RESUMEN
Covalent DNA-protein cross-links (DPCs) impede replication fork progression and threaten genome integrity. Using Xenopus egg extracts, we previously showed that replication fork collision with DPCs causes their proteolysis, followed by translesion DNA synthesis. We show here that when DPC proteolysis is blocked, the replicative DNA helicase CMG (CDC45, MCM2-7, GINS), which travels on the leading strand template, bypasses an intact leading strand DPC. Single-molecule imaging reveals that GINS does not dissociate from CMG during bypass and that CMG slows dramatically after bypass, likely due to uncoupling from the stalled leading strand. The DNA helicase RTEL1 facilitates bypass, apparently by generating single-stranded DNA beyond the DPC. The absence of RTEL1 impairs DPC proteolysis, suggesting that CMG must bypass the DPC to enable proteolysis. Our results suggest a mechanism that prevents inadvertent CMG destruction by DPC proteases, and they reveal CMG's remarkable capacity to overcome obstacles on its translocation strand.
Asunto(s)
ADN Helicasas/metabolismo , ADN Helicasas/fisiología , Reparación del ADN/fisiología , Animales , Proteínas de Ciclo Celular/metabolismo , ADN/metabolismo , Replicación del ADN , ADN de Cadena Simple , Proteínas de Unión al ADN/fisiología , Femenino , Masculino , Proteolisis , Imagen Individual de Molécula/métodos , Xenopus laevis/metabolismoRESUMEN
Lesions on DNA uncouple DNA synthesis from the replisome, generating stretches of unreplicated single-stranded DNA (ssDNA) behind the replication fork. These ssDNA gaps need to be filled in to complete DNA duplication. Gap-filling synthesis involves either translesion DNA synthesis (TLS) or template switching (TS). Controlling these processes, ubiquitylated PCNA recruits many proteins that dictate pathway choice, but the enzymes regulating PCNA ubiquitylation in vertebrates remain poorly defined. Here we report that the E3 ubiquitin ligase RFWD3 promotes ubiquitylation of proteins on ssDNA. The absence of RFWD3 leads to a profound defect in recruitment of key repair and signaling factors to damaged chromatin. As a result, PCNA ubiquitylation is inhibited without RFWD3, and TLS across different DNA lesions is drastically impaired. We propose that RFWD3 is an essential coordinator of the response to ssDNA gaps, where it promotes ubiquitylation to drive recruitment of effectors of PCNA ubiquitylation and DNA damage bypass.
Asunto(s)
Cromatina/metabolismo , Roturas del ADN de Cadena Simple , Reparación del ADN , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Línea Celular Tumoral , Cromatina/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Femenino , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Especificidad por Sustrato , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Xenopus laevisRESUMEN
DNA-protein crosslinks (DPCs) are bulky lesions that interfere with DNA metabolism and therefore threaten genomic integrity. Recent studies implicate the metalloprotease SPRTN in S phase removal of DPCs, but how SPRTN is targeted to DPCs during DNA replication is unknown. Using Xenopus egg extracts that recapitulate replication-coupled DPC proteolysis, we show that DPCs can be degraded by SPRTN or the proteasome, which act as independent DPC proteases. Proteasome recruitment requires DPC polyubiquitylation, which is partially dependent on the ubiquitin ligase activity of TRAIP. In contrast, SPRTN-mediated DPC degradation does not require DPC polyubiquitylation but instead depends on nascent strand extension to within a few nucleotides of the lesion, implying that polymerase stalling at the DPC activates SPRTN on both leading and lagging strand templates. Our results demonstrate that SPRTN and proteasome activities are coupled to DNA replication by distinct mechanisms that promote replication across immovable protein barriers.
Asunto(s)
Reparación del ADN , Replicación del ADN , ADN/biosíntesis , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/metabolismo , Animales , ADN/química , ADN/genética , Femenino , Masculino , Conformación de Ácido Nucleico , Complejo de la Endopetidasa Proteasomal/genética , Dominios y Motivos de Interacción de Proteínas , Proteolisis , Células Sf9 , Relación Estructura-Actividad , Ubiquitinación , Proteínas de Xenopus/genética , Xenopus laevis/genéticaRESUMEN
DNA-protein crosslinks (DPCs) obstruct essential DNA transactions, posing a serious threat to genome stability and functionality. DPCs are proteolytically processed in a ubiquitin- and DNA replication-dependent manner by SPRTN and the proteasome but can also be resolved via targeted SUMOylation. However, the mechanistic basis of SUMO-mediated DPC resolution and its interplay with replication-coupled DPC repair remain unclear. Here, we show that the SUMO-targeted ubiquitin ligase RNF4 defines a major pathway for ubiquitylation and proteasomal clearance of SUMOylated DPCs in the absence of DNA replication. Importantly, SUMO modifications of DPCs neither stimulate nor inhibit their rapid DNA replication-coupled proteolysis. Instead, DPC SUMOylation provides a critical salvage mechanism to remove DPCs formed after DNA replication, as DPCs on duplex DNA do not activate interphase DNA damage checkpoints. Consequently, in the absence of the SUMO-RNF4 pathway cells are able to enter mitosis with a high load of unresolved DPCs, leading to defective chromosome segregation and cell death. Collectively, these findings provide mechanistic insights into SUMO-driven pathways underlying replication-independent DPC resolution and highlight their critical importance in maintaining chromosome stability and cellular fitness.
Asunto(s)
Reparación del ADN , Replicación del ADN , Proteínas Nucleares/metabolismo , Transducción de Señal , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Factores de Transcripción/metabolismo , Inestabilidad Genómica , Humanos , Unión Proteica , Procesamiento Proteico-Postraduccional , Sumoilación , Ubiquitina/metabolismo , UbiquitinaciónRESUMEN
Giardiasis is a diarrheal disease caused by the unicellular parasite Giardia intestinalis, for which metronidazole is the main treatment option. The parasite is dependent on exogenous deoxyribonucleosides for DNA replication and thus is also potentially vulnerable to deoxyribonucleoside analogs. Here, we characterized the G. intestinalis thymidine kinase, a divergent member of the thymidine kinase 1 family that consists of two weakly homologous parts within one polypeptide. We found that the recombinantly expressed enzyme is monomeric, with 100-fold higher catalytic efficiency for thymidine compared to its second-best substrate, deoxyuridine, and is furthermore subject to feedback inhibition by dTTP. This efficient substrate discrimination is in line with the lack of thymidylate synthase and dUTPase in the parasite, which makes deoxy-UMP a dead-end product that is potentially harmful if converted to deoxy-UTP. We also found that the antiretroviral drug azidothymidine (AZT) was an equally good substrate as thymidine and was active against WT as well as metronidazole-resistant G. intestinalis trophozoites. This drug inhibited DNA synthesis in the parasite and efficiently decreased cyst production in vitro, which suggests that it could reduce infectivity. AZT also showed a good effect in G. intestinalis-infected gerbils, reducing both the number of trophozoites in the small intestine and the number of viable cysts in the stool. Taken together, these results suggest that the absolute dependency of the parasite on thymidine kinase for its DNA synthesis can be exploited by AZT, which has promise as a future medication effective against metronidazole-refractory giardiasis.
Asunto(s)
Replicación del ADN , Giardia lamblia , Proteínas Protozoarias , Timidina Quinasa , Zidovudina , Animales , Descubrimiento de Drogas , Gerbillinae , Giardia lamblia/enzimología , Giardia lamblia/genética , Giardiasis/tratamiento farmacológico , Metronidazol/uso terapéutico , Proteínas Protozoarias/antagonistas & inhibidores , Proteínas Protozoarias/genética , Timidina , Timidina Quinasa/antagonistas & inhibidores , Timidina Quinasa/genética , Zidovudina/farmacologíaRESUMEN
Telomeric regions of the genome are inherently difficult-to-replicate due to their propensity to generate DNA secondary structures and form nucleoprotein complexes that can impede DNA replication fork progression. Precisely how cells respond to DNA replication stalling within a telomere remains poorly characterized, largely due to the methodological difficulties in analysing defined stalling events in molecular detail. Here, we utilized a site-specific DNA replication barrier mediated by the 'Tus/Ter' system to define the consequences of DNA replication perturbation within a single telomeric locus. Through molecular genetic analysis of this defined fork-stalling event, coupled with the use of a genome-wide genetic screen, we identified an important role for the SUMO-like domain protein, Esc2, in limiting genome rearrangements at a telomere. Moreover, we showed that these rearrangements are driven by the combined action of the Mph1 helicase and the homologous recombination machinery. Our findings demonstrate that chromosomal context influences cellular responses to a stalled replication fork and reveal protective factors that are required at telomeric loci to limit DNA replication stress-induced chromosomal instability.
Asunto(s)
ARN Helicasas DEAD-box/genética , Replicación del ADN/genética , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Telómero/genética , Proteínas de Ciclo Celular , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Recombinación Homóloga/genética , Conformación de Ácido Nucleico , Saccharomyces cerevisiae/genéticaRESUMEN
Proliferating cells acquire genome alterations during the act of DNA replication. This leads to mutation accumulation and somatic cell mosaicism in multicellular organisms, and is also implicated as an underlying cause of aging and tumorigenesis. The molecular mechanisms of DNA replication-associated genome rearrangements are poorly understood, largely due to methodological difficulties in analyzing specific replication forks in vivo. To provide an insight into this process, we analyzed the mutagenic consequences of replication fork stalling at a single, site-specific replication barrier (the Escherichia coli Tus/Ter complex) engineered into the yeast genome. We demonstrate that transient stalling at this barrier induces a distinct pattern of genome rearrangements in the newly replicated region behind the stalled fork, which primarily consist of localized losses and duplications of DNA sequences. These genetic alterations arise through the aberrant repair of a single-stranded DNA gap, in a process that is dependent on Exo1- and Shu1-dependent homologous recombination repair (HRR). Furthermore, aberrant processing of HRR intermediates, and elevated HRR-associated mutagenesis, is detectable in a yeast model of the human cancer predisposition disorder, Bloom's syndrome. Our data reveal a mechanism by which cellular responses to stalled replication forks can actively generate genomic alterations and genetic diversity in normal proliferating cells.
Asunto(s)
Replicación del ADN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ADN de Hongos/genética , ADN de Hongos/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Genes Reporteros , Ingeniería Genética , Humanos , Modelos Biológicos , Mutagénesis , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reparación del ADN por Recombinación , Origen de Réplica , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
DNA-protein crosslinks (DPCs) are toxic lesions that inhibit DNA related processes. Post-translational modifications (PTMs), including SUMOylation and ubiquitylation, play a central role in DPC resolution, but whether other PTMs are also involved remains elusive. Here, we identify a DPC repair pathway orchestrated by poly-ADP-ribosylation (PARylation). Using Xenopus egg extracts, we show that DPCs on single-stranded DNA gaps can be targeted for degradation via a replication-independent mechanism. During this process, DPCs are initially PARylated by PARP1 and subsequently ubiquitylated and degraded by the proteasome. Notably, PARP1-mediated DPC resolution is required for resolving topoisomerase 1-DNA cleavage complexes (TOP1ccs) induced by camptothecin. Using the Flp-nick system, we further reveal that in the absence of PARP1 activity, the TOP1cc-like lesion persists and induces replisome disassembly when encountered by a DNA replication fork. In summary, our work uncovers a PARP1-mediated DPC repair pathway that may underlie the synergistic toxicity between TOP1 poisons and PARP inhibitors.
Asunto(s)
Reparación del ADN , Replicación del ADN , ADN-Topoisomerasas de Tipo I , Poli(ADP-Ribosa) Polimerasa-1 , Poli ADP Ribosilación , Animales , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genética , ADN-Topoisomerasas de Tipo I/metabolismo , Xenopus laevis , Ubiquitinación , Humanos , ADN/metabolismo , Daño del ADN , Camptotecina/farmacología , Procesamiento Proteico-Postraduccional , ADN de Cadena Simple/metabolismo , Proteínas de Xenopus/metabolismoRESUMEN
Progression of DNA replication depends on the ability of the replisome complex to overcome nucleoprotein barriers. During eukaryotic replication, the CMG helicase translocates along the leading-strand template and unwinds the DNA double helix. While proteins bound to the leading-strand template efficiently block the helicase, the impact of lagging-strand protein obstacles on helicase translocation and replisome progression remains controversial. Here, we show that CMG and replisome progressions are impaired when proteins crosslinked to the lagging-strand template enhance the stability of duplex DNA. In contrast, proteins that exclusively interact with the lagging-strand template influence neither the translocation of isolated CMG nor replisome progression in Xenopus egg extracts. Our data imply that CMG completely excludes the lagging-strand template from the helicase central channel while unwinding DNA at the replication fork, which clarifies how two CMG helicases could freely cross one another during replication initiation and termination.
Asunto(s)
ADN Helicasas/química , Replicación del ADN , Animales , Línea Celular , ADN/química , ADN/metabolismo , ADN Helicasas/metabolismo , Cinética , Unión Proteica , Dominios Proteicos , Spodoptera , Xenopus laevisRESUMEN
Site-specific arrest of DNA replication is a useful tool for analyzing cellular responses to DNA replication perturbation. The E. coli Tus-Ter replication barrier can be reconstituted in eukaryotic cells as a system to engineer an unscheduled collision between a replication fork and an "alien" impediment to DNA replication. To further develop this system as a versatile tool, we describe a set of reagents and a detailed protocol that can be used to engineer Tus-Ter barriers into any locus in the budding yeast genome. Because the Tus-Ter complex is a bipartite system with intrinsic DNA replication-blocking activity, the reagents and protocols developed and validated in yeast could also be optimized to engineer site-specific replication fork barriers into other eukaryotic cell types.
Asunto(s)
Replicación del ADN , Escherichia coli/genética , Saccharomyces cerevisiae/genética , Ciclo Celular/genética , Escherichia coli/metabolismo , Citometría de Flujo , Genes Reporteros , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/metabolismo , Transformación GenéticaRESUMEN
The high-affinity binding of the Tus protein to specific 21-bp sequences, called Ter, causes site-specific, and polar, DNA replication fork arrest in E coli. The Tus-Ter complex serves to coordinate DNA replication with chromosome segregation in this organism. A number of recent and ongoing studies have demonstrated that Tus-Ter can be used as a heterologous tool to generate site-specific perturbation of DNA replication when reconstituted in eukaryotes. Here, we review these recent findings and explore the molecular mechanism by which Tus-Ter mediates replication fork (RF) arrest in the budding yeast, S. cerevisiae. We propose that Tus-Ter is a versatile, genetically tractable, and regulatable RF blocking system that can be utilized for disrupting DNA replication in a diverse range of host cells.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Proteínas de Escherichia coli/genética , Reparación del ADN por Recombinación , Saccharomyces cerevisiae/metabolismoRESUMEN
Replication fork (RF) pausing occurs at both 'programmed' sites and non-physiological barriers (for example, DNA adducts). Programmed RF pausing is required for site-specific DNA replication termination in Escherichia coli, and this process requires the binding of the polar terminator protein, Tus, to specific DNA sequences called Ter. Here, we demonstrate that Tus-Ter modules also induce polar RF pausing when engineered into the Saccharomyces cerevisiae genome. This heterologous RF barrier is distinct from a number of previously characterized, protein-mediated, RF pause sites in yeast, as it is neither Tof1-dependent nor counteracted by the Rrm3 helicase. Although the yeast replisome can overcome RF pausing at Tus-Ter modules, this event triggers site-specific homologous recombination that requires the RecQ helicase, Sgs1, for its timely resolution. We propose that Tus-Ter can be utilized as a versatile, site-specific, heterologous DNA replication-perturbing system, with a variety of potential applications.
Asunto(s)
Replicación del ADN/fisiología , Escherichia coli/genética , ADN Helicasas/metabolismo , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Repair of DNA double-strand breaks via homologous recombination can produce double Holliday junctions (dHJs) that require enzymatic separation. Topoisomerase IIIα (TopIIIα) together with RMI1 disentangles the final hemicatenane intermediate obtained once dHJs have converged. How binding of RMI1 to TopIIIα influences it to behave as a hemicatenane dissolvase, rather than as an enzyme that relaxes DNA topology, is unknown. Here, we present the crystal structure of human TopIIIα complexed to the first oligonucleotide-binding domain (OB fold) of RMI1. TopIII assumes a toroidal type 1A topoisomerase fold. RMI1 attaches to the edge of the gate in TopIIIα through which DNA passes. RMI1 projects a 23-residue loop into the TopIIIα gate, thereby influencing the dynamics of its opening and closing. Our results provide a mechanistic rationale for how RMI1 stabilizes TopIIIα-gate opening to enable dissolution and illustrate how binding partners modulate topoisomerase function.