RESUMEN
DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.
Asunto(s)
Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN , Reparación del ADN , Exodesoxirribonucleasas , Humanos , Roturas del ADN de Doble Cadena/efectos de la radiación , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , ADN/metabolismo , ADN/genética , Ubiquitinación , Proteínas de Ciclo CelularRESUMEN
Most genotoxic anticancer agents fail in tumors with intact DNA repair. Therefore, trabectedin, anagent more toxic to cells with active DNA repair, specifically transcription-coupled nucleotide excision repair (TC-NER), provides therapeutic opportunities. To unlock the potential of trabectedin and inform its application in precision oncology, an understanding of the mechanism of the drug's TC-NER-dependent toxicity is needed. Here, we determine that abortive TC-NER of trabectedin-DNA adducts forms persistent single-strand breaks (SSBs) as the adducts block the second of the two sequential NER incisions. We map the 3'-hydroxyl groups of SSBs originating from the first NER incision at trabectedin lesions, recording TC-NER on a genome-wide scale. Trabectedin-induced SSBs primarily occur in transcribed strands of active genes and peak near transcription start sites. Frequent SSBs are also found outside gene bodies, connecting TC-NER to divergent transcription from promoters. This work advances the use of trabectedin for precision oncology and for studying TC-NER and transcription.
Asunto(s)
Reparación por Escisión , Neoplasias , Humanos , Trabectedina , Transcripción Genética , Medicina de Precisión , Reparación del ADN , Daño del ADN , ADN/genética , Nucleótidos , Roturas del ADNRESUMEN
The posttranslational modifier ubiquitin regulates most cellular processes. Its ability to form polymeric chains of distinct linkages is key to its diverse functionality. Yet, we still lack the experimental tools to induce linkage-specific polyubiquitylation of a protein of interest in cells. Here, we introduce a set of engineered ubiquitin protein ligases and matching ubiquitin acceptor tags for the rapid, inducible linear (M1-), K48-, or K63-linked polyubiquitylation of proteins in yeast and mammalian cells. By applying the so-called "Ubiquiton" system to proteasomal targeting and the endocytic pathway, we validate this tool for soluble cytoplasmic and nuclear as well as chromatin-associated and integral membrane proteins and demonstrate how it can be used to control the localization and stability of its targets. We expect that the Ubiquiton system will serve as a versatile, broadly applicable research tool to explore the signaling functions of polyubiquitin chains in many biological contexts.
Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , Animales , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Poliubiquitina/genética , Poliubiquitina/metabolismo , Transducción de Señal , Complejo de la Endopetidasa Proteasomal/metabolismo , Ubiquitinación , Mamíferos/metabolismoRESUMEN
Reactive aldehydes are produced by normal cellular metabolism or after alcohol consumption, and they accumulate in human tissues if aldehyde clearance mechanisms are impaired. Their toxicity has been attributed to the damage they cause to genomic DNA and the subsequent inhibition of transcription and replication. However, whether interference with other cellular processes contributes to aldehyde toxicity has not been investigated. We demonstrate that formaldehyde induces RNA-protein crosslinks (RPCs) that stall the ribosome and inhibit translation in human cells. RPCs in the messenger RNA (mRNA) are recognized by the translating ribosomes, marked by atypical K6-linked ubiquitylation catalyzed by the RING-in-between-RING (RBR) E3 ligase RNF14, and subsequently resolved by the ubiquitin- and ATP-dependent unfoldase VCP. Our findings uncover an evolutionary conserved formaldehyde-induced stress response pathway that protects cells against RPC accumulation in the cytoplasm, and they suggest that RPCs contribute to the cellular and tissue toxicity of reactive aldehydes.
Asunto(s)
ARN , Ubiquitina-Proteína Ligasas , Humanos , ARN/metabolismo , Ubiquitinación , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Formaldehído/toxicidad , Aldehídos/toxicidad , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
The actin cytoskeleton is of fundamental importance for numerous cellular processes, including intracellular transport, cell plasticity, and cell migration. However, functions of filamentous actin (F-actin) in the nucleus remain understudied due to the comparatively low abundance of nuclear actin and the resulting experimental limitations to its visualization. Owing to recent technological advances such as super-resolution microscopy and the development of nuclear-specific actin probes, essential roles of the actin cytoskeleton in the context of genome maintenance are now emerging. In addition to the contributions of monomeric actin as a component of multiple important nuclear protein complexes, nuclear actin has been found to undergo polymerization in response to DNA damage and DNA replication stress. Consequently, nuclear F-actin plays important roles in the regulation of intra-nuclear mobility of repair and replication foci as well as the maintenance of nuclear shape, two important aspects of efficient stress tolerance. Beyond actin itself, there is accumulating evidence for the participation of multiple actin-binding proteins (ABPs) in the surveillance of genome integrity, including nucleation factors and motor proteins of the myosin family. Here we summarize recent findings highlighting the importance of actin cytoskeletal factors within the nucleus in key genome maintenance pathways.
Asunto(s)
Actinas , Cromatina , Humanos , Actinas/metabolismo , Cromatina/metabolismo , Núcleo Celular/metabolismo , Citoesqueleto/metabolismo , Inestabilidad GenómicaRESUMEN
In eukaryotes, the posttranslational modifier ubiquitin is used to regulate the amounts, interactions, or activities of proteins in diverse pathways and signaling networks. Its effects are mediated by monoubiquitin or polyubiquitin chains of varying geometries. We describe the design, validation, and application of a series of avidity-based probes against the ubiquitylated forms of the DNA replication clamp, proliferating cell nuclear antigen (PCNA), in budding yeast. Directed against total ubiquitylated PCNA or specifically K63-polyubiquitylated PCNA, the probes are tunable in their activities and can be used either as biosensors or as inhibitors of the PCNA-dependent DNA damage bypass pathway. Used in live cells, the probes revealed the timing of PCNA ubiquitylation during damage bypass and a particular susceptibility of the ribosomal DNA locus to the activation of the pathway. Our approach is applicable to a wide range of ubiquitin-conjugated proteins, thus representing a generalizable strategy for the design of biosensors for specific (poly)ubiquitylated forms of individual substrates.
Asunto(s)
Daño del ADN , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación , ADN Ribosómico , UbiquitinaRESUMEN
The actin cytoskeleton is of fundamental importance for cellular structure and plasticity. However, abundance and function of filamentous actin in the nucleus are still controversial. Here we show that the actin-based molecular motor myosin VI contributes to the stabilization of stalled or reversed replication forks. In response to DNA replication stress, myosin VI associates with stalled replication intermediates and cooperates with the AAA ATPase Werner helicase interacting protein 1 (WRNIP1) in protecting these structures from DNA2-mediated nucleolytic attack. Using functionalized affinity probes to manipulate myosin VI levels in a compartment-specific manner, we provide evidence for the direct involvement of myosin VI in the nucleus and against a contribution of the abundant cytoplasmic pool during the replication stress response.
Asunto(s)
Replicación del ADN , Proteínas de Unión al ADN , Proteínas de Unión al ADN/metabolismo , Actinas/metabolismo , Núcleo Celular/metabolismoRESUMEN
The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.
Asunto(s)
Proteínas de Ciclo Celular , Proteínas Cromosómicas no Histona , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Proteómica , Compartimentos de Replicación ViralRESUMEN
In healthy vessels, endothelial cells maintain a stable, differentiated, and growth-arrested phenotype for years. Upon injury, a rapid phenotypic switch facilitates proliferation to restore tissue perfusion. Here we report the identification of the endothelial cell-enriched long non-coding RNA (lncRNA) PCAT19, which contributes to the proliferative switch and acts as a safeguard for the endothelial genome. PCAT19 is enriched in confluent, quiescent endothelial cells and binds to the full replication protein A (RPA) complex in a DNA damage- and cell-cycle-related manner. Our results suggest that PCAT19 limits the phosphorylation of RPA2, primarily on the serine 33 (S33) residue, and thereby facilitates an appropriate DNA damage response while slowing cell cycle progression. Reduction in PCAT19 levels in response to either loss of cell contacts or knockdown promotes endothelial proliferation and angiogenesis. Collectively, PCAT19 acts as a dynamic guardian of the endothelial genome and facilitates rapid switching from quiescence to proliferation.
Asunto(s)
ARN Largo no Codificante , Fosforilación , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Células Endoteliales/metabolismo , ADN/metabolismo , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismoRESUMEN
A polyubiquitin chain can adopt a variety of shapes, depending on how the ubiquitin monomers are joined. However, the relevance of linkage for the signaling functions of polyubiquitin chains is often poorly understood because of our inability to control or manipulate this parameter in vivo. Here, we present a strategy for reprogramming polyubiquitin chain linkage by means of tailor-made, linkage- and substrate-selective ubiquitin ligases. Using the polyubiquitylation of the budding yeast replication factor PCNA in response to DNA damage as a model case, we show that altering the features of a polyubiquitin chain in vivo can change the fate of the modified substrate. We also provide evidence for redundancy between distinct but structurally similar linkages, and we demonstrate by proof-of-principle experiments that the method can be generalized to targets beyond PCNA. Our study illustrates a promising approach toward the in vivo analysis of polyubiquitin signaling.
Asunto(s)
Poliubiquitina , Ubiquitina-Proteína Ligasas , ADN , Daño del ADN , Poliubiquitina/genética , Antígeno Nuclear de Célula en Proliferación/genética , Ubiquitina/genética , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
Ubiquitin and its relatives are major players in many biological pathways, and a variety of experimental tools based on biological chemistry or protein engineering is available for their manipulation. One popular approach is the use of linear fusions between the modifier and a protein of interest. Such artificial constructs can facilitate the understanding of the role of ubiquitin in biological processes and can be exploited to control protein stability, interactions and degradation. Here we summarize the basic design considerations and discuss the advantages as well as limitations associated with their use. Finally, we will refer to several published case studies highlighting the principles of how they provide insight into pathways ranging from membrane protein trafficking to the control of epigenetic modifications.
Asunto(s)
Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina , Ubiquitina , Humanos , Estabilidad Proteica , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Ubiquitina/genética , Epigénesis GenéticaRESUMEN
The minichromosome maintenance (MCM) helicase physically interacts with the recombination proteins Rad51 and Rad52 from yeast to human cells. We show, in Saccharomyces cerevisiae, that these interactions occur within a nuclease-insoluble scaffold enriched in replication/repair factors. Rad51 accumulates in a MCM- and DNA-binding-independent manner and interacts with MCM helicases located outside of the replication origins and forks. MCM, Rad51, and Rad52 accumulate in this scaffold in G1 and are released during the S phase. In the presence of replication-blocking lesions, Cdc7 prevents their release from the scaffold, thus maintaining the interactions. We identify a rad51 mutant that is impaired in its ability to bind to MCM but not to the scaffold. This mutant is proficient in recombination but partially defective in single-stranded DNA (ssDNA) gap filling and replication fork progression through damaged DNA. Therefore, cells accumulate MCM/Rad51/Rad52 complexes at specific nuclear scaffolds in G1 to assist stressed forks through non-recombinogenic functions.
Asunto(s)
Replicación del ADN , ADN de Cadena Simple/metabolismo , Recombinación Homóloga/genética , Complejos Multiproteicos/metabolismo , Recombinasa Rad51/metabolismo , Saccharomyces cerevisiae/metabolismo , Ciclo Celular/genética , Núcleo Celular/metabolismo , Daño del ADN/genética , Reparación del ADN/genética , Metilmetanosulfonato , Modelos Biológicos , Unión Proteica , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , SolubilidadRESUMEN
Dealing with DNA lesions during genome replication is particularly challenging because damaged replication templates interfere with the progression of the replicative DNA polymerases and thereby endanger the stability of the replisome. A variety of mechanisms for the recovery of replication forks exist, but both bacteria and eukaryotic cells also have the option of continuing replication downstream of the lesion, leaving behind a daughter-strand gap in the newly synthesized DNA. In this review, we address the significance of these single-stranded DNA structures as sites of DNA damage sensing and processing at a distance from ongoing genome replication. We describe the factors controlling the emergence of daughter-strand gaps from stalled replication intermediates, the benefits and risks of their expansion and repair via translesion synthesis or recombination-mediated template switching, and the mechanisms by which they activate local as well as global replication stress signals. Our growing understanding of daughter-strand gaps not only identifies them as targets of fundamental genome maintenance mechanisms, but also suggests that proper control over their activities has important practical implications for treatment strategies and resistance mechanisms in cancer therapy.
Asunto(s)
Daño del ADN , Reparación del ADN , Replicación del ADN , Transducción de Señal , Animales , ADN/metabolismo , Eucariontes/genética , Eucariontes/metabolismo , HumanosRESUMEN
Telomeres are bound by dedicated proteins, which protect them from DNA damage and regulate telomere length homeostasis. In the nematode Caenorhabditis elegans, a comprehensive understanding of the proteins interacting with the telomere sequence is lacking. Here, we harnessed a quantitative proteomics approach to identify TEBP-1 and TEBP-2, two paralogs expressed in the germline and embryogenesis that associate to telomeres in vitro and in vivo. tebp-1 and tebp-2 mutants display strikingly distinct phenotypes: tebp-1 mutants have longer telomeres than wild-type animals, while tebp-2 mutants display shorter telomeres and a Mortal Germline. Notably, tebp-1;tebp-2 double mutant animals have synthetic sterility, with germlines showing signs of severe mitotic and meiotic arrest. Furthermore, we show that POT-1 forms a telomeric complex with TEBP-1 and TEBP-2, which bridges TEBP-1/-2 with POT-2/MRT-1. These results provide insights into the composition and organization of a telomeric protein complex in C. elegans.
Asunto(s)
Proteínas de Caenorhabditis elegans/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Complejos Multiproteicos/metabolismo , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Animales , Animales Modificados Genéticamente , Secuencia de Bases , Sitios de Unión/genética , Caenorhabditis elegans/genética , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/clasificación , Proteínas de Caenorhabditis elegans/genética , ADN/genética , Proteínas de Unión al ADN/genética , Células Germinativas/metabolismo , Microscopía Fluorescente/métodos , Complejos Multiproteicos/genética , Mutación , Filogenia , Unión Proteica , Isoformas de Proteínas/clasificación , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Telómero/genética , Proteínas de Unión a Telómeros/clasificación , Proteínas de Unión a Telómeros/genéticaRESUMEN
In contrast to our extensive knowledge on covalent small ubiquitin-like modifier (SUMO) target proteins, we are limited in our understanding of non-covalent SUMO-binding proteins. We identify interactors of different SUMO isoforms-monomeric SUMO1, monomeric SUMO2, or linear trimeric SUMO2 chains-using a mass spectrometry-based proteomics approach. We identify 379 proteins that bind to different SUMO isoforms, mainly in a preferential manner. Interestingly, XRCC4 is the only DNA repair protein in our screen with a preference for SUMO2 trimers over mono-SUMO2, as well as the only protein in our screen that belongs to the non-homologous end joining (NHEJ) DNA double-strand break repair pathway. A SUMO interaction motif (SIM) in XRCC4 regulates its recruitment to sites of DNA damage and phosphorylation of S320 by DNA-PKcs. Our data highlight the importance of non-covalent and covalent sumoylation for DNA double-strand break repair via the NHEJ pathway and provide a resource of SUMO isoform interactors.
Asunto(s)
Reparación del ADN por Unión de Extremidades/genética , Reparación del ADN/genética , Unión Proteica/genética , Proteína SUMO-1/metabolismo , HumanosRESUMEN
Mapping the genome-wide distribution of DNA lesions is key to understanding damage signalling and DNA repair in the context of genome and chromatin structure. Analytical tools based on high-throughput next-generation sequencing have revolutionized our progress with such investigations, and numerous methods are now available for various base lesions and modifications as well as for DNA double-strand breaks. Considering that single-strand breaks are by far the most common type of lesion and arise not only from exposure to exogenous DNA-damaging agents, but also as obligatory intermediates of DNA replication, recombination and repair, it is surprising that our insight into their genome-wide patterns, that is the 'SSBreakome', has remained rather obscure until recently, due to a lack of suitable mapping technology. Here we briefly review classical methods for analysing single-strand breaks and discuss and compare in detail a series of recently developed high-resolution approaches for the genome-wide mapping of these lesions, their advantages and limitations and how they have already provided valuable insight into the impact of this type of damage on the genome.
Asunto(s)
Mapeo Cromosómico/métodos , Roturas del ADN de Cadena Simple , Reparación del ADN , ADN/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ADN/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , Replicación del ADN , Genoma Humano/genética , HumanosRESUMEN
DNA damage tolerance relies on homologous recombination (HR) and translesion synthesis (TLS) mechanisms to fill in the ssDNA gaps generated during passing of the replication fork over DNA lesions in the template. Whereas TLS requires specialized polymerases able to incorporate a dNTP opposite the lesion and is error-prone, HR uses the sister chromatid and is mostly error-free. We report that the HR protein Rad52-but not Rad51 and Rad57-acts in concert with the TLS machinery (Rad6/Rad18-mediated PCNA ubiquitylation and polymerases Rev1/Pol ζ) to repair MMS and UV light-induced ssDNA gaps through a non-recombinogenic mechanism, as inferred from the different phenotypes displayed in the absence of Rad52 and Rad54 (essential for MMS- and UV-induced HR); accordingly, Rad52 is required for efficient DNA damage-induced mutagenesis. In addition, Rad52, Rad51, and Rad57, but not Rad54, facilitate Rad6/Rad18 binding to chromatin and subsequent DNA damage-induced PCNA ubiquitylation. Therefore, Rad52 facilitates the tolerance process not only by HR but also by TLS through Rad51/Rad57-dependent and -independent processes, providing a novel role for the recombination proteins in maintaining genome integrity.
Asunto(s)
Daño del ADN , Reparación del ADN , Replicación del ADN , Proteína Recombinante y Reparadora de ADN Rad52 , ADN de Cadena Simple/genética , ADN Polimerasa Dirigida por ADN/genéticaRESUMEN
GLOE-Seq is a next-generation sequencing method for the genome-wide mapping of 3'-OH termini, either resulting from single- or double-strand breaks or introduced by enzymatic conversion of lesions or modified nucleotides. This protocol provides instructions for isolation of genomic DNA from budding yeast or mammalian cells, preparation of libraries for sequencing, and data analysis by the associated computational pipeline, GLOE-Pipe. It is optimized for the Illumina next-generation sequencing platform and can be adapted to intact genomic DNA of any origin. For complete details on the use and execution of this protocol, please refer to Sriramachandran et al. (2020).
Asunto(s)
Mapeo Cromosómico/métodos , Roturas del ADN de Cadena Simple , Replicación del ADN/genética , Biblioteca de Genes , Análisis de Secuencia de ADN/métodos , Animales , Técnicas de Cultivo de Célula , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Saccharomycetales/genéticaRESUMEN
DNA single-strand breaks (SSBs) are among the most common lesions in the genome, arising spontaneously and as intermediates of many DNA transactions. Nevertheless, in contrast to double-strand breaks (DSBs), their distribution in the genome has hardly been addressed in a meaningful way. We now present a technique based on genome-wide ligation of 3'-OH ends followed by sequencing (GLOE-Seq) and an associated computational pipeline designed for capturing SSBs but versatile enough to be applied to any lesion convertible into a free 3'-OH terminus. We demonstrate its applicability to mapping of Okazaki fragments without prior size selection and provide insight into the relative contributions of DNA ligase 1 and ligase 3 to Okazaki fragment maturation in human cells. In addition, our analysis reveals biases and asymmetries in the distribution of spontaneous SSBs in yeast and human chromatin, distinct from the patterns of DSBs.