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1.
Mol Cell ; 75(3): 605-619.e6, 2019 08 08.
Artículo en Inglés | MEDLINE | ID: mdl-31255466

RESUMEN

Accurate DNA replication is essential to preserve genomic integrity and prevent chromosomal instability-associated diseases including cancer. Key to this process is the cells' ability to stabilize and restart stalled replication forks. Here, we show that the EXD2 nuclease is essential to this process. EXD2 recruitment to stressed forks suppresses their degradation by restraining excessive fork regression. Accordingly, EXD2 deficiency leads to fork collapse, hypersensitivity to replication inhibitors, and genomic instability. Impeding fork regression by inactivation of SMARCAL1 or removal of RECQ1's inhibition in EXD2-/- cells restores efficient fork restart and genome stability. Moreover, purified EXD2 efficiently processes substrates mimicking regressed forks. Thus, this work identifies a mechanism underpinned by EXD2's nuclease activity, by which cells balance fork regression with fork restoration to maintain genome stability. Interestingly, from a clinical perspective, we discover that EXD2's depletion is synthetic lethal with mutations in BRCA1/2, implying a non-redundant role in replication fork protection.


Asunto(s)
ADN Helicasas/genética , Replicación del ADN/genética , Exodesoxirribonucleasas/genética , RecQ Helicasas/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Inestabilidad Genómica/genética , Células HeLa , Humanos , Neoplasias/genética , Mutaciones Letales Sintéticas/genética
2.
Nucleic Acids Res ; 51(12): 6337-6354, 2023 07 07.
Artículo en Inglés | MEDLINE | ID: mdl-37224534

RESUMEN

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here, we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e. Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that ß-actin interacts with RPA directly in vitro, and in vivo a hyper-depolymerizing ß-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing ß-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.


Asunto(s)
Actinas , Replicación del ADN , Actinas/genética , Proteína de Replicación A/genética , Proteína de Replicación A/metabolismo , ADN de Cadena Simple/genética , Chaperonas Moleculares/genética
3.
Nat Commun ; 14(1): 2428, 2023 04 27.
Artículo en Inglés | MEDLINE | ID: mdl-37105990

RESUMEN

Telomerase-independent cancer proliferation via the alternative lengthening of telomeres (ALT) relies upon two distinct, largely uncharacterized, break-induced-replication (BIR) processes. How cancer cells initiate and regulate these terminal repair mechanisms is unknown. Here, we establish that the EXD2 nuclease is recruited to ALT telomeres to direct their maintenance. We demonstrate that EXD2 loss leads to telomere shortening, elevated telomeric sister chromatid exchanges, C-circle formation as well as BIR-mediated telomeric replication. We discover that EXD2 fork-processing activity triggers a switch between RAD52-dependent and -independent ALT-associated BIR. The latter is suppressed by EXD2 but depends specifically on the fork remodeler SMARCAL1 and the MUS81 nuclease. Thus, our findings suggest that processing of stalled replication forks orchestrates elongation pathway choice at ALT telomeres. Finally, we show that co-depletion of EXD2 with BLM, DNA2 or POLD3 confers synthetic lethality in ALT cells, identifying EXD2 as a potential druggable target for ALT-reliant cancers.


Asunto(s)
Neoplasias , Telomerasa , Humanos , Homeostasis del Telómero , Replicación del ADN , Acortamiento del Telómero , Reparación del ADN , Telomerasa/genética , Telómero/genética , Telómero/metabolismo , Neoplasias/genética , ADN Helicasas/genética , ADN Helicasas/metabolismo
4.
bioRxiv ; 2023 Jan 12.
Artículo en Inglés | MEDLINE | ID: mdl-36711944

RESUMEN

Accurate genome replication is essential for all life and a key mechanism of disease prevention, underpinned by the ability of cells to respond to replicative stress (RS) and protect replication forks. These responses rely on the formation of Replication Protein A (RPA)-single stranded (ss) DNA complexes, yet this process remains largely uncharacterized. Here we establish that actin nucleation-promoting factors (NPFs) associate with replication forks, promote efficient DNA replication and facilitate association of RPA with ssDNA at sites of RS. Accordingly, their loss leads to deprotection of ssDNA at perturbed forks, impaired ATR activation, global replication defects and fork collapse. Supplying an excess of RPA restores RPA foci formation and fork protection, suggesting a chaperoning role for actin nucleators (ANs) (i.e., Arp2/3, DIAPH1) and NPFs (i.e, WASp, N-WASp) in regulating RPA availability upon RS. We also discover that ß-actin interacts with RPA directly in vitro , and in vivo a hyper-depolymerizing ß-actin mutant displays a heightened association with RPA and the same dysfunctional replication phenotypes as loss of ANs/NPFs, which contrasts with the phenotype of a hyper-polymerizing ß-actin mutant. Thus, we identify components of actin polymerization pathways that are essential for preventing ectopic nucleolytic degradation of perturbed forks by modulating RPA activity.

5.
Nat Genet ; 55(8): 1311-1323, 2023 08.
Artículo en Inglés | MEDLINE | ID: mdl-37524790

RESUMEN

SF3B1 hotspot mutations are associated with a poor prognosis in several tumor types and lead to global disruption of canonical splicing. Through synthetic lethal drug screens, we identify that SF3B1 mutant (SF3B1MUT) cells are selectively sensitive to poly (ADP-ribose) polymerase inhibitors (PARPi), independent of hotspot mutation and tumor site. SF3B1MUT cells display a defective response to PARPi-induced replication stress that occurs via downregulation of the cyclin-dependent kinase 2 interacting protein (CINP), leading to increased replication fork origin firing and loss of phosphorylated CHK1 (pCHK1; S317) induction. This results in subsequent failure to resolve DNA replication intermediates and G2/M cell cycle arrest. These defects are rescued through CINP overexpression, or further targeted by a combination of ataxia-telangiectasia mutated and PARP inhibition. In vivo, PARPi produce profound antitumor effects in multiple SF3B1MUT cancer models and eliminate distant metastases. These data provide the rationale for testing the clinical efficacy of PARPi in a biomarker-driven, homologous recombination proficient, patient population.


Asunto(s)
Neoplasias , Inhibidores de Poli(ADP-Ribosa) Polimerasas , Humanos , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Inhibidores de Poli(ADP-Ribosa) Polimerasas/uso terapéutico , Mutación , Factores de Transcripción/genética , Neoplasias/tratamiento farmacológico , Neoplasias/genética , Proteína BRCA1/genética , Línea Celular Tumoral , Factores de Empalme de ARN/genética , Fosfoproteínas/genética
6.
Cancer Res ; 82(21): 3962-3973, 2022 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-36273494

RESUMEN

Gastric cancer represents the third leading cause of global cancer mortality and an area of unmet clinical need. Drugs that target the DNA damage response, including ATR inhibitors (ATRi), have been proposed as novel targeted agents in gastric cancer. Here, we sought to evaluate the efficacy of ATRi in preclinical models of gastric cancer and to understand how ATRi resistance might emerge as a means to identify predictors of ATRi response. A positive selection genome-wide CRISPR-Cas9 screen identified candidate regulators of ATRi resistance in gastric cancer. Loss-of-function mutations in either SMG8 or SMG9 caused ATRi resistance by an SMG1-mediated mechanism. Although ATRi still impaired ATR/CHK1 signaling in SMG8/9-defective cells, other characteristic responses to ATRi exposure were not seen, such as changes in ATM/CHK2, γH2AX, phospho-RPA, or 53BP1 status or changes in the proportions of cells in S- or G2-M-phases of the cell cycle. Transcription/replication conflicts (TRC) elicited by ATRi exposure are a likely cause of ATRi sensitivity, and SMG8/9-defective cells exhibited a reduced level of ATRi-induced TRCs, which could contribute to ATRi resistance. These observations suggest ATRi elicits antitumor efficacy in gastric cancer but that drug resistance could emerge via alterations in the SMG8/9/1 pathway. SIGNIFICANCE: These findings reveal how cancer cells acquire resistance to ATRi and identify pathways that could be targeted to enhance the overall effectiveness of these inhibitors.


Asunto(s)
Antineoplásicos , Neoplasias Gástricas , Humanos , Antineoplásicos/farmacología , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Inhibidores de Proteínas Quinasas , Proteínas Serina-Treonina Quinasas , Péptidos y Proteínas de Señalización Intracelular/metabolismo
7.
Biochem Soc Trans ; 37(Pt 4): 926-30, 2009 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-19614620

RESUMEN

The Cdc (cell division cycle) 45 protein has a central role in the regulation of the initiation and elongation stages of eukaryotic chromosomal DNA replication. In addition, it is the main target for a Chk1 (checkpoint kinase 1)-dependent Cdc25/CDK2 (cyclin-dependent kinase 2)-independent DNA damage checkpoint signal transduction pathway following low doses of BPDE (benzo[a]pyrene dihydrodiol epoxide) treatment, which causes DNA damage similar to UV-induced adducts. Cdc45 interacts physically and functionally with the putative eukaryotic replicative DNA helicase, the MCM (mini-chromosome maintenance) complex, and forms a helicase active 'supercomplex', the CMG [Cdc45-MCM2-7-GINS (go-ichi-ni-san)] complex. These known protein-protein interactions, as well as unknown interactions and post-translational modifications, may be important for the regulation of Cdc45 and the initiation of DNA replication following DNA damage. Future studies will help to elucidate the molecular basis of this newly identified S-phase checkpoint pathway which has Cdc45 as a target.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , Ciclo Celular/fisiología , Daño del ADN/fisiología , Regulación de la Expresión Génica , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Daño del ADN/genética , Replicación del ADN/genética , Replicación del ADN/fisiología , Inestabilidad Genómica/genética , Inestabilidad Genómica/fisiología , Humanos , Modelos Biológicos
8.
Nat Commun ; 8: 15983, 2017 07 17.
Artículo en Inglés | MEDLINE | ID: mdl-28714477

RESUMEN

Failure to restart replication forks stalled at genomic regions that are difficult to replicate or contain endogenous DNA lesions is a hallmark of BRCA2 deficiency. The nucleolytic activity of MUS81 endonuclease is required for replication fork restart under replication stress elicited by exogenous treatments. Here we investigate whether MUS81 could similarly facilitate DNA replication in the context of BRCA2 abrogation. Our results demonstrate that replication fork progression in BRCA2-deficient cells requires MUS81. Failure to complete genome replication and defective checkpoint surveillance enables BRCA2-deficient cells to progress through mitosis with under-replicated DNA, which elicits severe chromosome interlinking in anaphase. MUS81 nucleolytic activity is required to activate compensatory DNA synthesis during mitosis and to resolve mitotic interlinks, thus facilitating chromosome segregation. We propose that MUS81 provides a mechanism of replication stress tolerance, which sustains survival of BRCA2-deficient cells and can be exploited therapeutically through development of specific inhibitors of MUS81 nuclease activity.


Asunto(s)
Proteína BRCA2/genética , Segregación Cromosómica/genética , Daño del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , ADN/metabolismo , Endonucleasas/genética , Anafase , Línea Celular Tumoral , Células HeLa , Humanos , Mitosis
10.
Nat Cell Biol ; 18(3): 271-280, 2016 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-26807646

RESUMEN

Repair of DNA double-strand breaks (DSBs) by homologous recombination (HR) is critical for survival and genome stability of individual cells and organisms, but also contributes to the genetic diversity of species. A vital step in HR is MRN-CtIP-dependent end resection, which generates the 3' single-stranded DNA overhangs required for the subsequent strand exchange reaction. Here, we identify EXD2 (also known as EXDL2) as an exonuclease essential for DSB resection and efficient HR. EXD2 is recruited to chromatin in a damage-dependent manner and confers resistance to DSB-inducing agents. EXD2 functionally interacts with the MRN complex to accelerate resection through its 3'-5' exonuclease activity, which efficiently processes double-stranded DNA substrates containing nicks. Finally, we establish that EXD2 stimulates both short- and long-range DSB resection, and thus, together with MRE11, is required for efficient HR. This establishes a key role for EXD2 in controlling the initial steps of chromosomal break repair.


Asunto(s)
Proteínas Portadoras/metabolismo , Roturas del ADN de Doble Cadena , Reparación del ADN/genética , ADN/metabolismo , Cromatina/metabolismo , Recombinación Homóloga/fisiología , Humanos , Proteínas Nucleares/metabolismo , Fase S/genética
11.
Cell Cycle ; 14(19): 3040-4, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-26266709

RESUMEN

Faithful chromosome segregation is critical in preventing genome loss or damage during cell division. Failure to properly disentangle catenated sister chromatids can lead to the formation of bulky or ultrafine anaphase bridges, and ultimately genome instability. In this review we present an overview of the current state of knowledge of how sister chromatid decatenation is carried out, with particular focus on the role of TOP2A and TOPBP1 in this process.


Asunto(s)
Cromátides/metabolismo , Anafase/genética , Anafase/fisiología , Animales , Antígenos de Neoplasias/genética , Antígenos de Neoplasias/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Ciclo Celular/genética , Ciclo Celular/fisiología , Segregación Cromosómica/genética , Segregación Cromosómica/fisiología , ADN-Topoisomerasas de Tipo II/genética , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Humanos , Mitosis/genética , Mitosis/fisiología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Unión a Poli-ADP-Ribosa
12.
Nat Commun ; 6: 6572, 2015 Mar 12.
Artículo en Inglés | MEDLINE | ID: mdl-25762097

RESUMEN

During mitosis, sister chromatids must be faithfully segregated to ensure that daughter cells receive one copy of each chromosome. However, following replication they often remain entangled. Topoisomerase IIα (TOP2A) has been proposed to resolve such entanglements, but the mechanisms governing TOP2A recruitment to these structures remain poorly understood. Here, we identify TOPBP1 as a novel interactor of TOP2A, and reveal that it is required for TOP2A recruitment to ultra-fine anaphase bridges (UFBs) in mitosis. The C-terminal region of TOPBP1 interacts with TOP2A, and TOPBP1 recruitment to UFBs requires its BRCT domain 5. Depletion of TOPBP1 leads to accumulation of UFBs, the majority of which arise from centromeric loci. Accordingly, expression of a TOPBP1 mutant that is defective in TOP2A binding phenocopies TOP2A depletion. These findings provide new mechanistic insights into how TOP2A promotes resolution of UFBs during mitosis, and highlights a pivotal role for TOPBP1 in this process.


Asunto(s)
Anafase , Antígenos de Neoplasias/metabolismo , Proteínas Portadoras/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/metabolismo , Regulación de la Expresión Génica , Proteínas Nucleares/metabolismo , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Centrómero/ultraestructura , Cromátides/química , Cromosomas/ultraestructura , ADN/química , Regulación Neoplásica de la Expresión Génica , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Células HeLa , Humanos , Microscopía Fluorescente , Mitosis , Mutación , Proteínas de Unión a Poli-ADP-Ribosa , Unión Proteica , Estructura Terciaria de Proteína
13.
FEBS J ; 280(19): 4888-902, 2013 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-23910567

RESUMEN

The replication factor Cdc45 has essential functions in the initiation and elongation steps of eukaryotic DNA replication and plays an important role in the intra-S-phase checkpoint. Its interactions with other replication proteins during the cell cycle and after intra-S-phase checkpoint activation are only partially characterized. In the present study, we show that the C terminal part of Cdc45 may mediate its interactions with Claspin. The interactions of human Cdc45 with the three replication factors Claspin, replication protein A and DNA polymerase δ are maximal during the S phase. Following UVC-induced DNA damage, Cdc45-Claspin complex formation is reduced, whereas the binding of Cdc45 to replication protein A is not affected. We also show that treatment of cells with UCN-01 and phosphatidylinositol 3-kinase-like kinase inhibitors does not rescue the UV-induced destabilization of Cdc45-Claspin interactions, suggesting that the loss of the interaction between Cdc45 and Claspin occurs upstream of ataxia telangiectasia and Rad 3-related activation in the intra-S-phase checkpoint.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Línea Celular , Daño del ADN/genética , Daño del ADN/efectos de la radiación , Replicación del ADN/genética , Replicación del ADN/efectos de la radiación , Humanos
14.
PLoS One ; 7(4): e35537, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22536402

RESUMEN

Eukaryotic DNA replication is a dynamic process requiring the co-operation of specific replication proteins. We measured the mobility of eGFP-Cdc45 by Fluorescence Correlation Spectroscopy (FCS) in vivo in asynchronous cells and in cells synchronized at the G1/S transition and during S phase. Our data show that eGFP-Cdc45 mobility is faster in G1/S transition compared to S phase suggesting that Cdc45 is part of larger protein complex formed in S phase. Furthermore, the size of complexes containing Cdc45 was estimated in asynchronous, G1/S and S phase-synchronized cells using gel filtration chromatography; these findings complemented the in vivo FCS data. Analysis of the mobility of eGFP-Cdc45 and the size of complexes containing Cdc45 and eGFP-Cdc45 after UVC-mediated DNA damage revealed no significant changes in diffusion rates and complex sizes using FCS and gel filtration chromatography analyses. This suggests that after UV-damage, Cdc45 is still present in a large multi-protein complex and that its mobility within living cells is consistently similar following UVC-mediated DNA damage.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular , Proteínas Recombinantes de Fusión/metabolismo , Proteínas de Ciclo Celular/aislamiento & purificación , Cromatina/metabolismo , Cromatina/efectos de la radiación , Cromatografía en Gel , Daño del ADN , Difusión , Proteínas Fluorescentes Verdes/metabolismo , Células HeLa , Humanos , Complejos Multiproteicos/aislamiento & purificación , Complejos Multiproteicos/metabolismo , Unión Proteica , Transporte de Proteínas , Espectrometría de Fluorescencia , Rayos Ultravioleta
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