in vitro. In summary, this study offers the first definition of the APE2 proteostasis network and lays the foundation for future studies pertaining to the posttranslational modification regulation and functions of APE2 in genome integrity and cancer etiology/treatment.
Topics: Humans; Ubiquitination; DNA-(Apurinic or Apyrimidinic Site) Lyase; Ubiquitin; Protein Processing, Post-Translational; Ubiquitin-Protein Ligases; HEK293 Cells; Proteasome Endopeptidase Complex; Proteolysis; Endonucleases; Multifunctional Enzymes
PubMed: 38705397
DOI: 10.1016/j.jbc.2024.107337