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The Biochemical Journal Jan 2021Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of... (Review)
Review
Genomic integrity is most threatened by double-strand breaks, which, if left unrepaired, lead to carcinogenesis or cell death. The cell generates a network of protein-protein signaling interactions that emanate from the DNA damage which are now recognized as a rich basis for anti-cancer therapy development. Deciphering the structures of signaling proteins has been an uphill task owing to their large size and complex domain organization. Recent advances in mammalian protein expression/purification and cryo-EM-based structure determination have led to significant progress in our understanding of these large multidomain proteins. This review is an overview of the structural principles that underlie some of the key signaling proteins that function at the double-strand break site. We also discuss some plausible ideas that could be considered for future structural approaches to visualize and build a more complete understanding of protein dynamics at the break site.
Topics: Acid Anhydride Hydrolases; Animals; Ataxia Telangiectasia Mutated Proteins; Cell Cycle Proteins; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; DNA-Binding Proteins; Humans; MRE11 Homologue Protein; Nuclear Proteins; Protein Processing, Post-Translational; Signal Transduction; Tumor Suppressor Proteins; Tumor Suppressor p53-Binding Protein 1; Ubiquitin-Protein Ligases
PubMed: 33439989
DOI: 10.1042/BCJ20200066 -
Genes Jan 2020DNA damage leads to genome instability by interfering with DNA replication. Cells possess several damage bypass pathways that mitigate the effects of DNA damage during... (Review)
Review
DNA damage leads to genome instability by interfering with DNA replication. Cells possess several damage bypass pathways that mitigate the effects of DNA damage during replication. These pathways include translesion synthesis and template switching. These pathways are regulated largely through post-translational modifications of proliferating cell nuclear antigen (PCNA), an essential replication accessory factor. Mono-ubiquitylation of PCNA promotes translesion synthesis, and K63-linked poly-ubiquitylation promotes template switching. This article will discuss the mechanisms of how these post-translational modifications of PCNA control these bypass pathways from a structural and biochemical perspective. We will focus on the structure and function of the E3 ubiquitin ligases Rad18 and Rad5 that facilitate the mono-ubiquitylation and poly-ubiquitylation of PCNA, respectively. We conclude by reviewing alternative ideas about how these post-translational modifications of PCNA regulate the assembly of the multi-protein complexes that promote damage bypass pathways.
Topics: DNA Damage; DNA Helicases; Fungal Proteins; Humans; Lysine; Models, Molecular; Proliferating Cell Nuclear Antigen; Protein Conformation; Protein Processing, Post-Translational; Ubiquitin-Protein Ligases; Ubiquitination; Yeasts
PubMed: 32013080
DOI: 10.3390/genes11020138 -
Journal of Thermal Biology Jul 2022Wood frogs, Rana sylvatica, endure the freezing of ∼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also...
Wood frogs, Rana sylvatica, endure the freezing of ∼65% of total body water while overwintering in cold climates, enduring not only internal ice formation but also long-term anoxia due to cessation of heartbeat and breathing. Thawing restores perfusion but rapid reoxygenation can increase vulnerability to reactive oxygen species and induce oxidative damage. This study provides a first assessment of antioxidant capacity, DNA damage, and DNA repair responses comparing freeze/thaw and anoxia/reoxygenation in liver and skeletal muscle of wood frogs. Oxidation of guanine resides in DNA did not change under either stress but total antioxidant capacity rose in both tissues under anoxia. Relative expression of eight proteins involved in double-stranded break repair (Mre11, Rad50, phospho-p95, XLF, DNA ligase IV, XRCC4, Ku70, Rad51) were assessed in both tissues. Freezing suppressed Ku70 and Rad51 in liver and Rad51 in muscle but levels rose again after thawing. Anoxia exposure suppressed XLF, Ku70 and Rad51 proteins in muscle. However, in liver, anoxia exposure led to elevated Mre11, Ku70 and DNA ligase IV, the former two belonging to the MRN complex that binds DNA and marks sites of double stranded breaks (DSBs). Large increases in Mre11 and Ku70 expression suggested DSB damage in liver under anoxia but not during freezing, whereas muscle was resistant to DSB damage under both stresses. These data indicate that DNA damage is minimal during whole body freezing due to tissue and stress specific regulation of antioxidant capacity and DNA damage repair to preserve genomic integrity.
Topics: Animals; Antioxidants; DNA; DNA Damage; DNA Ligase ATP; Freezing; Hypoxia; Muscle, Skeletal; Ranidae
PubMed: 35701025
DOI: 10.1016/j.jtherbio.2022.103274 -
Biochimica Et Biophysica Acta.... May 2022Mitochondrion is a double membrane organelle that is responsible for cellular respiration and production of most of the ATP in eukaryotic cells. Mitochondrial DNA... (Review)
Review
Mitochondrion is a double membrane organelle that is responsible for cellular respiration and production of most of the ATP in eukaryotic cells. Mitochondrial DNA (mtDNA) is the genetic material carried by mitochondria, which encodes some essential subunits of respiratory complexes independent of nuclear DNA. Normally, mtDNA binds to certain proteins to form a nucleoid that is stable in mitochondria. Nevertheless, a variety of physiological or pathological stresses can cause mtDNA damage, and the accumulation of damaged mtDNA in mitochondria leads to mitochondrial dysfunction, which triggers the occurrence of mitochondrial diseases in vivo. In response to mtDNA damage, cell initiates multiple pathways including mtDNA repair, degradation, clearance and release, to recover mtDNA, and maintain mitochondrial quality and cell homeostasis. In this review, we provide our current understanding of the fate of damaged mtDNA, focus on the pathways and mechanisms of removing damaged mtDNA in the cell.
Topics: DNA Damage; DNA Repair; DNA, Mitochondrial; Extracellular Vesicles; Humans; Mitochondria; Mitochondrial Diseases; Mitophagy; Signal Transduction; Ubiquitin-Protein Ligases
PubMed: 35131372
DOI: 10.1016/j.bbamcr.2022.119233 -
Nature Structural & Molecular Biology Oct 2023DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the...
DNA replication introduces thousands of RNA primers into the lagging strand that need to be removed for replication to be completed. In Escherichia coli when the replicative DNA polymerase Pol IIIα terminates at a previously synthesized RNA primer, DNA Pol I takes over and continues DNA synthesis while displacing the downstream RNA primer. The displaced primer is subsequently excised by an endonuclease, followed by the sealing of the nick by a DNA ligase. Yet how the sequential actions of Pol IIIα, Pol I polymerase, Pol I endonuclease and DNA ligase are coordinated is poorly defined. Here we show that each enzymatic activity prepares the DNA substrate for the next activity, creating an efficient four-point molecular handover. The cryogenic-electron microscopy structure of Pol I bound to a DNA substrate with both an upstream and downstream primer reveals how it displaces the primer in a manner analogous to the monomeric helicases. Moreover, we find that in addition to its flap-directed nuclease activity, the endonuclease domain of Pol I also specifically cuts at the RNA-DNA junction, thus marking the end of the RNA primer and creating a 5' end that is a suitable substrate for the ligase activity of LigA once all RNA has been removed.
Topics: DNA Polymerase III; DNA; DNA Replication; RNA; DNA Ligases; DNA Ligase ATP; Endonucleases
PubMed: 37620586
DOI: 10.1038/s41594-023-01071-y -
Seminars in Cancer Biology Nov 2022Monoubiquitination of histone H2B on lysine 120 (H2Bub1) is implicated in the control of multiple essential processes, including transcription, DNA damage repair and... (Review)
Review
Monoubiquitination of histone H2B on lysine 120 (H2Bub1) is implicated in the control of multiple essential processes, including transcription, DNA damage repair and mitotic chromosome segregation. Accordingly, aberrant regulation of H2Bub1 can induce transcriptional reprogramming and genome instability that may promote oncogenesis. Remarkably, alterations of the ubiquitin ligases and deubiquitinating enzymes regulating H2Bub1 are emerging as ubiquitous features in cancer, further supporting the possibility that the misregulation of H2Bub1 is an underlying mechanism contributing to cancer pathogenesis. To date, aberrant H2Bub1 dynamics have been reported in multiple cancer types and are associated with transcriptional changes that promote oncogenesis in a cancer type-specific manner. Owing to the multi-functional nature of H2Bub1, misregulation of its writers and erasers may drive disease initiation and progression through additional synergistic processes. Accordingly, understanding the molecular determinants and pathogenic impacts associated with aberrant H2Bub1 regulation may reveal novel drug targets and therapeutic vulnerabilities that can be exploited to develop innovative precision medicine strategies that better combat cancer. In this review, we present the normal functions of H2Bub1 in the control of DNA-associated processes and describe the pathogenic implications associated with its misregulation in cancer. We further discuss the challenges coupled with the development of therapeutic strategies targeting H2Bub1 misregulation and expose the potential benefits of designing treatments that synergistically exploit the multiple functionalities of H2Bub1 to improve treatment selectivity and efficacy.
Topics: Humans; Histones; Ubiquitination; Neoplasms; Ubiquitin-Protein Ligases; Carcinogenesis
PubMed: 34953650
DOI: 10.1016/j.semcancer.2021.12.007 -
Experimental & Molecular Medicine Oct 2022Antitumor therapeutic strategies that fundamentally rely on the induction of DNA damage to eradicate and inhibit the growth of cancer cells are integral approaches to... (Review)
Review
Antitumor therapeutic strategies that fundamentally rely on the induction of DNA damage to eradicate and inhibit the growth of cancer cells are integral approaches to cancer therapy. Although DNA-damaging therapies advance the battle with cancer, resistance, and recurrence following treatment are common. Thus, searching for vulnerabilities that facilitate the action of DNA-damaging agents by sensitizing cancer cells is an active research area. Therefore, it is crucial to decipher the detailed molecular events involved in DNA damage responses (DDRs) to DNA-damaging agents in cancer. The tumor suppressor p53 is active at the hub of the DDR. Researchers have identified an increasing number of genes regulated by p53 transcriptional functions that have been shown to be critical direct or indirect mediators of cell fate, cell cycle regulation, and DNA repair. Posttranslational modifications (PTMs) primarily orchestrate and direct the activity of p53 in response to DNA damage. Many molecules mediating PTMs on p53 have been identified. The anticancer potential realized by targeting these molecules has been shown through experiments and clinical trials to sensitize cancer cells to DNA-damaging agents. This review briefly acknowledges the complexity of DDR pathways/networks. We specifically focus on p53 regulators, protein kinases, and E3/E4 ubiquitin ligases and their anticancer potential.
Topics: Humans; Tumor Suppressor Protein p53; DNA Damage; DNA Repair; Protein Kinases; Ubiquitin-Protein Ligases; Neoplasms
PubMed: 36207426
DOI: 10.1038/s12276-022-00863-4 -
Nucleic Acids Research Jan 2023Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III...
Double-strand DNA breaks (DSBs) are toxic to cells, and improper repair can cause chromosomal abnormalities that initiate and drive cancer progression. DNA ligases III and IV (LIG3, LIG4) have long been credited for repair of DSBs in mammals, but recent evidence suggests that DNA ligase I (LIG1) has intrinsic end-joining (EJ) activity that can compensate for their loss. To test this model, we employed in vitro biochemical assays to compare EJ by LIG1 and LIG3. The ligases join blunt-end and 3'-overhang-containing DNA substrates with similar catalytic efficiency, but LIG1 joins 5'-overhang-containing DNA substrates ∼20-fold less efficiently than LIG3 under optimal conditions. LIG1-catalyzed EJ is compromised at a physiological concentration of Mg2+, but its activity is restored by increased molecular crowding. In contrast to LIG1, LIG3 efficiently catalyzes EJ reactions at a physiological concentration of Mg2+ with or without molecular crowding. Under all tested conditions, LIG3 has greater affinity than LIG1 for DNA ends. Remarkably, LIG3 can ligate both strands of a DSB during a single binding encounter. The weaker DNA binding affinity of LIG1 causes significant abortive ligation that is sensitive to molecular crowding and DNA terminal structure. These results provide new insights into mechanisms of alternative nonhomologous EJ.
Topics: Animals; Humans; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA Ligase ATP; DNA Repair; Magnesium; Mammals
PubMed: 36625284
DOI: 10.1093/nar/gkac1263 -
Molecular Cell Jun 2021Agents that induce DNA damage can cure some cancers. However, the side effects of chemotherapy are severe because of the indiscriminate action of DNA-damaging agents on... (Review)
Review
Agents that induce DNA damage can cure some cancers. However, the side effects of chemotherapy are severe because of the indiscriminate action of DNA-damaging agents on both healthy and cancerous cells. DNA repair pathway inhibition provides a less toxic and targeted alternative to chemotherapy. A compelling DNA repair target is the Fanconi anemia (FA) E3 ligase core complex due to its critical-and likely singular-role in the efficient removal of specific DNA lesions. FA pathway inactivation has been demonstrated to specifically kill some types of cancer cells without the addition of exogenous DNA damage, including cells that lack BRCA1, BRCA2, ATM, or functionally related genes. In this perspective, we discuss the genetic and biochemical evidence in support of the FA core complex as a compelling drug target for cancer therapy. In particular, we discuss the genetic, biochemical, and structural data that could rapidly advance our capacity to identify and implement the use of FA core complex inhibitors in the clinic.
Topics: Ataxia Telangiectasia Mutated Proteins; BRCA1 Protein; BRCA2 Protein; DNA Damage; DNA Repair; Enzyme Inhibitors; Fanconi Anemia; Fanconi Anemia Complementation Group Proteins; Gene Expression Regulation, Neoplastic; Humans; Molecular Targeted Therapy; Morpholines; Pyrones; RNA, Small Interfering; Signal Transduction; Synthetic Lethal Mutations; Ubiquitin-Protein Ligases; Ubiquitins
PubMed: 33984284
DOI: 10.1016/j.molcel.2021.04.023 -
Proceedings of the National Academy of... Apr 2024The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1...
The S-phase checkpoint involving CHK1 is essential for fork stability in response to fork stalling. PARP1 acts as a sensor of replication stress and is required for CHK1 activation. However, it is unclear how the activity of PARP1 is regulated. Here, we found that UFMylation is required for the efficient activation of CHK1 by UFMylating PARP1 at K548 during replication stress. Inactivation of UFL1, the E3 enzyme essential for UFMylation, delayed CHK1 activation and inhibits nascent DNA degradation during replication blockage as seen in PARP1-deficient cells. An in vitro study indicated that PARP1 is UFMylated at K548, which enhances its catalytic activity. Correspondingly, a PARP1 UFMylation-deficient mutant (K548R) and pathogenic mutant (F553L) compromised CHK1 activation, the restart of stalled replication forks following replication blockage, and chromosome stability. Defective PARP1 UFMylation also resulted in excessive nascent DNA degradation at stalled replication forks. Finally, we observed that PARP1 UFMylation-deficient knock-in mice exhibited increased sensitivity to replication stress caused by anticancer treatments. Thus, we demonstrate that PARP1 UFMylation promotes CHK1 activation and replication fork stability during replication stress, thus safeguarding genome integrity.
Topics: DNA Replication; Animals; Poly (ADP-Ribose) Polymerase-1; Checkpoint Kinase 1; Mice; Humans; DNA Damage; Ubiquitin-Protein Ligases
PubMed: 38657044
DOI: 10.1073/pnas.2322520121