ABSTRACT
Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond-scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N and C termini of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point toward specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process.
Subject(s)
DNA Topoisomerases, Type II , Molecular Dynamics Simulation , Allosteric Regulation , DNA Topoisomerases, Type II/metabolism , DNA Topoisomerases, Type II/chemistry , DNA Topoisomerases, Type II/genetics , Saccharomyces cerevisiae/enzymology , Saccharomyces cerevisiae/metabolism , Protein Domains , Models, MolecularABSTRACT
Type II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction cycle. Curiously, most type II topoisomerases (topos II, IV and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase IIß (hTOP2ß) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss elevates the enzyme's propensity for DNA damage. The unstructured C-terminal domains (CTDs) of hTOP2ß strongly potentiate strand passage activity in ATPase-less enzymes, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleavage in vitro, as well as in vivo. By contrast, aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are present. Our findings are consistent with the proposal that type II topoisomerases acquired ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.
Subject(s)
DNA Topoisomerases, Type II , DNA , Humans , Adenosine Triphosphatases/genetics , Adenosine Triphosphate , DNA/genetics , DNA Topoisomerases, Type II/genetics , Etoposide/pharmacology , DNA DamageABSTRACT
Type II topoisomerases (TopoIIs) are ubiquitous enzymes that are involved in crucial nuclear processes such as genome organization, chromosome segregation, and other DNA metabolic processes. These enzymes function as large, homodimeric complexes that undergo a complex cycle of binding and hydrolysis of two ATP molecules in their ATPase domains, which regulates the capture and passage of one DNA double-helix through a second, cleaved DNA molecule. This process requires the transmission of information about the state of the bound nucleotide over vast ranges in the TopoII complex. How this information is transmitted at the molecular level to regulate TopoII functions and how protein substitutions disrupt these mechanisms remains largely unknown. Here, we employed extensive microsecond scale molecular dynamics simulations of the yeast TopoII enzyme in multiple nucleotide-bound states and with amino acid substitutions near both the N- and C-terminals of the complex. Simulation results indicate that the ATPase domains are remarkably flexible on the sub-microsecond timescale and that these dynamics are modulated by the identity of the bound nucleotides and both local and distant amino acid substitutions. Network analyses point towards specific allosteric networks that transmit information about the hydrolysis cycle throughout the complex, which include residues in both the protein and the bound DNA molecule. Amino acid substitutions weaken many of these pathways. Together, our results provide molecular-level details on how the TopoII catalytic cycle is controlled through nucleotide binding and hydrolysis and how mutations may disrupt this process.
ABSTRACT
The catalytic cycle of topoisomerase 2 (TOP2) enzymes proceeds via a transient DNA double-strand break (DSB) intermediate termed the TOP2 cleavage complex (TOP2cc), in which the TOP2 protein is covalently bound to DNA. Anticancer agents such as etoposide operate by stabilizing TOP2ccs, ultimately generating genotoxic TOP2-DNA protein cross-links that require processing and repair. Here, we identify RAD54 like 2 (RAD54L2) as a factor promoting TOP2cc resolution. We demonstrate that RAD54L2 acts through a novel mechanism together with zinc finger protein associated with tyrosyl-DNA phosphodiesterase 2 (TDP2) and TOP2 (ZATT/ZNF451) and independent of TDP2. Our work suggests a model wherein RAD54L2 recognizes sumoylated TOP2 and, using its ATPase activity, promotes TOP2cc resolution and prevents DSB exposure. These findings suggest RAD54L2-mediated TOP2cc resolution as a potential mechanism for cancer therapy resistance and highlight RAD54L2 as an attractive candidate for drug discovery.
Subject(s)
DNA Adducts , DNA-Binding Proteins , Humans , DNA Adducts/genetics , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Phosphoric Diester Hydrolases/genetics , DNA Topoisomerases, Type II/genetics , DNA/genetics , Genomic Instability , DNA Helicases/geneticsABSTRACT
Type II topoisomerases transiently cleave duplex DNA as part of a strand passage mechanism that helps control chromosomal organization and superstructure. Aberrant DNA cleavage can result in genomic instability, and how topoisomerase activity is controlled to prevent unwanted breaks is poorly understood. Using a genetic screen, we identified mutations in the beta isoform of human topoisomerase II (hTOP2ß) that render the enzyme hypersensitive to the chemotherapeutic agent etoposide. Several of these variants were unexpectedly found to display hypercleavage behavior in vitro and to be capable of inducing cell lethality in a DNA repair-deficient background; surprisingly, a subset of these mutations were also observed in TOP2B sequences from cancer genome databases. Using molecular dynamics simulations and computational network analyses, we found that many of the mutations obtained from the screen map to interfacial points between structurally coupled elements, and that dynamical modeling could be used to identify other damage-inducing TOP2B alleles present in cancer genome databases. This work establishes that there is an innate link between DNA cleavage predisposition and sensitivity to topoisomerase II poisons, and that certain sequence variants of human type II topoisomerases found in cancer cells can act as DNA-damaging agents. Our findings underscore the potential for hTOP2ß to function as a clastogen capable of generating DNA damage that may promote or support cellular transformation.
Subject(s)
Mutagens , Neoplasms , Humans , Topoisomerase II Inhibitors/pharmacology , Etoposide/pharmacology , DNA Topoisomerases, Type II/genetics , DNA Topoisomerases, Type II/metabolism , DNA Damage , DNAABSTRACT
Type II topoisomerases effect topological changes in DNA by cutting a single duplex, passing a second duplex through the break, and resealing the broken strand in an ATP-coupled reaction. Curiously, most type II topoisomerases (topos II, IV, and VI) catalyze DNA transformations that are energetically favorable, such as the removal of superhelical strain; why ATP is required for such reactions is unknown. Here, using human topoisomerase II ß (hTOP2ß) as a model, we show that the ATPase domains of the enzyme are not required for DNA strand passage, but that their loss leads to increased DNA nicking and double strand break formation by the enzyme. The unstructured C-terminal domains (CTDs) of hTOP2ß strongly potentiate strand passage activity in the absence of the ATPase regions, as do cleavage-prone mutations that confer hypersensitivity to the chemotherapeutic agent etoposide. The presence of either the CTD or the mutations lead ATPase-less enzymes to promote even greater levels of DNA cleava in ge vitro , as well as in vivo . By contrast, the aberrant cleavage phenotypes of these topo II variants is significantly repressed when the ATPase domains are restored. Our findings are consistent with the proposal that type II topoisomerases acquired an ATPase function to maintain high levels of catalytic activity while minimizing inappropriate DNA damage.
ABSTRACT
During a normal topoisomerase II (TOP2) reaction, the enzyme forms a covalent enzyme DNA intermediate consisting of a 5' phosphotyrosyl linkage between the enzyme and DNA. While the enzyme typically rejoins the transient breakage after strand passage, a variety of conditions including drugs targeting TOP2 can inhibit DNA resealing, leading to enzyme-mediated DNA damage. A critical aspect of the repair of TOP2-mediated damage is the removal of the TOP2 protein covalently bound to DNA. While proteolysis plays a role in repairing this damage, nucleolytic enzymes must remove the phosphotyrosyl-linked peptide bound to DNA. The MRN complex has been shown to participate in the removal of TOP2 protein from DNA following cellular treatment with TOP2 poisons. In this report we used an optimized ICE (In vivo Complex of Enzyme) assay to measure covalent TOP2/DNA complexes. In agreement with previous independent reports, we find that the absence or inhibition of the MRE11 endonuclease results in elevated levels of both TOP2α and TOP2ß covalent complexes. We also examined levels of TOP2 covalent complexes in cells treated with the proteasome inhibitor MG132. Although MRE11 inhibition plus MG132 was not synergistic in etoposide-treated cells, ectopic overexpression of MRE11 resulted in removal of TOP2 even in the presence of MG132. We also found that VCP/p97 inhibition led to elevated TOP2 covalent complexes and prevented the removal of TOP2 covalent complexes by MRE11 overexpression. Our results demonstrate the existence of multiple pathways for proteolytic processing of TOP2 prior to nucleolytic processing, and that MRE11 can process TOP2 covalent complexes even when the proteasome is inhibited. The interactions between VCP/p97 and proteolytic processing of TOP2 covalent complexes merit additional investigation.
ABSTRACT
Topoisomerases nick and reseal DNA to relieve torsional stress associated with transcription and replication and to resolve structures such as knots and catenanes. Stabilization of the yeast Top2 cleavage intermediates is mutagenic in yeast, but whether this extends to higher eukaryotes is less clear. Chemotherapeutic topoisomerase poisons also elevate cleavage, resulting in mutagenesis. Here, we describe p.K743N mutations in human topoisomerase hTOP2α and link them to a previously undescribed mutator phenotype in cancer. Overexpression of the orthologous mutant protein in yeast generated a characteristic pattern of 2- to 4-base pair (bp) duplications resembling those in tumors with p.K743N. Using mutant strains and biochemical analysis, we determined the genetic requirements of this mutagenic process and showed that it results from trapping of the mutant yeast yTop2 cleavage complex. In addition to 2- to 4-bp duplications, hTOP2α p.K743N is also associated with deletions that are absent in yeast. We call the combined pattern of duplications and deletions ID_TOP2α. All seven tumors carrying the hTOP2α p.K743N mutation showed ID_TOP2α, while it was absent from all other tumors examined (n = 12,269). Each tumor with the ID_TOP2α signature had indels in several known cancer genes, which included frameshift mutations in tumor suppressors PTEN and TP53 and an activating insertion in BRAF. Sequence motifs found at ID_TOP2α mutations were present at 80% of indels in cancer-driver genes, suggesting that ID_TOP2α mutagenesis may contribute to tumorigenesis. The results reported here shed further light on the role of topoisomerase II in genome instability.
Subject(s)
DNA Topoisomerases, Type II/genetics , Mutation , Neoplasms/genetics , Neoplasms/pathology , Phenotype , Alleles , Amino Acid Substitution , Base Sequence , Cell Survival , DNA Damage , DNA Mutational Analysis , DNA Topoisomerases, Type II/metabolism , Gene Duplication , Gene Rearrangement , Genetic Predisposition to Disease , Genotype , Humans , INDEL Mutation , Mutagenesis , Neoplasms/metabolism , Oncogenes , Poly-ADP-Ribose Binding Proteins/genetics , Sequence DeletionABSTRACT
Topoisomerases are enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination. All cells have two major forms of DNA topoisomerases: type I enzymes, which make single-stranded cuts in DNA, and type II enzymes, which cut and decatenate double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents. Provided in this article are protocols to assess activities of topoisomerases and their inhibitors. Included are an assay for topoisomerase I activity based on relaxation of supercoiled DNA; an assay for topoisomerase II based on the decatenation of double-stranded DNA; and approaches for enriching and quantifying DNA-protein covalent complexes formed as obligatory intermediates in the reactions of type I and II topoisomerases with DNA; and assays for measuring DNA cleavage in vitro. Topoisomerases are not the only proteins that form covalent adducts with DNA in living cells, and the approaches described here are likely to find use in characterizing other protein-DNA adducts and exploring their utility as targets for therapy. © 2021 Wiley Periodicals LLC. Basic Protocol 1: Assay of topoisomerase I activity Basic Protocol 2: Assay of topoisomerase II activity Basic Protocol 3: In vivo determination of topoisomerase covalent complexes using the in vivo complex of enzyme (ICE) assay Support Protocol 1: Preparation of mouse tissue for determination of topoisomerase covalent complexes using the ICE assay Support Protocol 2: Using recombinant topoisomerase standard for absolute quantification of cellular TOP2CC Basic Protocol 4: Quantification of topoisomerase-DNA covalent complexes by RADAR/ELISA: The rapid approach to DNA adduct recovery (RADAR) combined with the enzyme-linked immunosorbent assay (ELISA) Basic Protocol 5: Analysis of protein-DNA covalent complexes by RADAR/Western Support Protocol 3: Adduct-Seq to characterize adducted DNA Support Protocol 4: Nuclear fractionation and RNase treatment to reduce sample complexity Basic Protocol 6: Determination of DNA cleavage by purified topoisomerase I Basic Protocol 7: Determination of inhibitor effects on DNA cleavage by topoisomerase II using a plasmid linearization assay Alternate Protocol: Gel electrophoresis determination of topoisomerase II cleavage.
Subject(s)
DNA Topoisomerases, Type II , DNA, Superhelical , Animals , DNA Cleavage , DNA Topoisomerases , DNA Topoisomerases, Type II/metabolism , Mice , PlasmidsABSTRACT
Topoisomerases form transient covalent DNA cleavage complexes to perform their reactions. Topoisomerase I cleavage complexes (TOP1ccs) are trapped by camptothecin and TOP2ccs by etoposide. Proteolysis of the trapped topoisomerase DNA-protein cross-links (TOP-DPCs) is a key step for some pathways to repair these lesions. We describe a pathway that features a prominent role of the small ubiquitin-like modifier (SUMO) modification for both TOP1- and TOP2-DPC repair. Both undergo rapid and sequential SUMO-2/3 and SUMO-1 modifications in human cells. The SUMO ligase PIAS4 is required for these modifications. RNF4, a SUMO-targeted ubiquitin ligase (STUbL), then ubiquitylates the TOP-DPCs for their subsequent degradation by the proteasome. This pathway is conserved in yeast with Siz1 and Slx5-Slx8, the orthologs of human PIAS4 and RNF4.
Subject(s)
DNA Topoisomerases , Proteasome Endopeptidase Complex , Small Ubiquitin-Related Modifier Proteins , Ubiquitin , DNA/metabolism , DNA Topoisomerases/metabolism , Humans , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Proteasome Endopeptidase Complex/metabolism , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae Proteins/genetics , Small Ubiquitin-Related Modifier Proteins/metabolism , Transcription Factors/metabolism , Ubiquitin/metabolism , Ubiquitin-Protein Ligases/genetics , Ubiquitin-Protein Ligases/metabolismABSTRACT
Topoisomerase II (Top2) is an essential enzyme that resolves catenanes between sister chromatids as well as supercoils associated with the over- or under-winding of duplex DNA. Top2 alters DNA topology by making a double-strand break (DSB) in DNA and passing an intact duplex through the break. Each component monomer of the Top2 homodimer nicks one of the DNA strands and forms a covalent phosphotyrosyl bond with the 5' end. Stabilization of this intermediate by chemotherapeutic drugs such as etoposide leads to persistent and potentially toxic DSBs. We describe the isolation of a yeast top2 mutant (top2-F1025Y,R1128G) the product of which generates a stabilized cleavage intermediate in vitro. In yeast cells, overexpression of the top2-F1025Y,R1128G allele is associated with a mutation signature that is characterized by de novo duplications of DNA sequence that depend on the nonhomologous end-joining pathway of DSB repair. Top2-associated duplications are promoted by the clean removal of the enzyme from DNA ends and are suppressed when the protein is removed as part of an oligonucleotide. TOP2 cells treated with etoposide exhibit the same mutation signature, as do cells that overexpress the wild-type protein. These results have implications for genome evolution and are relevant to the clinical use of chemotherapeutic drugs that target Top2.
Subject(s)
DNA End-Joining Repair , DNA Topoisomerases, Type II/genetics , Gene Duplication , Saccharomyces cerevisiae Proteins/genetics , DNA Topoisomerases, Type II/metabolism , Etoposide , Rad52 DNA Repair and Recombination Protein/metabolism , Saccharomyces cerevisiae Proteins/metabolism , YeastsABSTRACT
DNA topoisomerases are essential for DNA metabolic processes such as replication and transcription. Since DNA is double stranded, the unwinding needed for these processes results in DNA supercoiling and catenation of replicated molecules. Changing the topology of DNA molecules to relieve supercoiling or resolve catenanes requires that DNA be transiently cut. While topoisomerases carry out these processes in ways that minimize the likelihood of genome instability, there are several ways that topoisomerases may fail. Topoisomerases can be induced to fail by therapeutic small molecules such as by fluoroquinolones that target bacterial topoisomerases, or a variety of anti-cancer agents that target the eukaryotic enzymes. Increasingly, there have been a large number of agents and processes, including natural products and their metabolites, DNA damage, and the intrinsic properties of the enzymes that can lead to long-lasting DNA breaks that subsequently lead to genome instability, cancer, and other diseases. Understanding the processes that can interfere with topoisomerases and how cells respond when topoisomerases fail will be important in minimizing the consequences when enzymes need to transiently interfere with DNA integrity.
Subject(s)
DNA Damage , DNA Topoisomerases/metabolism , Biological Products/pharmacology , DNA Repair/drug effects , Genomic Instability/drug effects , Genomic Instability/genetics , Humans , Small Molecule Libraries/pharmacologyABSTRACT
DNA topoisomerases carry out topological transformations of DNA by introducing transient DNA breaks. The covalent intermediate of topoisomerase reactions include the topoisomerase protein covalently bound to DNA by a phosphotyrosine intermediate. Anti-cancer drugs that target topoisomerases typically trap the covalent intermediate, and generate cytotoxic enzyme dependent DNA damage. More recently, structural alterations in DNA such as DNA damage have also been shown to trap covalent intermediates of topoisomerase reactions. Understanding the action of drugs that target topoisomerases as well as determining the importance of trapped topoisomerases on genome stability requires assays that can accurately and sensitively measure levels of topoisomerase/DNA complexes. This chapter describes two approaches that have been developed to quantitate topoisomerase DNA complexes. These assays termed ICE (in vivo complex of enzymes) and RADAR (rapid approach to DNA adduct recovery) rely on isolation of genomic DNA under conditions that preserve proteins covalently bound to DNA. Covalently bound proteins are then quantitated using antibodies directed against specific topoisomerases. We describe assays in both mammalian cells and the yeast Saccharomyces cerevisiae that can measure topoisomerase/DNA covalent complexes, and give examples that can be used to enhance the quantitative reliability of these assays.
Subject(s)
DNA Topoisomerases, Type I/metabolism , DNA/metabolism , Saccharomyces cerevisiae/enzymology , Animals , DNA Topoisomerases, Type I/chemistry , Humans , Mammals , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
Tyrosyl-DNA phosphodiesterase 1 (TDP1) can remove a wide variety of 3' and 5' terminal DNA adducts. Genetic studies in yeast identified TDP1 as a regulator of non-homologous end joining (NHEJ) fidelity in the repair of double-strand breaks (DSBs) lacking terminal adducts. In this communication, we show that TDP1 plays an important role in joining cohesive DSBs in human cells. To investigate the role of TDP1 in NHEJ in live human cells we used CRISPR/cas9 to produce TDP1-knockout (TDP1-KO) HEK-293 cells. As expected, human TDP1-KO cells were highly sensitive to topoisomerase poisons and ionizing radiation. Using a chromosomally-integrated NHEJ reporter substrate to compare end joining between wild type and TDP1-KO cells, we found that TDP1-KO cells have a 5-fold reduced ability to repair I-SceI-generated DSBs. Extracts prepared from TDP1-KO cells had reduced NHEJ activity in vitro, as compared to extracts from wild type cells. Analysis of end-joining junctions showed that TDP1 deficiency reduced end-joining fidelity, with a significant increase in insertion events, similar to previous observations in yeast. It has been reported that phosphorylation of TDP1 serine 81 (TDP1-S81) by ATM and DNA-PK stabilizes TDP1 and recruits TDP1 to sites of DNA damage. We found that end joining in TDP1-KO cells was partially restored by the non-phosphorylatable mutant TDP1-S81A, but not by the phosphomimetic TDP1-S81E. We previously reported that TDP1 physically interacted with XLF. In this study, we found that XLF binding by TDP1 was reduced 2-fold by the S81A mutation, and 10-fold by the S81E phosphomimetic mutation. Our results demonstrate a novel role for TDP1 in NHEJ in human cells. We hypothesize that TDP1 participation in human NHEJ is mediated by interaction with XLF, and that TDP1-XLF interactions and subsequent NHEJ events are regulated by phosphorylation of TDP1-S81.
Subject(s)
DNA Breaks, Double-Stranded , DNA End-Joining Repair , DNA Repair Enzymes/metabolism , DNA-Binding Proteins/metabolism , Phosphoric Diester Hydrolases/metabolism , Protein Processing, Post-Translational , Ataxia Telangiectasia Mutated Proteins/metabolism , DNA/metabolism , DNA-Activated Protein Kinase/metabolism , HEK293 Cells , Humans , Nuclear Proteins/metabolism , Phosphorylation , Protein Interaction Domains and MotifsABSTRACT
BACKGROUND AND AIMS: Anti-HMGB1 autoimmunity plays a role in systemic lupus erythematosus (SLE). Because SLE increases atherosclerosis, we asked whether the same autoimmunity might play a role in atherogenesis. METHODS: We looked for the induction of HMGB1-specific B and T cell responses by a western-type diet (WTD) in the Apoe(-/-) mouse model of atherosclerosis. We also determined whether modifying the responses modulates atherosclerosis. RESULTS: In the plasma of male Apoe(-/-) mice fed WTD, the level of anti-HMGB1 antibodies (Abs) was detected at â¼50 µg/ml, which was â¼6 times higher than that in either Apoe(-/-) mice fed a normal chow or Apoe(+/+) mice fed WTD (p ≤ 0.0005). The Abs were directed largely toward a novel, dominant epitope of HMGB1 named HMW4; accordingly, compared with chow-fed mice, WTD-fed Apoe(-/-) mice had more activated HMW4-reactive B and T cells (p = 0.005 and p = 0.01, respectively). Compared with mock-immunized mice, Apoe(-/-) mice immunized with HMW4 along with an immunogenic adjuvant showed proportional increases in anti-HMW4 IgG and IgM Abs, HMW4-reactive B-1 and B-2 cells, and HMW4-reactive Treg and Teff cells, which was associated with â¼30% increase in aortic arch lesions (p ≤ 0.01) by two methods. In contrast, Apoe(-/-) mice immunized with HMW4 using a tolerogenic adjuvant showed preferential increases in anti-HMW4 IgM (over IgG) Abs, HMW4-reactive B-1 (over B-2) cells, and HMW4-specific Treg (over Teff) cells, which was associated with â¼40% decrease in aortic arch lesions (p ≤ 0.03). CONCLUSIONS: Anti-HMGB1 autoimmunity may potentially play a role in atherogenesis.
Subject(s)
Atherosclerosis/genetics , Autoimmunity/genetics , Diet, Western/adverse effects , HMGB1 Protein/genetics , Animals , Antigens/blood , Aorta, Thoracic/pathology , B-Lymphocytes/immunology , Disease Models, Animal , Enzyme-Linked Immunosorbent Assay , Epitopes/chemistry , Inflammation , Lipids/blood , Lipids/chemistry , Male , Mice , Mice, Inbred C57BL , Mice, Knockout, ApoEABSTRACT
The repair of DNA double-strand breaks (DSB) is central to the maintenance of genomic integrity. In tumor cells, the ability to repair DSBs predicts response to radiation and many cytotoxic anti-cancer drugs. DSB repair pathways include homologous recombination and non-homologous end joining (NHEJ). NHEJ is a template-independent mechanism, yet many NHEJ repair products carry limited genetic changes, which suggests that NHEJ includes mechanisms to minimize error. Proteins required for mammalian NHEJ include Ku70/80, the DNA-dependent protein kinase (DNA-PKcs), XLF/Cernunnos and the XRCC4:DNA ligase IV complex. NHEJ also utilizes accessory proteins that include DNA polymerases, nucleases, and other end-processing factors. In yeast, mutations of tyrosyl-DNA phosphodiesterase (TDP1) reduced NHEJ fidelity. TDP1 plays an important role in repair of topoisomerase-mediated DNA damage and 3'-blocking DNA lesions, and mutation of the human TDP1 gene results in an inherited human neuropathy termed SCAN1. We found that human TDP1 stimulated DNA binding by XLF and physically interacted with XLF to form TDP1:XLF:DNA complexes. TDP1:XLF interactions preferentially stimulated TDP1 activity on dsDNA as compared to ssDNA. TDP1 also promoted DNA binding by Ku70/80 and stimulated DNA-PK activity. Because Ku70/80 and XLF are the first factors recruited to the DSB at the onset of NHEJ, our data suggest a role for TDP1 during the early stages of mammalian NHEJ.
Subject(s)
DNA End-Joining Repair , Phosphoric Diester Hydrolases/metabolism , Antigens, Nuclear/metabolism , DNA/metabolism , DNA Repair Enzymes/metabolism , DNA-Activated Protein Kinase/metabolism , DNA-Binding Proteins/metabolism , Humans , Ku AutoantigenABSTRACT
Anthracyclines are active clinical agents that have multiple mechanisms of cytotoxicity. Cardiotoxicity by anthracyclines limits the therapeutic potential of these agents, but mechanisms leading to cardiotoxicity remain controversial. Transgenic mice that lack mitochondrial topoisomerase I are hypersensitive to doxorubicin cardiotoxicity, providing support for cardiotoxicity arising from damage of mitochondrial DNA.
Subject(s)
Antibiotics, Antineoplastic/toxicity , Cardiotoxicity/physiopathology , DNA Damage , DNA, Mitochondrial/drug effects , Doxorubicin/toxicity , Heart/drug effects , Animals , Mice , Mice, TransgenicABSTRACT
DNA damage is considered to be a prime factor in several spinocerebellar neurodegenerative diseases; however, the DNA lesions underpinning disease etiology are unknown. We observed the endogenous accumulation of pathogenic topoisomerase-1 (Top1)-DNA cleavage complexes (Top1ccs) in murine models of ataxia telangiectasia and spinocerebellar ataxia with axonal neuropathy 1. We found that the defective DNA damage response factors in these two diseases cooperatively modulated Top1cc turnover in a non-epistatic and ATM kinase-independent manner. Furthermore, coincident neural inactivation of ATM and DNA single-strand break repair factors, including tyrosyl-DNA phosphodiesterase-1 or XRCC1, resulted in increased Top1cc formation and excessive DNA damage and neurodevelopmental defects. Notably, direct Top1 poisoning to elevate Top1cc levels phenocopied the neuropathology of the mouse models described above. Our results identify a critical endogenous pathogenic lesion associated with neurodegenerative syndromes arising from DNA repair deficiency, indicating that genome integrity is important for preventing disease in the nervous system.
Subject(s)
DNA Topoisomerases, Type I/genetics , Genomic Instability/genetics , Neurodegenerative Diseases/enzymology , Neurodegenerative Diseases/genetics , Animals , Cell Line , Cells, Cultured , DNA Damage/genetics , DNA Topoisomerases, Type I/deficiency , Disease Models, Animal , Humans , Mice , Mice, Knockout , Mice, Transgenic , Neural Stem Cells/enzymology , Neural Stem Cells/pathology , Neural Stem Cells/physiology , Neurodegenerative Diseases/pathology , SyndromeABSTRACT
Eukaryotic type II topoisomerases (Top2α and Top2ß) are homodimeric enzymes; they are essential for altering DNA topology by the formation of normally transient double strand DNA cleavage. Anticancer drugs (etoposide, doxorubicin, and mitoxantrone) and also Top2 oxidation and DNA helical alterations cause potentially irreversible Top2·DNA cleavage complexes (Top2cc), leading to Top2-linked DNA breaks. Top2cc are the therapeutic mechanism for killing cancer cells. Yet Top2cc can also generate recombination, translocations, and apoptosis in normal cells. The Top2 protein-DNA covalent complexes are excised (in part) by tyrosyl-DNA-phosphodiesterase 2 (TDP2/TTRAP/EAP2/VPg unlinkase). In this study, we show that irreversible Top2cc induced in suicidal substrates are not processed by TDP2 unless they first undergo proteolytic processing or denaturation. We also demonstrate that TDP2 is most efficient when the DNA attached to the tyrosyl is in a single-stranded configuration and that TDP2 can efficiently remove a tyrosine linked to a single misincorporated ribonucleotide or to polyribonucleotides, which expands the TDP2 catalytic profile with RNA substrates. The 1.6-Å resolution crystal structure of TDP2 bound to a substrate bearing a 5'-ribonucleotide defines a mechanism through which RNA can be accommodated in the TDP2 active site, albeit in a strained conformation.