ABSTRACT
DNA polymerase theta (Polθ) is a DNA helicase-polymerase protein that facilitates DNA repair and is synthetic lethal with homology-directed repair (HDR) factors. Thus, Polθ is a promising precision oncology drug-target in HDR-deficient cancers. Here, we characterize the binding and mechanism of action of a Polθ helicase (Polθ-hel) small-molecule inhibitor (AB25583) using cryo-EM. AB25583 exhibits 6 nM IC50 against Polθ-hel, selectively kills BRCA1/2-deficient cells, and acts synergistically with olaparib in cancer cells harboring pathogenic BRCA1/2 mutations. Cryo-EM uncovers predominantly dimeric Polθ-hel:AB25583 complex structures at 3.0-3.2 Å. The structures reveal a binding-pocket deep inside the helicase central-channel, which underscores the high specificity and potency of AB25583. The cryo-EM structures in conjunction with biochemical data indicate that AB25583 inhibits the ATPase activity of Polθ-hel helicase via an allosteric mechanism. These detailed structural data and insights about AB25583 inhibition pave the way for accelerating drug development targeting Polθ-hel in HDR-deficient cancers.
Subject(s)
Cryoelectron Microscopy , DNA Helicases , DNA Polymerase theta , DNA-Directed DNA Polymerase , Humans , DNA Helicases/metabolism , DNA Helicases/chemistry , DNA Helicases/genetics , DNA Helicases/antagonists & inhibitors , DNA-Directed DNA Polymerase/metabolism , DNA-Directed DNA Polymerase/chemistry , DNA-Directed DNA Polymerase/genetics , BRCA2 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/chemistry , BRCA1 Protein/metabolism , BRCA1 Protein/genetics , BRCA1 Protein/chemistry , Piperazines/pharmacology , Piperazines/chemistry , Cell Line, Tumor , Phthalazines/pharmacology , Phthalazines/chemistry , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/chemistry , Models, Molecular , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/antagonists & inhibitors , Protein BindingABSTRACT
DNA replication is remarkably accurate with estimates of only a handful of mutations per human genome per cell division cycle. Replication stress caused by DNA lesions, transcription-replication conflicts, and other obstacles to the replication machinery must be efficiently overcome in ways that minimize errors and maximize completion of DNA synthesis. Replication fork reversal is one mechanism that helps cells tolerate replication stress. This process involves reannealing of parental template DNA strands and generation of a nascent-nascent DNA duplex. While fork reversal may be beneficial by facilitating DNA repair or template switching, it must be confined to the appropriate contexts to preserve genome stability. Many enzymes have been implicated in this process including ATP-dependent DNA translocases like SMARCAL1, ZRANB3, HLTF, and the helicase FBH1. In addition, the RAD51 recombinase is required. Many additional factors and regulatory activities also act to ensure reversal is beneficial instead of yielding undesirable outcomes. Finally, reversed forks must also be stabilized and often need to be restarted to complete DNA synthesis. Disruption or deregulation of fork reversal causes a variety of human diseases. In this review we will describe the latest models for reversal and key mechanisms of regulation.
Subject(s)
DNA Damage Tolerance , Animals , Humans , DNA/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , DNA Repair , Genomic InstabilityABSTRACT
Eukaryotes have one replicative helicase known as CMG, which centrally organizes and drives the replisome, and leads the way at the front of replication forks. Obtaining a deep mechanistic understanding of the dynamics of CMG is critical to elucidating how cells achieve the enormous task of efficiently and accurately replicating their entire genome once per cell cycle. Single-molecule techniques are uniquely suited to quantify the dynamics of CMG due to their unparalleled temporal and spatial resolution. Nevertheless, single-molecule studies of CMG motion have thus far relied on pre-formed CMG purified from cells as a complex, which precludes the study of the steps leading up to its activation. Here, we describe a hybrid ensemble and single-molecule assay that allowed imaging at the single-molecule level of the motion of fluorescently labeled CMG after fully reconstituting its assembly and activation from 36 different purified S. cerevisiae polypeptides. This assay relies on the double functionalization of the ends of a linear DNA substrate with two orthogonal attachment moieties, and can be adapted to study similarly complex DNA-processing mechanisms at the single-molecule level.
Subject(s)
Saccharomyces cerevisiae , Single Molecule Imaging , Saccharomyces cerevisiae/metabolism , Single Molecule Imaging/methods , DNA Helicases/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Fluorescent Dyes/chemistry , DNA Replication , DNA, Fungal/geneticsABSTRACT
Trypanosoma cruzi is the etiological agent of Chagas disease and a peculiar eukaryote with unique biological characteristics. DNA damage can block RNA polymerase, activating transcription-coupled nucleotide excision repair (TC-NER), a DNA repair pathway specialized in lesions that compromise transcription. If transcriptional stress is unresolved, arrested RNA polymerase can activate programmed cell death. Nonetheless, how this parasite modulates these processes is unknown. Here, we demonstrate that T. cruzi cell death after UV irradiation, a genotoxic agent that generates lesions resolved by TC-NER, depends on active transcription and is signaled mainly by an apoptotic-like pathway. Pre-treated parasites with α-amanitin, a selective RNA polymerase II inhibitor, become resistant to such cell death. Similarly, the gamma pre-irradiated cells are more resistant to UV when the transcription processes are absent. The Cockayne Syndrome B protein (CSB) recognizes blocked RNA polymerase and can initiate TC-NER. Curiously, CSB overexpression increases parasites' cell death shortly after UV exposure. On the other hand, at the same time after irradiation, the single-knockout CSB cells show resistance to the same treatment. UV-induced fast death is signalized by the exposition of phosphatidylserine to the outer layer of the membrane, indicating a cell death mainly by an apoptotic-like pathway. Furthermore, such death is suppressed in WT parasites pre-treated with inhibitors of ataxia telangiectasia and Rad3-related (ATR), a key DDR kinase. Signaling for UV radiation death may be related to R-loops since the overexpression of genes associated with the resolution of these structures suppress it. Together, results suggest that transcription blockage triggered by UV radiation activates an ATR-dependent apoptosis-like mechanism in T. cruzi, with the participation of CSB protein in this process.
Subject(s)
Ataxia Telangiectasia Mutated Proteins , DNA Damage , DNA Repair , R-Loop Structures , Transcription, Genetic , Trypanosoma cruzi , Ultraviolet Rays , Trypanosoma cruzi/metabolism , Ataxia Telangiectasia Mutated Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/genetics , Protozoan Proteins/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , Cell Death , Apoptosis , HumansABSTRACT
Loss of Heterozygosity (LOH) due to mitotic recombination is frequently associated with the development of various cancers (e.g. retinoblastoma). LOH is also an important source of genetic diversity, especially in organisms where meiosis is infrequent. Irc20 is a putative helicase, and E3 ubiquitin ligase involved in DNA double-strand break repair pathway. We analyzed genome-wide LOH events, gross chromosomal changes, small insertion-deletions and single nucleotide mutations in eleven S. cerevisiae mutation accumulation lines of irc20∆, which underwent 50 mitotic bottlenecks. LOH enhancement in irc20∆ was small (1.6 fold), but statistically significant as compared to the wild type. Short (≤ 1â¯kb) and long (> 10â¯kb) LOH tracts were significantly enhanced in irc20∆. Both interstitial and terminal LOH events were also significantly enhanced in irc20∆ compared to the wild type. LOH events in irc20∆ were more telomere proximal and away from centromeres compared to the wild type. Gross chromosomal changes, single nucleotide mutations and in-dels were comparable between irc20∆ and wild type. Locus based and genome-wide analysis of meiotic recombination showed that meiotic crossover frequencies are not altered in irc20∆. These results suggest Irc20 primarily regulates mitotic recombination and does not affect meiotic crossovers. Our results suggest that the IRC20 gene is important for regulating LOH frequency and distribution.
Subject(s)
Loss of Heterozygosity , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/metabolism , Saccharomyces cerevisiae Proteins/genetics , DNA Helicases/metabolism , DNA Helicases/genetics , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Mitosis , Mutation , DNA Repair , Meiosis , DNA Breaks, Double-StrandedABSTRACT
Chromodomain helicase DNA-binding protein (CHD) gene family, an ATP (adenosine triphosphate) -dependent chromatin remodeler family, is involved in multiple developmental process and tumor development. However, there have been none pan-cancer analyses of this family. The expression levels, survival profiles, mutation profiles and immune infiltration of the CHD family genes from TCGA and TARGET database were analyzed using online tools or R packages. Interestingly, all types of CHD gene expressions were associated with the prognosis of Neuroblastoma, Acute lymphoblastic leukemia-Phase 3 and Acute Myeloid Leukemia (All P < 0.05). Knock down of CHD7 and CHD9 in K562 (human erythromyeloblastoid leukemia) and HEC-1-B (human endometrial adenocarcinoma) cells significantly inhibit cell proliferation and migration (P < 0.05). Proliferation, colony formation and migration assays were performed in CHD7 and CHD9 knockdown K562 and HBC-1-B cell lines. Mechanisms were also analyzed by PPI and GO ontology for our experiments. Histone modification, especially the methylation of H3K4, might be involved in CHD7 and CHD9 related oncogenesis. Through bioinformatic analysis, we showed CHD genes significantly affected the prognosis of different tumor types, including childhood tumor. Our findings provide new insights into the function and mechanism of CHD gene family, especially in CHD7 and CHD9.
Subject(s)
Computational Biology , DNA Helicases , DNA-Binding Proteins , Neoplasms , Humans , Computational Biology/methods , DNA Helicases/genetics , DNA Helicases/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Neoplasms/genetics , Neoplasms/pathology , Cell Proliferation/genetics , Gene Expression Regulation, Neoplastic , Cell Movement/genetics , Prognosis , Cell Line, Tumor , MutationABSTRACT
The transmembrane protein ß-amyloid precursor protein (APP) is central to the pathophysiology of Alzheimer's disease (AD). The ß-amyloid hypothesis posits that aberrant processing of APP forms neurotoxic ß-amyloid aggregates, which lead to the cognitive impairments observed in AD. Although numerous additional factors contribute to AD, there is a need to better understand the synaptic function of APP. We have found that Drosophila APP-like (APPL) has both shared and non-shared roles at the synapse with Kismet (Kis), a chromatin helicase binding domain (CHD) protein. Kis is the homolog of CHD7 and CHD8, both of which are implicated in neurodevelopmental disorders including CHARGE Syndrome and autism spectrum disorders, respectively. Loss of function mutations in kis and animals expressing human APP and BACE in their central nervous system show reductions in the glutamate receptor subunit, GluRIIC, the GTPase Rab11, and the bone morphogenetic protein (BMP), pMad, at the Drosophila larval neuromuscular junction (NMJ). Similarly, processes like endocytosis, larval locomotion, and neurotransmission are deficient in these animals. Our pharmacological and epistasis experiments indicate that there is a functional relationship between Kis and APPL, but Kis does not regulate appl expression at the larval NMJ. Instead, Kis likely influences the synaptic localization of APPL, possibly by promoting rab11 transcription. These data identify a potential mechanistic connection between chromatin remodeling proteins and aberrant synaptic function in AD.
Subject(s)
Amyloid beta-Protein Precursor , Drosophila Proteins , Neuromuscular Junction , rab GTP-Binding Proteins , Animals , Neuromuscular Junction/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , rab GTP-Binding Proteins/metabolism , rab GTP-Binding Proteins/genetics , Amyloid beta-Protein Precursor/metabolism , Amyloid beta-Protein Precursor/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Synaptic Transmission , Synapses/metabolism , Receptors, Glutamate/metabolism , Receptors, Glutamate/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Humans , DNA Helicases/metabolism , DNA Helicases/genetics , Membrane Proteins , Nerve Tissue Proteins , Homeodomain Proteins , Receptors, Ionotropic GlutamateSubject(s)
Pulmonary Fibrosis , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/drug therapy , Humans , Transcription Factors/genetics , Transcription Factors/metabolism , Chromatin Assembly and Disassembly/genetics , Animals , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Mice , DNA Helicases/genetics , DNA Helicases/metabolismABSTRACT
An outbreak of mpox virus in May 2022 has spread over 110 nonpandemic regions in the world, posing a great threat to global health. Mpox virus E5, a helicase-primase, plays an essential role in DNA replication, but the molecular mechanisms are elusive. Here, we report seven structures of mpox virus E5 in a double hexamer (DH) and six in single hexamer in different conformations, indicating a rotation mechanism for helicase and a coupling action for primase. The DH is formed through the interface of zinc-binding domains, and the central channel density indicates potential double-stranded DNA (dsDNA), which helps to identify dsDNA binding residues Arg249, Lys286, Lys315, and Lys317. Our work is important not only for understanding poxviral DNA replication but also for the development of novel therapeutics for serious poxviral infections including smallpox virus and mpox virus.
Subject(s)
DNA Helicases , DNA Primase , DNA Primase/metabolism , DNA Primase/chemistry , DNA Helicases/metabolism , DNA Helicases/chemistry , Models, Molecular , Viral Proteins/metabolism , Viral Proteins/chemistry , Viral Proteins/genetics , Protein Multimerization , DNA Replication , Protein Binding , DNA, Viral/metabolismABSTRACT
Despite advancements in chemotherapy and the availability of novel therapies, the outcome of adult patients with B-cell acute lymphoblastic leukemia (B-ALL) remains unsatisfactory. Therefore, it is necessary to understand the molecular mechanisms underlying the progression of B-ALL. Brahma-related gene 1 (BRG1) is a poor prognostic factor for multiple cancers. Here, the expression of BRG1 was found to be higher in patients with B-ALL, irrespective of the molecular subtype, than in healthy individuals, and its overexpression was associated with a poor prognosis. Upregulation of BRG1 accelerated cell cycle progression into the S phase, resulting in increased cell proliferation, whereas its downregulation facilitated the apoptosis of B-ALL cells. Mechanistically, BRG1 occupies the transcriptional activation site of PPP2R1A, thereby inhibiting its expression and activating the PI3K/AKT signaling pathway to regulate the proto-oncogenes c-Myc and BCL-2. Consistently, silencing of BRG1 and administration of PFI-3 (a specific inhibitor targeting BRG1) significantly inhibited the progression of leukemia and effectively prolonged survival in cell-derived xenograft mouse models of B-ALL. Altogether, this study demonstrates that BRG1-induced overactivation of the PPP2R1A/PI3K/AKT signaling pathway plays an important role in promoting the progression of B-ALL. Therefore, targeting BRG1 represents a promising strategy for the treatment of B-ALL in adults.
Subject(s)
DNA Helicases , Disease Progression , Nuclear Proteins , Protein Phosphatase 2 , Transcription Factors , Animals , Female , Humans , Male , Mice , Apoptosis/drug effects , Apoptosis/genetics , Cell Line, Tumor , Cell Proliferation/drug effects , DNA Helicases/metabolism , DNA Helicases/genetics , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/genetics , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/pathology , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/metabolism , Precursor B-Cell Lymphoblastic Leukemia-Lymphoma/drug therapy , Protein Phosphatase 2/metabolism , Protein Phosphatase 2/genetics , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/drug effects , Transcription Factors/metabolism , Transcription Factors/genetics , Transcription, Genetic/drug effectsABSTRACT
Mutations of the SNF2 family ATPase HELLS and its activator CDCA7 cause immunodeficiency, centromeric instability, and facial anomalies syndrome, characterized by DNA hypomethylation at heterochromatin. It remains unclear why CDCA7-HELLS is the sole nucleosome remodeling complex whose deficiency abrogates the maintenance of DNA methylation. We here identify the unique zinc-finger domain of CDCA7 as an evolutionarily conserved hemimethylation-sensing zinc finger (HMZF) domain. Cryo-electron microscopy structural analysis of the CDCA7-nucleosome complex reveals that the HMZF domain can recognize hemimethylated CpG in the outward-facing DNA major groove within the nucleosome core particle, whereas UHRF1, the critical activator of the maintenance methyltransferase DNMT1, cannot. CDCA7 recruits HELLS to hemimethylated chromatin and facilitates UHRF1-mediated H3 ubiquitylation associated with replication-uncoupled maintenance DNA methylation. We propose that the CDCA7-HELLS nucleosome remodeling complex assists the maintenance of DNA methylation on chromatin by sensing hemimethylated CpG that is otherwise inaccessible to UHRF1 and DNMT1.
Subject(s)
CCAAT-Enhancer-Binding Proteins , DNA Methylation , Nucleosomes , Ubiquitin-Protein Ligases , Humans , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Nucleosomes/metabolism , Nucleosomes/genetics , CCAAT-Enhancer-Binding Proteins/metabolism , CCAAT-Enhancer-Binding Proteins/genetics , Cryoelectron Microscopy , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , Cell Cycle Proteins/chemistry , CpG Islands , Ubiquitination , Evolution, Molecular , DNA/metabolism , DNA/chemistry , DNA/genetics , Zinc Fingers , Chromatin/metabolism , Chromatin/genetics , DNA (Cytosine-5-)-Methyltransferase 1/metabolism , DNA (Cytosine-5-)-Methyltransferase 1/genetics , DNA Helicases/metabolism , DNA Helicases/genetics , DNA Helicases/chemistry , Nuclear Proteins/metabolism , Nuclear Proteins/genetics , Nuclear Proteins/chemistry , Eukaryota/genetics , Eukaryota/metabolism , Protein Binding , Histones/metabolism , Histones/genetics , Adenosine Triphosphatases/metabolism , Adenosine Triphosphatases/genetics , Adenosine Triphosphatases/chemistryABSTRACT
Mammalian TIP60 is a multi-functional enzyme with histone acetylation and histone dimer exchange activities. It plays roles in diverse cellular processes including transcription, DNA repair, cell cycle control, and embryonic development. Here we report the cryo-electron microscopy structures of the human TIP60 complex with the core subcomplex and TRRAP module refined to 3.2-Å resolution. The structures show that EP400 acts as a backbone integrating the motor module, the ARP module, and the TRRAP module. The RUVBL1-RUVBL2 hexamer serves as a rigid core for the assembly of EP400 ATPase and YL1 in the motor module. In the ARP module, an ACTL6A-ACTB heterodimer and an extra ACTL6A make hydrophobic contacts with EP400 HSA helix, buttressed by network interactions among DMAP1, EPC1, and EP400. The ARP module stably associates with the motor module but is flexibly tethered to the TRRAP module, exhibiting a unique feature of human TIP60. The architecture of the nucleosome-bound human TIP60 reveals an unengaged nucleosome that is located between the core subcomplex and the TRRAP module. Our work illustrates the molecular architecture of human TIP60 and provides architectural insights into how this complex is bound by the nucleosome.
Subject(s)
Cryoelectron Microscopy , Lysine Acetyltransferase 5 , Humans , Lysine Acetyltransferase 5/metabolism , Lysine Acetyltransferase 5/chemistry , Lysine Acetyltransferase 5/genetics , Nucleosomes/metabolism , Nucleosomes/ultrastructure , Nucleosomes/chemistry , DNA Helicases/metabolism , DNA Helicases/chemistry , Models, Molecular , ATPases Associated with Diverse Cellular Activities/metabolism , ATPases Associated with Diverse Cellular Activities/chemistry , ATPases Associated with Diverse Cellular Activities/genetics , Carrier Proteins/metabolism , Carrier Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/chemistry , Protein Binding , Protein Multimerization , Bromodomain Containing Proteins , Adaptor Proteins, Signal TransducingABSTRACT
Senataxin is an evolutionarily conserved DNA/RNA helicase, whose dysfunctions are linked to neurodegeneration and cancer. A main activity of this protein is the removal of R-loops, which are nucleic acid structures capable to promote DNA damage and replication stress. Here we found that Senataxin deficiency causes the release of damaged DNA into extranuclear bodies, called micronuclei, triggering the massive recruitment of cGAS, the apical sensor of the innate immunity pathway, and the downstream stimulation of interferon genes. Such cGAS-positive micronuclei are characterized by defective membrane envelope and are particularly abundant in cycling cells lacking Senataxin, but not after exposure to a DNA breaking agent or in absence of the tumor suppressor BRCA1 protein, a partner of Senataxin in R-loop removal. Micronuclei with a discontinuous membrane are normally cleared by autophagy, a process that we show is impaired in Senataxin-deficient cells. The formation of Senataxin-dependent inflamed micronuclei is promoted by the persistence of nuclear R-loops stimulated by the DSIF transcription elongation complex and the engagement of EXO1 nuclease activity on nuclear DNA. Coherently, high levels of EXO1 result in poor prognosis in a subset of tumors lacking Senataxin expression. Hence, R-loop homeostasis impairment, together with autophagy failure and unscheduled EXO1 activity, elicits innate immune response through micronuclei formation in cells lacking Senataxin.
Subject(s)
Autophagy , DNA Damage , DNA Helicases , Inflammation , Multifunctional Enzymes , Nucleotidyltransferases , R-Loop Structures , RNA Helicases , Humans , Autophagy/genetics , BRCA1 Protein/metabolism , BRCA1 Protein/genetics , BRCA1 Protein/deficiency , DNA Helicases/metabolism , DNA Helicases/genetics , DNA Helicases/deficiency , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/genetics , DNA Repair Enzymes/deficiency , Exodeoxyribonucleases/metabolism , Exodeoxyribonucleases/genetics , Immunity, Innate , Inflammation/pathology , Inflammation/metabolism , Inflammation/genetics , Multifunctional Enzymes/metabolism , Multifunctional Enzymes/genetics , Nucleotidyltransferases/metabolism , Nucleotidyltransferases/genetics , Phosphoproteins , RNA Helicases/metabolism , RNA Helicases/geneticsABSTRACT
Cockayne Syndrome B (CSB) is a hereditary multiorgan syndrome which-through largely unknown mechanisms-can affect the brain where it clinically presents with microcephaly, intellectual disability and demyelination. Using human induced pluripotent stem cell (hiPSC)-derived neural 3D models generated from CSB patient-derived and isogenic control lines, we here provide explanations for these three major neuropathological phenotypes. In our models, CSB deficiency is associated with (i) impaired cellular migration due to defective autophagy as an explanation for clinical microcephaly; (ii) altered neuronal network functionality and neurotransmitter GABA levels, which is suggestive of a disturbed GABA switch that likely impairs brain circuit formation and ultimately causes intellectual disability; and (iii) impaired oligodendrocyte maturation as a possible cause of the demyelination observed in children with CSB. Of note, the impaired migration and oligodendrocyte maturation could both be partially rescued by pharmacological HDAC inhibition.
Subject(s)
Cockayne Syndrome , Induced Pluripotent Stem Cells , Oligodendroglia , Humans , Induced Pluripotent Stem Cells/metabolism , Induced Pluripotent Stem Cells/cytology , Induced Pluripotent Stem Cells/pathology , Cockayne Syndrome/genetics , Cockayne Syndrome/metabolism , Cockayne Syndrome/pathology , Oligodendroglia/metabolism , Oligodendroglia/cytology , Cell Movement , DNA Repair Enzymes/metabolism , DNA Repair Enzymes/genetics , Neurons/metabolism , Neurons/pathology , Autophagy , Brain/metabolism , Brain/pathology , Poly-ADP-Ribose Binding Proteins/metabolism , Poly-ADP-Ribose Binding Proteins/genetics , gamma-Aminobutyric Acid/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , Microcephaly/pathology , Microcephaly/metabolism , Microcephaly/genetics , Demyelinating Diseases/pathology , Demyelinating Diseases/metabolism , Cell DifferentiationABSTRACT
RTEL1 is an essential DNA helicase which plays an important role in various aspects of genome stability, from telomere metabolism to DNA replication, repair and recombination. RTEL1 has been implicated in a number of genetic diseases and cancer development, including glioma, breast, lung and gastrointestinal tumors. RTEL1 is a FeS helicase but, in addition to the helicase core, it comprises a long C-terminal region which includes a number of folded domains connected by intrinsically disordered loops and mediates RTEL1 interaction with factors involved in pivotal cellular pathways. However, information on the architecture and the function of this region is still limited. We expressed and purified a variety of fragments encompassing the folded domains and the unstructured regions. We determined the crystal structure of the second repeat, confirming that it has a fold similar to the harmonin homology domains. SAXS data provide low-resolution information on all the fragments and suggest that the presence of the RING domain affects the overall architecture of the C-terminal region, making the structure significantly more compact. NMR data provide experimental information on the interaction between PCNA and the RTEL1 C-terminal region, revealing a putative low-affinity additional site of interaction. A biochemical analysis shows that the C-terminal region, in addition to a preference for telomeric RNA and DNA G-quadruplexes, has a high affinity for R-loops and D-loops, consistent with the role played by the RTEL1 helicase in homologous recombination, telomere maintenance and preventing replication-transcription conflicts. We further dissected the contribution of each domain in binding different substrates.
Subject(s)
DNA Helicases , Humans , DNA Helicases/chemistry , DNA Helicases/metabolism , DNA Helicases/genetics , Crystallography, X-Ray , Models, Molecular , Proliferating Cell Nuclear Antigen/chemistry , Proliferating Cell Nuclear Antigen/metabolism , Proliferating Cell Nuclear Antigen/genetics , Protein Domains , Scattering, Small AngleABSTRACT
Three-stranded DNA-RNA structures known as R-loops that form during papillomavirus transcription can cause transcription-replication conflicts and lead to DNA damage. We found that R-loops accumulated at the viral early promoter in human papillomavirus (HPV) episomal cells but were greatly reduced in cells with integrated HPV genomes. RNA-DNA helicases unwind R-loops and allow for transcription and replication to proceed. Depletion of the RNA-DNA helicase senataxin (SETX) using siRNAs increased the presence of R-loops at the viral early promoter in HPV-31 (CIN612) and HPV-16 (W12) episomal HPV cell lines. Depletion of SETX reduced viral transcripts in episomal HPV cell lines. The viral E2 protein, which binds with high affinity to specific palindromes near the promoter and origin, complexes with SETX, and both SETX and E2 are present at the viral p97 promoter in CIN612 and W12 cells. SETX overexpression increased E2 transcription activity on the p97 promoter. SETX depletion also significantly increased integration of viral genomes in CIN612 cells. Our results demonstrate that SETX resolves viral R-loops to proceed with HPV transcription and prevent genome integration.IMPORTANCEPapillomaviruses contain small circular genomes of approximately 8 kilobase pairs and undergo unidirectional transcription from the sense strand of the viral genome. Co-transcriptional R-loops were recently reported to be present at high levels in cells that maintain episomal HPV and were also detected at the early viral promoter. R-loops can inhibit transcription and DNA replication. The process that removes R-loops from the PV genome and the requisite enzymes are unknown. We propose a model in which the host RNA-DNA helicase senataxin assembles on the HPV genome to resolve R-loops in order to maintain the episomal status of the viral genome.
Subject(s)
DNA Helicases , Multifunctional Enzymes , Promoter Regions, Genetic , R-Loop Structures , RNA Helicases , Humans , RNA Helicases/genetics , RNA Helicases/metabolism , Multifunctional Enzymes/genetics , Multifunctional Enzymes/metabolism , DNA Helicases/metabolism , DNA Helicases/genetics , R-Loop Structures/genetics , Plasmids/genetics , Virus Replication , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Papillomaviridae/genetics , Genome, Viral , Oncogene Proteins, Viral/genetics , Oncogene Proteins, Viral/metabolism , Transcription, Genetic , Cell Line , DNA, Viral/geneticsABSTRACT
The simian virus 40 (SV40) replisome only encodes for its helicase; large T-antigen (L-Tag), while relying on the host for the remaining proteins, making it an intriguing model system. Despite being one of the earliest reconstituted eukaryotic systems, the interactions coordinating its activities and the identification of new factors remain largely unexplored. Herein, we in vitro reconstituted the SV40 replisome activities at the single-molecule level, including DNA unwinding by L-Tag and the single-stranded DNA-binding protein Replication Protein A (RPA), primer extension by DNA polymerase δ, and their concerted leading-strand synthesis. We show that RPA stimulates the processivity of L-Tag without altering its rate and that DNA polymerase δ forms a stable complex with L-Tag during leading-strand synthesis. Furthermore, similar to human and budding yeast Cdc45-MCM-GINS helicase, L-Tag uses the fork protection complex (FPC) and the mini-chromosome maintenance protein 10 (Mcm10) during synthesis. Hereby, we demonstrate that FPC increases this rate, and both FPC and Mcm10 increase the processivity by stabilizing stalled replisomes and increasing their chances of restarting synthesis. The detailed kinetics and novel factors of the SV40 replisome establish it as a closer mimic of the host replisome and expand its application as a model replication system.
Subject(s)
DNA Replication , Minichromosome Maintenance Proteins , Replication Protein A , Simian virus 40 , Simian virus 40/metabolism , Simian virus 40/genetics , Humans , Minichromosome Maintenance Proteins/metabolism , Minichromosome Maintenance Proteins/genetics , Replication Protein A/metabolism , DNA Polymerase III/metabolism , DNA Polymerase III/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , DNA Helicases/metabolism , DNA Helicases/genetics , DNA, Viral/metabolism , DNA, Viral/genetics , Virus Replication , Single Molecule Imaging , Antigens, Polyomavirus Transforming/metabolism , Antigens, Polyomavirus Transforming/genetics , DNA, Single-Stranded/metabolism , DNA-Directed DNA Polymerase , Multienzyme ComplexesABSTRACT
The SARS-CoV-2 helicase, non-structural protein 13 (Nsp13), plays an essential role in viral replication, translocating in the 5' â 3' direction as it unwinds double-stranded RNA/DNA. We investigated the impact of structurally distinct DNA lesions on DNA unwinding catalyzed by Nsp13. The selected lesions include two benzo[a]pyrene (B[a]P)-derived dG adducts, the UV-induced cyclobutane pyrimidine dimer (CPD), and the pyrimidine (6-4) pyrimidone (6-4PP) photolesion. The experimentally observed unwinding rate constants (kobs) and processivities (P) were examined. Relative to undamaged DNA, the kobs values were diminished by factors of up to ~15 for B[a]P adducts but only by factors of ~2-5 for photolesions. A minor-groove-oriented B[a]P adduct showed the smallest impact on P, which decreased by ~11% compared to unmodified DNA, while an intercalated one reduced P by ~67%. However, the photolesions showed a greater impact on the processivities; notably, the CPD, with the highest kobs value, exhibited the lowest P, which was reduced by ~90%. Our findings thus show that DNA unwinding efficiencies are lesion-dependent and most strongly inhibited by the CPD, leading to the conclusion that processivity is a better measure of DNA lesions' inhibitory effects than unwinding rate constants.
Subject(s)
DNA Helicases , SARS-CoV-2 , Viral Nonstructural Proteins , SARS-CoV-2/metabolism , Viral Nonstructural Proteins/metabolism , Viral Nonstructural Proteins/chemistry , DNA Helicases/metabolism , DNA Helicases/chemistry , DNA/metabolism , DNA/chemistry , Humans , DNA Damage , COVID-19/virology , Kinetics , Methyltransferases , RNA HelicasesABSTRACT
High spontaneous mutation rate is crucial for obtaining ideal phenotype and exploring the relationship between genes and phenotype. How to break the genetic stability of organisms and increase the mutation frequency has become a research hotspot. Here, we present a practical and controllable evolutionary tool (oMut-Cgts) based on dual genetic level modification engineering for Corynebacterium glutamicum. Firstly, the modification engineering of transcription and replication levels based on RNA polymerase α subunit and DNA helicase Cgl0854 as the 'dock' of cytidine deaminase (pmCDA1) significantly increased the mutation rate, proving that the localization of pmCDA1 around transient ssDNA is necessary for genome mutation. Then, the combined modification and optimization of engineering at dual genetic level achieved 1.02 × 104-fold increased mutation rate. The genome sequencing revealed that the oMut-Cgts perform uniform and efficient C:GâT:A transitions on a genome-wide scale. Furthermore, oMut-Cgts-mediated rapid evolution of C. glutamicum with stress (acid, oxidative and ethanol) tolerance proved that the tool has powerful functions in multi-dimensional biological engineering (rapid phenotype evolution, gene function mining and protein evolution). The strategies for rapid genome evolution provided in this study are expected to be applicable to a variety of applications in all prokaryotic cells.
Subject(s)
Corynebacterium glutamicum , Genome, Bacterial , Corynebacterium glutamicum/genetics , Genetic Engineering/methods , Cytidine Deaminase/genetics , Cytidine Deaminase/metabolism , Mutation Rate , Evolution, Molecular , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , DNA Helicases/genetics , DNA Helicases/metabolism , DNA Replication/genetics , MutationABSTRACT
Post-mitotic, non-proliferative dermal fibroblasts have crucial functions in maintenance and restoration of tissue homeostasis. They are involved in essential processes such as wound healing, pigmentation and hair growth, but also tumor development and aging-associated diseases. These processes are energetically highly demanding and error prone when mitochondrial damage occurs. However, mitochondrial function in fibroblasts and the influence of mitochondrial dysfunction on fibroblast-specific demands are still unclear. To address these questions, we created a mouse model in which accelerated cell-specific mitochondrial DNA (mtDNA) damage accumulates. We crossed mice carrying a dominant-negative mutant of the mitochondrial replicative helicase Twinkle (RosaSTOP system) with mice that express fibroblast-specific Cre Recombinase (Collagen1A2 CreERT) which can be activated by Tamoxifen (TwinkleFIBRO). Thus, we are able to induce mtDNA deletions and duplications in specific cells, a process which resembles the physiological aging process in humans, where this damage accumulates in all tissues. Upon proliferation in vitro, Tamoxifen induced Twinkle fibroblasts deplete most of their mitochondrial DNA which, although not disturbing the stoichiometry of the respiratory chain complexes, leads to reduced ROS production and mitochondrial membrane potential as well as an anti-inflammatory and anti-fibrotic profile of the cells. In Sodium Azide treated wildtype fibroblasts, without a functioning respiratory chain, we observe the opposite, a rather pro-inflammatory and pro-fibrotic signature. Upon accumulation of mitochondrial DNA mutations in vivo the TwinkleFIBRO mice are protected from fibrosis development induced by intradermal Bleomycin injections. This is due to dampened differentiation of the dermal fibroblasts into α-smooth-muscle-actin positive myofibroblasts in TwinkleFIBRO mice. We thus provide evidence for striking differences of the impact that mtDNA mutations have in contrast to blunted mitochondrial function in dermal fibroblasts and skin homeostasis. These data contribute to improved understanding of mitochondrial function and dysfunction in skin and provide mechanistic insight into potential targets to treat skin fibrosis in the future.