RESUMEN
Fanconi anemia (FA) is a hereditary disorder caused by mutations in any 1 of 22 FA genes. The disease is characterized by hypersensitivity to interstrand crosslink (ICL) inducers such as mitomycin C (MMC). In addition to promoting ICL repair, FA proteins such as RAD51, BRCA2, or FANCD2 protect stalled replication forks from nucleolytic degradation during replication stress, which may have a profound impact on FA pathophysiology. Recent studies showed that expression of the putative DNA/RNA helicase SLFN11 in cancer cells correlates with cell death on chemotherapeutic treatment. However, the underlying mechanisms of SLFN11-mediated DNA damage sensitivity remain unclear. Because SLFN11 expression is high in hematopoietic stem cells, we hypothesized that SLFN11 depletion might ameliorate the phenotypes of FA cells. Here we report that SLFN11 knockdown in the FA patient-derived FANCD2-deficient PD20 cell line improved cell survival on treatment with ICL inducers. FANCD2-/-SLFN11-/- HAP1 cells also displayed phenotypic rescue, including reduced levels of MMC-induced chromosome breakage compared with FANCD2-/- cells. Importantly, we found that SLFN11 promotes extensive fork degradation in FANCD2-/- cells. The degradation process is mediated by the nucleases MRE11 or DNA2 and depends on the SLFN11 ATPase activity. This observation was accompanied by an increased RAD51 binding at stalled forks, consistent with the role of RAD51 antagonizing nuclease recruitment and subsequent fork degradation. Suppression of SLFN11 protects nascent DNA tracts even in wild-type cells. We conclude that SLFN11 destabilizes stalled replication forks, and this function may contribute to the attrition of hematopoietic stem cells in FA.
Asunto(s)
Replicación del ADN , Anemia de Fanconi/patología , Proteínas Nucleares/metabolismo , Puntos de Control del Ciclo Celular , Línea Celular , Rotura Cromosómica , Reactivos de Enlaces Cruzados/farmacología , ADN Helicasas/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Técnicas de Silenciamiento del Gen , Humanos , Proteína Homóloga de MRE11/metabolismo , Modelos Biológicos , Mutación/genética , Fenotipo , ARN Interferente Pequeño/metabolismo , Recombinasa Rad51/metabolismoRESUMEN
G-quadruplexes represent unique roadblocks to DNA replication, which tends to stall at these secondary structures. Although G-quadruplexes can be found throughout the genome, telomeres, due to their G-richness, are particularly predisposed to forming these structures and thus represent difficult-to-replicate regions. Here, we demonstrate that exonuclease 1 (EXO1) plays a key role in the resolution of, and replication through, telomeric G-quadruplexes. When replication forks encounter G-quadruplexes, EXO1 resects the nascent DNA proximal to these structures to facilitate fork progression and faithful replication. In the absence of EXO1, forks accumulate at stabilized G-quadruplexes and ultimately collapse. These collapsed forks are preferentially repaired via error-prone end joining as depletion of EXO1 diverts repair away from error-free homology-dependent repair. Such aberrant repair leads to increased genomic instability, which is exacerbated at chromosome termini in the form of dysfunction and telomere loss.
Asunto(s)
Enzimas Reparadoras del ADN/fisiología , Replicación del ADN , Exodesoxirribonucleasas/fisiología , G-Cuádruplex , Telómero/química , Aminoquinolinas/farmacología , Línea Celular , Reparación del ADN por Unión de Extremidades , Reparación del ADN , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , G-Cuádruplex/efectos de los fármacos , Técnicas de Inactivación de Genes , Células HeLa , Humanos , Neoplasias/metabolismo , Neoplasias/mortalidad , Ácidos Picolínicos/farmacología , PronósticoRESUMEN
The ATR-CHK1 axis stabilizes stalled replication forks and prevents their collapse into DNA double-strand breaks (DSBs). Here, we show that fork collapse in Atr-deleted cells is mediated through the combined effects the sumo targeted E3-ubiquitin ligase RNF4 and activation of the AURKA-PLK1 pathway. As indicated previously, Atr-deleted cells exhibited a decreased ability to restart DNA replication following fork stalling in comparison with control cells. However, suppression of RNF4, AURKA, or PLK1 returned the reinitiation of replication in Atr-deleted cells to near wild-type levels. In RNF4-depleted cells, this rescue directly correlated with the persistence of sumoylation of chromatin-bound factors. Notably, RNF4 repression substantially suppressed the accumulation of DSBs in ATR-deficient cells, and this decrease in breaks was enhanced by concomitant inhibition of PLK1. DSBs resulting from ATR inhibition were also observed to be dependent on the endonuclease scaffold protein SLX4, suggesting that RNF4 and PLK1 either help activate the SLX4 complex or make DNA replication fork structures accessible for subsequent SLX4-dependent cleavage. Thus, replication fork collapse following ATR inhibition is a multistep process that disrupts replisome function and permits cleavage of the replication fork.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/metabolismo , Células 3T3 , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de Ciclo Celular/genética , Cromatina/metabolismo , Roturas del ADN de Doble Cadena , Ratones , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/genética , Recombinasas/metabolismo , Sumoilación , Factores de Transcripción/genética , Ubiquitina-Proteína Ligasas , Quinasa Tipo Polo 1RESUMEN
Deoxyribonucleic acid (DNA) replication can be divided into three major steps: initiation, elongation and termination. Each time a human cell divides, these steps must be reiteratively carried out. Disruption of DNA replication can lead to genomic instability, with the accumulation of point mutations or larger chromosomal anomalies such as rearrangements. While cancer is the most common class of disease associated with genomic instability, several congenital diseases with dysfunctional DNA replication give rise to similar DNA alterations. In this review, we discuss all congenital diseases that arise from pathogenic variants in essential replication genes across the spectrum of aberrant replisome assembly, origin activation and DNA synthesis. For each of these conditions, we describe their clinical phenotypes as well as molecular studies aimed at determining the functional mechanisms of disease, including the assessment of genomic stability. By comparing and contrasting these diseases, we hope to illuminate how the disruption of DNA replication at distinct steps affects human health in a surprisingly cell-type-specific manner.
Asunto(s)
Replicación del ADN/genética , Inestabilidad Genómica/genética , Neoplasias/genética , RecQ Helicasas/genética , Craneosinostosis/genética , Craneosinostosis/inmunología , Replicación del ADN/inmunología , Inestabilidad Genómica/inmunología , Humanos , Mutación/genética , Neoplasias/epidemiología , Fenotipo , RecQ Helicasas/inmunologíaRESUMEN
Small ubiquitin-like modifier (SUMO)-targeted E3 ubiquitin ligases (STUbLs) are specialized enzymes that recognize SUMOylated proteins and attach ubiquitin to them. They therefore connect the cellular SUMOylation and ubiquitination circuits. STUbLs participate in diverse molecular processes that span cell cycle regulated events, including DNA repair, replication, mitosis, and transcription. They operate during unperturbed conditions and in response to challenges, such as genotoxic stress. These E3 ubiquitin ligases modify their target substrates by catalyzing ubiquitin chains that form different linkages, resulting in proteolytic or non-proteolytic outcomes. Often, STUbLs function in compartmentalized environments, such as the nuclear envelope or kinetochore, and actively aid in nuclear relocalization of damaged DNA and stalled replication forks to promote DNA repair or fork restart. Furthermore, STUbLs reside in the same vicinity as SUMO proteases and deubiquitinases (DUBs), providing spatiotemporal control of their targets. In this review, we focus on the molecular mechanisms by which STUbLs help to maintain genome stability across different species.
Asunto(s)
Inestabilidad Genómica/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Sumoilación/genética , Ubiquitina-Proteína Ligasas/genética , Ubiquitina/genética , Animales , Reparación del ADN/genética , HumanosRESUMEN
Overexpression of the flap endonuclease FEN1 has been observed in a variety of cancer types and is a marker for poor prognosis. To better understand the cellular consequences of FEN1 overexpression we utilized a model of its Saccharomyces cerevisiae homolog, RAD27. In this system, we discovered that flap endonuclease overexpression impedes replication fork progression and leads to an accumulation of cells in mid-S phase. This was accompanied by increased phosphorylation of the checkpoint kinase Rad53 and histone H2A-S129. RAD27 overexpressing cells were hypersensitive to treatment with DNA damaging agents, and defective in ubiquitinating the replication clamp proliferating cell nuclear antigen (PCNA) at lysine 164. These effects were reversed when the interaction between overexpressed Rad27 and PCNA was ablated, suggesting that the observed phenotypes were linked to problems in DNA replication. RAD27 overexpressing cells also exhibited an unexpected dependence on the SUMO ligases SIZ1 and MMS21 for viability. Importantly, we found that overexpression of FEN1 in human cells also led to phosphorylation of CHK1, CHK2, RPA32 and histone H2AX, all markers of genome instability. Our data indicate that flap endonuclease overexpression is a driver of genome instability in yeast and human cells that impairs DNA replication in a manner dependent on its interaction with PCNA.
Asunto(s)
Daño del ADN , Endonucleasas de ADN Solapado/metabolismo , Inestabilidad Genómica , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Neoplasias Pulmonares/enzimología , Carcinoma Pulmonar de Células Pequeñas/enzimología , Sumoilación , UbiquitinaciónRESUMEN
In this issue of Molecular Cell, Fachinetti et al. provide the first comprehensive map of replication termination sites (TERs) in Saccharomyces cerevisiae (Fachinetti et al., 2010). Strikingly, the majority of TERs are occupied by topoisomerase 2, which shields these regions against genomic instability.
RESUMEN
Ubiquitination of the replication clamp proliferating cell nuclear antigen (PCNA) at the conserved residue lysine (K)164 triggers postreplicative repair (PRR) to fill single-stranded gaps that result from stalled DNA polymerases. However, it has remained elusive as to whether cells engage PRR in response to replication defects that do not directly impair DNA synthesis. To experimentally address this question, we performed synthetic genetic array (SGA) analysis with a ubiquitination-deficient K164 to arginine (K164R) mutant of PCNA against a library of S. cerevisiae temperature-sensitive alleles. The SGA signature of the K164R allele showed a striking correlation with profiles of mutants deficient in various aspects of lagging strand replication, including rad27Δ and elg1Δ. Rad27 is the primary flap endonuclease that processes 5' flaps generated during lagging strand replication, whereas Elg1 has been implicated in unloading PCNA from chromatin. We observed chronic ubiquitination of PCNA at K164 in both rad27Δ and elg1Δ mutants. Notably, only rad27Δ cells exhibited a decline in cell viability upon elimination of PRR pathways, whereas elg1Δ mutants were not affected. We further provide evidence that K164 ubiquitination suppresses replication stress resulting from defective flap processing during Okazaki fragment maturation. Accordingly, ablation of PCNA ubiquitination increased S phase checkpoint activation, indicated by hyperphosphorylation of the Rad53 kinase. Furthermore, we demonstrate that alternative flap processing by overexpression of catalytically active exonuclease 1 eliminates PCNA ubiquitination. This suggests a model in which unprocessed flaps may directly participate in PRR signaling. Our findings demonstrate that PCNA ubiquitination at K164 in response to replication stress is not limited to DNA synthesis defects but extends to DNA processing during lagging strand replication.
Asunto(s)
Reparación del ADN , Replicación del ADN , ADN/metabolismo , Exodesoxirribonucleasas/genética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/genética , UbiquitinaciónRESUMEN
Minichromosome maintenance protein 10 (Mcm10) is required for DNA replication in all eukaryotes. Although the exact contribution of Mcm10 to genome replication remains heavily debated, early reports suggested that it promotes DNA unwinding and origin firing. These ideas have been solidified by recent studies that propose a role for Mcm10 in helicase activation. Whereas the molecular underpinnings of this activation step have yet to be revealed, structural data on Mcm10 provide further insight into a possible mechanism of action. The essential role in DNA replication initiation is not mutually exclusive with additional functions that Mcm10 may have as part of the elongation machinery. Here, we review the recent findings regarding the role of Mcm10 in DNA replication and discuss existing controversies.
Asunto(s)
Replicación del ADN , ADN de Hongos/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Animales , Ciclo Celular , Cromatina/genética , Cromatina/metabolismo , ADN/genética , Proteínas de Mantenimiento de Minicromosoma/genética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Estructura Terciaria de Proteína , Origen de Réplica , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , UbiquitinaciónRESUMEN
Minichromsome maintenance protein 10 (Mcm10) is an essential replication factor that is required for the activation of the Cdc45:Mcm2-7:GINS helicase. Mcm10's ability to bind both ds and ssDNA appears vital for this function. In addition, Mcm10 interacts with multiple players at the replication fork, including DNA polymerase-α and proliferating cell nuclear antigen with which it cooperates during DNA elongation. Mcm10 lacks enzymatic function, but instead provides the replication apparatus with an oligomeric scaffold that likely acts in the coordination of DNA unwinding and DNA synthesis. Not surprisingly, loss of Mcm10 engages checkpoint, DNA repair and SUMO-dependent rescue pathways that collectively counteract replication stress and chromosome breakage. Here, we review Mcm10's structure and function and explain how it contributes to the maintenance of genome integrity.
Asunto(s)
Proteínas de Mantenimiento de Minicromosoma/fisiología , Secuencia de Aminoácidos , Animales , Secuencia Conservada , Daño del ADN , Reparación del ADN , Replicación del ADN , Humanos , Proteínas de Mantenimiento de Minicromosoma/química , Neoplasias/genética , Unión Proteica , Multimerización de ProteínaRESUMEN
Accurate replication of the genome requires the evolutionarily conserved minichromosome maintenance protein, Mcm10. Although the details of the precise role of Mcm10 in DNA replication are still debated, it interacts with the Mcm2-7 core helicase, the lagging strand polymerase, DNA polymerase-α and the replication clamp, proliferating cell nuclear antigen. Loss of these interactions caused by the depletion of Mcm10 leads to chromosome breakage and cell cycle checkpoint activation. However, whether Mcm10 has an active role in DNA damage prevention is unknown. Here, we present data that establish a novel role of the N-terminus of Mcm10 in resisting DNA damage. We show that Mcm10 interacts with the Mec3 subunit of the 9-1-1 clamp in response to replication stress evoked by UV irradiation or nucleotide shortage. We map the interaction domain with Mec3 within the N-terminal region of Mcm10 and demonstrate that its truncation causes UV light sensitivity. This sensitivity is not further enhanced by a deletion of MEC3, arguing that MCM10 and MEC3 operate in the same pathway. Since Rad53 phosphorylation in response to UV light appears to be normal in N-terminally truncated mcm10 mutants, we propose that Mcm10 may have a role in replication fork restart or DNA repair.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Daño del ADN , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/química , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Mantenimiento de Minicromosoma/química , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Eliminación de SecuenciaRESUMEN
Eukaryotic genome stability is maintained by a complex and diverse set of molecular processes. One class of enzymes that promotes proper DNA repair, replication and cell cycle progression comprises small ubiquitin-like modifier (SUMO)-targeted E3 ligases, or STUbLs. Previously, we reported a role for the budding yeast STUbL synthetically lethal with sgs1 (Slx) 5/8 in preventing G2/M-phase arrest in a minichromosome maintenance protein 10 (Mcm10)-deficient model of replication stress. Here, we extend these studies to human cells, examining the requirement for the human STUbL RING finger protein 4 (RNF4) in MCM10 mutant cancer cells. We find that MCM10 and RNF4 independently promote origin firing but regulate DNA synthesis epistatically and, unlike in yeast, the negative genetic interaction between RNF4 and MCM10 causes cells to accumulate in G1-phase. When MCM10 is deficient, RNF4 prevents excessive DNA under-replication at hard-to-replicate regions that results in large DNA copy number alterations and severely reduced viability. Overall, our findings highlight that STUbLs participate in species-specific mechanisms to maintain genome stability, and that human RNF4 is required for origin activation in the presence of chronic replication stress.
Asunto(s)
Reparación del ADN , Inestabilidad Genómica , Humanos , Replicación del ADN , Mitosis , Saccharomyces cerevisiae/genética , Proteínas Nucleares/genética , Factores de TranscripciónRESUMEN
Natural killer (NK) cell deficiency (NKD) is a rare disease in which NK cell function is reduced, leaving affected individuals susceptible to repeated viral infections and cancer. Recently, a patient with NKD was identified carrying compound heterozygous variants of MCM10 (minichromosome maintenance protein 10), an essential gene required for DNA replication, that caused a significant decrease in the amount of functional MCM10. NKD in this patient presented as loss of functionally mature late-stage NK cells. To understand how MCM10 deficiency affects NK cell development, we generated MCM10 heterozygous (MCM10+/-) induced pluripotent stem cell (iPSC) lines. Analyses of these cell lines demonstrated that MCM10 was haploinsufficient, similar to results in other human cell lines. Reduced levels of MCM10 in mutant iPSCs was associated with impaired clonogenic survival and increased genomic instability, including micronuclei formation and telomere erosion. The severity of these phenotypes correlated with the extent of MCM10 depletion. Significantly, MCM10+/- iPSCs displayed defects in NK cell differentiation, exhibiting reduced yields of hematopoietic stem cells (HSCs). Although MCM10+/- HSCs were able to give rise to lymphoid progenitors, these did not generate mature NK cells. The lack of mature NK cells coincided with telomere erosion, suggesting that NKD caused by these MCM10 variants arose from the accumulation of genomic instability including degradation of chromosome ends.
Asunto(s)
Células Madre Pluripotentes Inducidas , Humanos , Diferenciación Celular , Genes Esenciales , Inestabilidad Genómica , Células Asesinas Naturales , Proteínas de Mantenimiento de MinicromosomaRESUMEN
Current approaches to define chemical-genetic interactions (CGIs) in human cell lines are resource-intensive. We designed a scalable chemical-genetic screening platform by generating a DNA damage response (DDR)-focused custom sgRNA library targeting 1011 genes with 3033 sgRNAs. We performed five proof-of-principle compound screens and found that the compounds' known modes-of-action (MoA) were enriched among the compounds' CGIs. These scalable screens recapitulated expected CGIs at a comparable signal-to-noise ratio (SNR) relative to genome-wide screens. Furthermore, time-resolved CGIs, captured by sequencing screens at various time points, suggested an unexpected, late interstrand-crosslinking (ICL) repair pathway response to camptothecin-induced DNA damage. Our approach can facilitate screening compounds at scale with 20-fold fewer resources than commonly used genome-wide libraries and produce biologically informative CGI profiles.
Asunto(s)
Sistemas CRISPR-Cas , ARN Guía de Sistemas CRISPR-Cas , Humanos , Genoma , Pruebas Genéticas , Daño del ADNRESUMEN
Innovative methods to retrieve proteins associated with actively replicating DNA have provided a glimpse into the molecular dynamics of replication fork stalling. We report that a combination of density-based replisome enrichment by isolating proteins on nascent DNA (iPOND2) and label-free quantitative mass spectrometry (iPOND2-DRIPPER) substantially increases both replication factor yields and the dynamic range of protein quantification. Replication protein abundance in retrieved nascent DNA is elevated up to 300-fold over post-replicative controls, and recruitment of replication stress factors upon fork stalling is observed at similar levels. The increased sensitivity of iPOND2-DRIPPER permits direct measurement of ubiquitination events without intervening retrieval of diglycine tryptic fragments of ubiquitin. Using this approach, we find that stalled replisomes stimulate the recruitment of a diverse cohort of DNA repair factors, including those associated with poly-K63-ubiquitination. Finally, we uncover the temporally controlled association of stalled replisomes with nuclear pore complex components and nuclear cytoskeleton networks.
Asunto(s)
Replicación del ADN , Ubiquitinación , Humanos , Reparación del ADN , ADN/metabolismoRESUMEN
DNA replication requires precise regulation achieved through post-translational modifications, including ubiquitination and SUMOylation. These modifications are linked by the SUMO-targeted E3 ubiquitin ligases (STUbLs). Ring finger protein 4 (RNF4), one of only two mammalian STUbLs, participates in double-strand break repair and resolving DNA-protein cross-links. However, its role in DNA replication has been poorly understood. Using CRISPR/Cas9 genetic screens, we discovered an unexpected dependency of RNF4 mutants on ubiquitin specific peptidase 7 (USP7) for survival in TP53-null retinal pigment epithelial cells. TP53-/-/RNF4-/-/USP7-/- triple knockout (TKO) cells displayed defects in DNA replication that cause genomic instability. These defects were exacerbated by the proteasome inhibitor bortezomib, which limited the nuclear ubiquitin pool. A shortage of free ubiquitin suppressed the ataxia telangiectasia and Rad3-related (ATR)-mediated checkpoint response, leading to increased cell death. In conclusion, RNF4 and USP7 work cooperatively to sustain a functional level of nuclear ubiquitin to maintain the integrity of the genome.
Asunto(s)
Replicación del ADN , Ubiquitina , Animales , Peptidasa Específica de Ubiquitina 7/genética , Procesamiento Proteico-Postraduccional , Ubiquitinación , MamíferosRESUMEN
Activation of the Fanconi anemia (FA) pathway after treatment with mitomycin C (MMC) is essential for preventing chromosome translocations termed "radials." When replication forks stall at MMC-induced interstrand crosslinks (ICLs), the FA pathway is activated to orchestrate ICL unhooking and repair of the DNA break intermediates. However, in FA-deficient cells, how ICL-associated breaks are resolved in a manner that leads to radials is unclear. Here, we demonstrate that MMC-induced radials are dependent on DNA polymerase theta (POLθ)-mediated alternative end joining (A-EJ). Specifically, we show that radials observed in FANCD2-/- cells are dependent on POLθ and DNA ligase III and occur independently of classical non-homologous end joining. Furthermore, treatment of FANCD2-/- cells with POLθ inhibitors abolishes radials and leads to the accumulation of breaks co-localizing with common fragile sites. Uniformly, these observations implicate A-EJ in radial formation and provide mechanistic insights into the treatment of FA pathway-deficient cancers with POLθ inhibitors.
Asunto(s)
Anemia de Fanconi , Humanos , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Cromosomas/metabolismo , Reparación del ADN por Unión de Extremidades , Mitomicina , Reparación del ADNRESUMEN
Ubiquitination of proliferating cell nuclear antigen (PCNA) at lysine 164 (K164) activates DNA damage tolerance pathways. Currently, we lack a comprehensive understanding of how PCNA K164 ubiquitination promotes genome stability. To evaluate this, we generated stable cell lines expressing PCNAK164R from the endogenous PCNA locus. Our data reveal that the inability to ubiquitinate K164 causes perturbations in global DNA replication. Persistent replication stress generates under-replicated regions and is exacerbated by the DNA polymerase inhibitor aphidicolin. We show that these phenotypes are due, in part, to impaired Fanconi anemia group D2 protein (FANCD2)-dependent mitotic DNA synthesis (MiDAS) in PCNAK164R cells. FANCD2 mono-ubiquitination is significantly reduced in PCNAK164R mutants, leading to reduced chromatin association and foci formation, both prerequisites for FANCD2-dependent MiDAS. Furthermore, K164 ubiquitination coordinates direct PCNA/FANCD2 colocalization in mitotic nuclei. Here, we show that PCNA K164 ubiquitination maintains human genome stability by promoting FANCD2-dependent MiDAS to prevent the accumulation of under-replicated DNA.
Asunto(s)
Reparación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Humanos , ADN/metabolismo , Daño del ADN , Replicación del ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Inestabilidad Genómica , Antígeno Nuclear de Célula en Proliferación/metabolismo , UbiquitinaciónRESUMEN
Pooled lentiviral CRISPR-Cas9 screens are utilized for assessing the differential sensitivity or resistance of many single-gene knockouts to a compound. Here, we present a scalable approach for high-throughput compound screening by utilizing a small custom library. We describe steps to perform a proof-of-principle chemical screen in non-transformed hTERT RPE-1 TP53-/- cells with higher coverage and greater timepoint resolution compared to genome-wide screens. This approach can be adapted for use in various cell lines, compounds, and other focused sgRNA libraries.
Asunto(s)
Sistemas CRISPR-Cas , Pruebas Genéticas , Humanos , Biblioteca de Genes , Genoma , Técnicas de Inactivación de GenesRESUMEN
Embryonic development is dictated by tight regulation of DNA replication, cell division and differentiation. Mutations in DNA repair and replication genes disrupt this equilibrium, giving rise to neurodevelopmental disease characterized by microcephaly, short stature and chromosomal breakage. Here, we identify biallelic variants in two components of the RAD18-SLF1/2-SMC5/6 genome stability pathway, SLF2 and SMC5, in 11 patients with microcephaly, short stature, cardiac abnormalities and anemia. Patient-derived cells exhibit a unique chromosomal instability phenotype consisting of segmented and dicentric chromosomes with mosaic variegated hyperploidy. To signify the importance of these segmented chromosomes, we have named this disorder Atelís (meaning - incomplete) Syndrome. Analysis of Atelís Syndrome cells reveals elevated levels of replication stress, partly due to a reduced ability to replicate through G-quadruplex DNA structures, and also loss of sister chromatid cohesion. Together, these data strengthen the functional link between SLF2 and the SMC5/6 complex, highlighting a distinct role for this pathway in maintaining genome stability.