RESUMO
Covalent DNA-protein crosslinks (DPCs) are pervasive DNA lesions that interfere with essential chromatin processes such as transcription or replication. This review strives to provide an overview of the sources and principles of cellular DPC formation. DPCs are caused by endogenous reactive metabolites and various chemotherapeutic agents. However, in certain conditions DPCs also arise physiologically in cells. We discuss the cellular mechanisms resolving these threats to genomic integrity. Detection and repair of DPCs require not only the action of canonical DNA repair pathways but also the activity of specialized proteolytic enzymes-including proteases of the SPRTN/Wss1 family-to degrade the crosslinked protein. Loss of DPC repair capacity has dramatic consequences, ranging from genome instability in yeast and worms to cancer predisposition and premature aging in mice and humans.
Assuntos
Reparo do DNA , Proteínas de Saccharomyces cerevisiae , Animais , DNA/genética , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Camundongos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
How mis-regulated chromatin directly impacts human immune disorders is poorly understood. Speckled Protein 140 (SP140) is an immune-restricted PHD and bromodomain-containing epigenetic "reader," and SP140 loss-of-function mutations associate with Crohn's disease (CD), multiple sclerosis (MS), and chronic lymphocytic leukemia (CLL). However, the relevance of these mutations and mechanisms underlying SP140-driven pathogenicity remains unexplored. Using a global proteomic strategy, we identified SP140 as a repressor of topoisomerases (TOPs) that maintains heterochromatin and macrophage fate. In humans and mice, SP140 loss resulted in unleashed TOP activity, de-repression of developmentally silenced genes, and ultimately defective microbe-inducible macrophage transcriptional programs and bacterial killing that drive intestinal pathology. Pharmacological inhibition of TOP1/2 rescued these defects. Furthermore, exacerbated colitis was restored with TOP1/2 inhibitors in Sp140-/- mice, but not wild-type mice, in vivo. Collectively, we identify SP140 as a TOP repressor and reveal repurposing of TOP inhibition to reverse immune diseases driven by SP140 loss.
Assuntos
Doença de Crohn , Animais , Humanos , Camundongos , Antígenos Nucleares , Doença de Crohn/genética , Doença de Crohn/patologia , Epigênese Genética , Regulação da Expressão Gênica , Macrófagos/patologia , Proteômica , Fatores de TranscriçãoRESUMO
The ongoing pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is currently affecting millions of lives worldwide. Large retrospective studies indicate that an elevated level of inflammatory cytokines and pro-inflammatory factors are associated with both increased disease severity and mortality. Here, using multidimensional epigenetic, transcriptional, in vitro, and in vivo analyses, we report that topoisomerase 1 (TOP1) inhibition suppresses lethal inflammation induced by SARS-CoV-2. Therapeutic treatment with two doses of topotecan (TPT), an FDA-approved TOP1 inhibitor, suppresses infection-induced inflammation in hamsters. TPT treatment as late as 4 days post-infection reduces morbidity and rescues mortality in a transgenic mouse model. These results support the potential of TOP1 inhibition as an effective host-directed therapy against severe SARS-CoV-2 infection. TPT and its derivatives are inexpensive clinical-grade inhibitors available in most countries. Clinical trials are needed to evaluate the efficacy of repurposing TOP1 inhibitors for severe coronavirus disease 2019 (COVID-19) in humans.
Assuntos
Tratamento Farmacológico da COVID-19 , DNA Topoisomerases Tipo I/metabolismo , SARS-CoV-2/metabolismo , Inibidores da Topoisomerase I/farmacologia , Topotecan/farmacologia , Animais , COVID-19/enzimologia , COVID-19/patologia , Chlorocebus aethiops , Humanos , Inflamação/tratamento farmacológico , Inflamação/enzimologia , Inflamação/patologia , Inflamação/virologia , Mesocricetus , Camundongos , Camundongos Transgênicos , Células THP-1 , Células VeroRESUMO
In many eukaryotes, Argonaute proteins, guided by short RNA sequences, defend cells against transposons and viruses. In the eubacterium Thermus thermophilus, the DNA-guided Argonaute TtAgo defends against transformation by DNA plasmids. Here, we report that TtAgo also participates in DNA replication. In vivo, TtAgo binds 15- to 18-nt DNA guides derived from the chromosomal region where replication terminates and associates with proteins known to act in DNA replication. When gyrase, the sole T. thermophilus type II topoisomerase, is inhibited, TtAgo allows the bacterium to finish replicating its circular genome. In contrast, loss of gyrase and TtAgo activity slows growth and produces long sausage-like filaments in which the individual bacteria are linked by DNA. Finally, wild-type T. thermophilus outcompetes an otherwise isogenic strain lacking TtAgo. We propose that the primary role of TtAgo is to help T. thermophilus disentangle the catenated circular chromosomes generated by DNA replication.
Assuntos
Proteínas Argonautas/metabolismo , Proteínas de Bactérias/metabolismo , DNA Girase/metabolismo , Replicação do DNA/genética , DNA/metabolismo , Thermus thermophilus/metabolismo , Proteínas Argonautas/genética , Proteínas de Bactérias/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Cromossomos/metabolismo , Ciprofloxacina/farmacologia , DNA/genética , Replicação do DNA/efeitos dos fármacos , Endonucleases/metabolismo , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Proteínas Recombinantes , Recombinação Genética/efeitos dos fármacos , Recombinação Genética/genética , Imagem Individual de Molécula , Espectrometria de Massas em Tandem , Thermus thermophilus/genética , Thermus thermophilus/crescimento & desenvolvimento , Thermus thermophilus/ultraestrutura , Inibidores da Topoisomerase II/farmacologiaRESUMO
DNA replication in eukaryotes generates DNA supercoiling, which may intertwine (braid) daughter chromatin fibers to form precatenanes, posing topological challenges during chromosome segregation. The mechanisms that limit precatenane formation remain unclear. By making direct torque measurements, we demonstrate that the intrinsic mechanical properties of chromatin play a fundamental role in dictating precatenane formation and regulating chromatin topology. Whereas a single chromatin fiber is torsionally soft, a braided fiber is torsionally stiff, indicating that supercoiling on chromatin substrates is preferentially directed in front of the fork during replication. We further show that topoisomerase II relaxation displays a strong preference for a single chromatin fiber over a braided fiber. These results suggest a synergistic coordination-the mechanical properties of chromatin inherently suppress precatenane formation during replication elongation by driving DNA supercoiling ahead of the fork, where supercoiling is more efficiently removed by topoisomerase II. VIDEO ABSTRACT.
Assuntos
Cromatina/química , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Torque , Cromatina/metabolismo , Replicação do DNA , DNA Super-Helicoidal/química , Células HeLa , Humanos , Pinças Ópticas , Saccharomyces cerevisiaeRESUMO
Cohesin extrusion is thought to play a central role in establishing the architecture of mammalian genomes. However, extrusion has not been visualized in vivo, and thus, its functional impact and energetics are unknown. Using ultra-deep Hi-C, we show that loop domains form by a process that requires cohesin ATPases. Once formed, however, loops and compartments are maintained for hours without energy input. Strikingly, without ATP, we observe the emergence of hundreds of CTCF-independent loops that link regulatory DNA. We also identify architectural "stripes," where a loop anchor interacts with entire domains at high frequency. Stripes often tether super-enhancers to cognate promoters, and in B cells, they facilitate Igh transcription and recombination. Stripe anchors represent major hotspots for topoisomerase-mediated lesions, which promote chromosomal translocations and cancer. In plasmacytomas, stripes can deregulate Igh-translocated oncogenes. We propose that higher organisms have coopted cohesin extrusion to enhance transcription and recombination, with implications for tumor development.
Assuntos
Trifosfato de Adenosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Genoma , Animais , Linfócitos B/citologia , Linfócitos B/metabolismo , Fator de Ligação a CCCTC/genética , Fator de Ligação a CCCTC/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Linhagem Celular , Proteoglicanas de Sulfatos de Condroitina/genética , Proteoglicanas de Sulfatos de Condroitina/metabolismo , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/química , Proteínas Cromossômicas não Histona/genética , Cromossomos/metabolismo , Proteínas de Ligação a DNA , Humanos , Camundongos , Mutagênese , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Transcrição Gênica , CoesinasRESUMO
When DNA is unwound during replication, it becomes overtwisted and forms positive supercoils in front of the translocating DNA polymerase. Unless removed or dissipated, this superhelical tension can impede replication elongation. Topoisomerases, including gyrase and topoisomerase IV in bacteria, are required to relax positive supercoils ahead of DNA polymerase but may not be sufficient for replication. Here, we find that GapR, a chromosome structuring protein in Caulobacter crescentus, is required to complete DNA replication. GapR associates in vivo with positively supercoiled chromosomal DNA, and our biochemical and structural studies demonstrate that GapR forms a dimer-of-dimers that fully encircles overtwisted DNA. Further, we show that GapR stimulates gyrase and topo IV to relax positive supercoils, thereby enabling DNA replication. Analogous chromosome structuring proteins that locate to the overtwisted DNA in front of replication forks may be present in other organisms, similarly helping to recruit and stimulate topoisomerases during DNA replication.
Assuntos
Cromossomos Bacterianos/fisiologia , DNA Bacteriano/química , DNA Super-Helicoidal/metabolismo , Proteínas de Bactérias/metabolismo , Caulobacter crescentus/metabolismo , Caulobacter crescentus/fisiologia , Estruturas Cromossômicas/fisiologia , Cromossomos Bacterianos/metabolismo , DNA/fisiologia , Replicação do DNA/fisiologia , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/metabolismo , DNA Topoisomerases Tipo II/fisiologia , DNA Bacteriano/fisiologia , Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica/genética , CinéticaRESUMO
In this study, we show that evolutionarily conserved chromosome loop anchors bound by CCCTC-binding factor (CTCF) and cohesin are vulnerable to DNA double strand breaks (DSBs) mediated by topoisomerase 2B (TOP2B). Polymorphisms in the genome that redistribute CTCF/cohesin occupancy rewire DNA cleavage sites to novel loop anchors. While transcription- and replication-coupled genomic rearrangements have been well documented, we demonstrate that DSBs formed at loop anchors are largely transcription-, replication-, and cell-type-independent. DSBs are continuously formed throughout interphase, are enriched on both sides of strong topological domain borders, and frequently occur at breakpoint clusters commonly translocated in cancer. Thus, loop anchors serve as fragile sites that generate DSBs and chromosomal rearrangements. VIDEO ABSTRACT.
Assuntos
Fragilidade Cromossômica , Quebras de DNA de Cadeia Dupla , Neoplasias/genética , Animais , Linfócitos B/metabolismo , Fator de Ligação a CCCTC , Linhagem Celular Tumoral , DNA Topoisomerases Tipo II/metabolismo , Proteínas de Ligação a DNA/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas de Ligação a Poli-ADP-Ribose , Proteínas Repressoras/metabolismoRESUMO
The topological state of chromosomes determines their mechanical properties, dynamics, and function. Recent work indicated that interphase chromosomes are largely free of entanglements. Here, we use Hi-C, polymer simulations, and multi-contact 3C and find that, by contrast, mitotic chromosomes are self-entangled. We explore how a mitotic self-entangled state is converted into an unentangled interphase state during mitotic exit. Most mitotic entanglements are removed during anaphase/telophase, with remaining ones removed during early G1, in a topoisomerase-II-dependent process. Polymer models suggest a two-stage disentanglement pathway: first, decondensation of mitotic chromosomes with remaining condensin loops produces entropic forces that bias topoisomerase II activity toward decatenation. At the second stage, the loops are released, and the formation of new entanglements is prevented by lower topoisomerase II activity, allowing the establishment of unentangled and territorial G1 chromosomes. When mitotic entanglements are not removed in experiments and models, a normal interphase state cannot be acquired.
Assuntos
Cromossomos , DNA Topoisomerases Tipo II , DNA Topoisomerases Tipo II/genética , Cromossomos/genética , Mitose/genética , Interfase/genética , PolímerosRESUMO
SLX4, disabled in the Fanconi anemia group P, is a scaffolding protein that coordinates the action of structure-specific endonucleases and other proteins involved in the replication-coupled repair of DNA interstrand cross-links. Here, we show that SLX4 dimerization and SUMO-SIM interactions drive the assembly of SLX4 membraneless compartments in the nucleus called condensates. Super-resolution microscopy reveals that SLX4 forms chromatin-bound clusters of nanocondensates. We report that SLX4 compartmentalizes the SUMO-RNF4 signaling pathway. SENP6 and RNF4 regulate the assembly and disassembly of SLX4 condensates, respectively. SLX4 condensation per se triggers the selective modification of proteins by SUMO and ubiquitin. Specifically, SLX4 condensation induces ubiquitylation and chromatin extraction of topoisomerase 1 DNA-protein cross-links. SLX4 condensation also induces the nucleolytic degradation of newly replicated DNA. We propose that the compartmentalization of proteins by SLX4 through site-specific interactions ensures the spatiotemporal control of protein modifications and nucleolytic reactions during DNA repair.
Assuntos
Reparo do DNA , Ubiquitina , Ubiquitinação , Ubiquitina/metabolismo , DNA/metabolismo , CromatinaRESUMO
High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Neoplasias/enzimologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Animais , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , DNA Super-Helicoidal/biossíntese , DNA Super-Helicoidal/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Células K562 , Complexos Multienzimáticos , Neoplasias/genética , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , RatosRESUMO
As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.
Assuntos
Proliferação de Células , Montagem e Desmontagem da Cromatina , Neoplasias Colorretais/enzimologia , DNA Topoisomerases Tipo I/metabolismo , Fase G1 , Mitose , RNA Polimerase II/metabolismo , Transcrição Gênica , Proliferação de Células/efeitos dos fármacos , Sequenciamento de Cromatina por Imunoprecipitação , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA Topoisomerases Tipo I/genética , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Inibidores de MTOR/farmacologia , Mitose/efeitos dos fármacos , RNA Polimerase II/genéticaRESUMO
Replication fork reversal is a global response to replication stress in mammalian cells, but precisely how it occurs remains poorly understood. Here, we show that, upon replication stress, DNA topoisomerase IIalpha (TOP2A) is recruited to stalled forks in a manner dependent on the SNF2-family DNA translocases HLTF, ZRANB3, and SMARCAL1. This is accompanied by an increase in TOP2A SUMOylation mediated by the SUMO E3 ligase ZATT and followed by recruitment of a SUMO-targeted DNA translocase, PICH. Disruption of the ZATT-TOP2A-PICH axis results in accumulation of partially reversed forks and enhanced genome instability. These results suggest that fork reversal occurs via a sequential two-step process. First, HLTF, ZRANB3, and SMARCAL1 initiate limited fork reversal, creating superhelical strain in the newly replicated sister chromatids. Second, TOP2A drives extensive fork reversal by resolving the resulting topological barriers and via its role in recruiting PICH to stalled forks.
Assuntos
DNA Helicases/metabolismo , Replicação do DNA , DNA Topoisomerases Tipo II/metabolismo , Genoma Humano , Instabilidade Genômica , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , DNA Helicases/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células HEK293 , Células HeLa , Humanos , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Topoisomerase 1 (Top1) controls DNA topology, relieves DNA supercoiling during replication and transcription, and is critical for mitotic progression to the G1 phase. Tyrosyl-DNA phosphodiesterase 1 (TDP1) mediates the removal of trapped Top1-DNA covalent complexes (Top1cc). Here, we identify CDK1-dependent phosphorylation of TDP1 at residue S61 during mitosis. A TDP1 variant defective for S61 phosphorylation (TDP1-S61A) is trapped on the mitotic chromosomes, triggering DNA damage and mitotic defects. Moreover, we show that Top1cc repair in mitosis occurs via a MUS81-dependent DNA repair mechanism. Replication stress induced by camptothecin or aphidicolin leads to TDP1-S61A enrichment at common fragile sites, which over-stimulates MUS81-dependent chromatid breaks, anaphase bridges, and micronuclei, ultimately culminating in the formation of 53BP1 nuclear bodies during G1 phase. Our findings provide new insights into the cell cycle-dependent regulation of TDP1 dynamics for the repair of trapped Top1-DNA covalent complexes during mitosis that prevents genomic instability following replication stress.
RESUMO
Programmed fork pausing is a complex process allowing cells to arrest replication forks at specific loci in a polar manner. Studies in budding yeast and other model organisms indicate that such replication fork barriers do not act as roadblocks passively impeding fork progression but rather elicit complex interactions between fork and barrier components. In this issue of Genes & Development, Shyian and colleagues (pp. 87-98) show that in budding yeast, the fork protection complex Tof1-Csm3 interacts physically with DNA topoisomerase I (Top1) at replication forks through the C-terminal domain of Tof1. Fork pausing at the ribosomal DNA (rDNA) replication fork barrier (RFB) is impaired in the absence of Top1 or in a tof1 mutant that does not bind Top1, but the function of Top1 can be partially compensated for by Top2. Together, these data indicate that topoisomerases play an unexpected role in the regulation of programmed fork pausing in Saccharomyces cerevisiae.
Assuntos
DNA Topoisomerases Tipo I , Proteínas de Saccharomyces cerevisiae , Proteínas de Ciclo Celular , Replicação do DNA , Proteínas de Ligação a DNARESUMO
Replication forks temporarily or terminally pause at hundreds of hard-to-replicate regions around the genome. A conserved pair of budding yeast replisome components Tof1-Csm3 (fission yeast Swi1-Swi3 and human TIMELESS-TIPIN) act as a "molecular brake" and promote fork slowdown at proteinaceous replication fork barriers (RFBs), while the accessory helicase Rrm3 assists the replisome in removing protein obstacles. Here we show that the Tof1-Csm3 complex promotes fork pausing independently of Rrm3 helicase by recruiting topoisomerase I (Top1) to the replisome. Topoisomerase II (Top2) partially compensates for the pausing decrease in cells when Top1 is lost from the replisome. The C terminus of Tof1 is specifically required for Top1 recruitment to the replisome and fork pausing but not for DNA replication checkpoint (DRC) activation. We propose that forks pause at proteinaceous RFBs through a "sTOP" mechanism ("slowing down with topoisomerases I-II"), which we show also contributes to protecting cells from topoisomerase-blocking agents.
Assuntos
Replicação do DNA/genética , DNA Topoisomerases/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/metabolismo , DNA Topoisomerases Tipo I/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mutação , Transporte Proteico , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMO
Topoisomerase II (TOP2) relieves torsional stress by forming transient cleavage complex intermediates (TOP2ccs) that contain TOP2-linked DNA breaks (DSBs). While TOP2ccs are normally reversible, they can be "trapped" by chemotherapeutic drugs such as etoposide and subsequently converted into irreversible TOP2-linked DSBs. Here, we have quantified etoposide-induced trapping of TOP2ccs, their conversion into irreversible TOP2-linked DSBs, and their processing during DNA repair genome-wide, as a function of time. We find that while TOP2 chromatin localization and trapping is independent of transcription, it requires pre-existing binding of cohesin to DNA. In contrast, the conversion of trapped TOP2ccs to irreversible DSBs during DNA repair is accelerated 2-fold at transcribed loci relative to non-transcribed loci. This conversion is dependent on proteasomal degradation and TDP2 phosphodiesterase activity. Quantitative modeling shows that only two features of pre-existing chromatin structure-namely, cohesin binding and transcriptional activity-can be used to predict the kinetics of TOP2-induced DSBs.
Assuntos
Quebras de DNA de Cadeia Dupla , DNA Topoisomerases Tipo II/química , DNA/genética , Complexos Multiproteicos/química , Proteínas de Ligação a Poli-ADP-Ribose/química , Quebra Cromossômica , Cromossomos/genética , DNA/química , Reparo do DNA/genética , DNA Topoisomerases Tipo II/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Etoposídeo/química , Conversão Gênica/genética , Células HCT116 , Humanos , Cinética , Complexos Multiproteicos/genética , Proteínas de Ligação a Poli-ADP-Ribose/genética , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia , Torção Mecânica , Transcrição Gênica , Translocação Genética/genéticaRESUMO
Displacement loops (D-loops) are pivotal intermediates of homologous recombination (HR), a universal DNA double strand break (DSB) repair pathway. We developed a versatile assay for the physical detection of D-loops in vivo, which enabled studying the kinetics of their formation and defining the activities controlling their metabolism. Nascent D-loops are detected within 2 h of DSB formation and extended in a delayed fashion in a genetic system designed to preclude downstream repair steps. The majority of nascent D-loops are disrupted by two pathways: one supported by the Srs2 helicase and the other by the Mph1 helicase and the Sgs1-Top3-Rmi1 helicase-topoisomerase complex. Both pathways operate without significant overlap and are delineated by the Rad54 paralog Rdh54 in an ATPase-independent fashion. This study uncovers a layer of quality control of HR relying on nascent D-loop dynamics.
Assuntos
Dano ao DNA , DNA Fúngico/genética , Reparo de DNA por Recombinação , Saccharomyces cerevisiae/genética , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , DNA Topoisomerases/genética , DNA Topoisomerases/metabolismo , DNA Fúngico/química , DNA Fúngico/metabolismo , Cinética , Conformação de Ácido Nucleico , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Relação Estrutura-AtividadeRESUMO
The convergence of two DNA replication forks creates unique problems during DNA replication termination. In E. coli and SV40, the release of torsional strain by type II topoisomerases is critical for converging replisomes to complete DNA synthesis, but the pathways that mediate fork convergence in eukaryotes are unknown. We studied the convergence of reconstituted yeast replication forks that include all core replisome components and both type I and type II topoisomerases. We found that most converging forks stall at a very late stage, indicating a role for additional factors. We showed that the Pif1 and Rrm3 DNA helicases promote efficient fork convergence and completion of DNA synthesis, even in the absence of type II topoisomerase. Furthermore, Rrm3 and Pif1 are also important for termination of plasmid DNA replication in vivo. These findings identify a eukaryotic pathway for DNA replication termination that is distinct from previously characterized prokaryotic mechanisms.
Assuntos
DNA Helicases/genética , Replicação do DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , DNA Topoisomerases/genética , Escherichia coli/genética , Eucariotos/genética , Instabilidade Genômica , Plasmídeos/genéticaRESUMO
The mTOR pathway integrates both extracellular and intracellular signals and serves as a central regulator of cell metabolism, growth, survival, and stress responses. Neurotropic viruses, such as herpes simplex virus-1 (HSV-1), also rely on cellular AKT-mTORC1 signaling to achieve viral latency. Here, we define a novel genotoxic response whereby spatially separated signals initiated by extracellular neurotrophic factors and nuclear DNA damage are integrated by the AKT-mTORC1 pathway. We demonstrate that endogenous DNA double-strand breaks (DSBs) mediated by Topoisomerase 2ß-DNA cleavage complex (TOP2ßcc) intermediates are required to achieve AKT-mTORC1 signaling and maintain HSV-1 latency in neurons. Suppression of host DNA-repair pathways that remove TOP2ßcc trigger HSV-1 reactivation. Moreover, perturbation of AKT phosphorylation dynamics by downregulating the PHLPP1 phosphatase led to AKT mis-localization and disruption of DSB-induced HSV-1 reactivation. Thus, the cellular genome integrity and environmental inputs are consolidated and co-opted by a latent virus to balance lifelong infection with transmission.