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1.
Mol Cell ; 77(3): 528-541.e8, 2020 02 06.
Artículo en Inglés | MEDLINE | ID: mdl-31759821

RESUMEN

Formation of co-transcriptional R-loops underlies replication fork stalling upon head-on transcription-replication encounters. Here, we demonstrate that RAD51-dependent replication fork reversal induced by R-loops is followed by the restart of semiconservative DNA replication mediated by RECQ1 and RECQ5 helicases, MUS81/EME1 endonuclease, RAD52 strand-annealing factor, the DNA ligase IV (LIG4)/XRCC4 complex, and the non-catalytic subunit of DNA polymerase δ, POLD3. RECQ5 disrupts RAD51 filaments assembled on stalled forks after RECQ1-mediated reverse branch migration, preventing a new round of fork reversal and facilitating fork cleavage by MUS81/EME1. MUS81-dependent DNA breaks accumulate in cells lacking RAD52 or LIG4 upon induction of R-loop formation, suggesting that RAD52 acts in concert with LIG4/XRCC4 to catalyze fork religation, thereby mediating replication restart. The resumption of DNA synthesis after R-loop-associated fork stalling also requires active transcription, the restoration of which depends on MUS81, RAD52, LIG4, and the transcription elongation factor ELL. These findings provide mechanistic insights into transcription-replication conflict resolution.


Asunto(s)
Replicación del ADN/fisiología , Estructuras R-Loop/genética , Recombinasa Rad51/metabolismo , Línea Celular Tumoral , ADN Ligasas/metabolismo , ADN Polimerasa III/metabolismo , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Células HeLa , Humanos , Estructuras R-Loop/fisiología , Recombinasa Rad51/genética , Recombinasa Rad51/fisiología , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , RecQ Helicasas/metabolismo , RecQ Helicasas/fisiología , Transcripción Genética/genética
2.
Cell Rep ; 24(10): 2629-2642.e5, 2018 09 04.
Artículo en Inglés | MEDLINE | ID: mdl-30184498

RESUMEN

Interstrand cross-links (ICLs) are toxic DNA lesions interfering with DNA metabolism that are induced by widely used anticancer drugs. They have long been considered absolute roadblocks for replication forks, implicating complex DNA repair processes at stalled or converging replication forks. Recent evidence challenged this view, proposing that single forks traverse ICLs by yet elusive mechanisms. Combining ICL immunolabeling and single-molecule approaches in human cells, we now show that ICL induction leads to global replication fork slowing, involving forks not directly challenged by ICLs. Active fork slowing is linked to rapid recruitment of RAD51 to replicating chromatin and to RAD51/ZRANB3-mediated fork reversal. This global modulation of fork speed and architecture requires ATR activation, promotes single-fork ICL traverse-here, directly visualized by electron microscopy-and prevents chromosomal breakage by untimely ICL processing. We propose that global fork slowing by remodeling provides more time for template repair and promotes bypass of residual lesions, limiting fork-associated processing.


Asunto(s)
Rotura Cromosómica , Daño del ADN/genética , Replicación del ADN/genética , ADN/metabolismo , Western Blotting , Línea Celular Tumoral , Ensayo Cometa , ADN/genética , ADN/ultraestructura , Daño del ADN/fisiología , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Microscopía Electrónica , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo
3.
EMBO Rep ; 19(7)2018 07.
Artículo en Inglés | MEDLINE | ID: mdl-29739811

RESUMEN

In both yeast and mammals, the topoisomerase poison camptothecin (CPT) induces fork reversal, which has been proposed to stabilize replication forks, thus providing time for the repair of CPT-induced lesions and supporting replication restart. We show that Tel1, the Saccharomyces cerevisiae orthologue of human ATM kinase, stabilizes CPT-induced reversed forks by counteracting their nucleolytic degradation by the MRX complex. Tel1-lacking cells are hypersensitive to CPT specifically and show less reversed forks in the presence of CPT The lack of Mre11 nuclease activity restores wild-type levels of reversed forks in CPT-treated tel1Δ cells without affecting fork reversal in wild-type cells. Moreover, Mrc1 inactivation prevents fork reversal in wild-type, tel1Δ, and mre11 nuclease-deficient cells and relieves the hypersensitivity of tel1Δ cells to CPT Altogether, our data indicate that Tel1 counteracts Mre11 nucleolytic activity at replication forks that undergo Mrc1-mediated reversal in the presence of CPT.


Asunto(s)
Proteínas de Ciclo Celular/genética , Replicación del ADN/genética , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de la Ataxia Telangiectasia Mutada/genética , Camptotecina/farmacología , Reparación del ADN/genética , Replicación del ADN/efectos de los fármacos , ADN-Topoisomerasas/genética , Humanos , Mutación , Saccharomyces cerevisiae/genética
4.
Methods Mol Biol ; 1672: 261-294, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29043630

RESUMEN

The DNA replication process can be heavily perturbed by several different conditions of genotoxic stress, particularly relevant for cancer onset and therapy. The combination of psoralen crosslinking and electron microscopy has proven instrumental to reveal the fine architecture of in vivo DNA replication intermediates and to uncover their remodeling upon specific conditions of genotoxic stress. The replication structures are stabilized in vivo (by psoralen crosslinking) prior to extraction and enrichment procedures, allowing their visualization at the transmission electron microscope. This chapter outlines the procedures required to visualize and interpret in vivo replication intermediates of eukaryotic genomic DNA, and includes an improved method for enrichment of replication intermediates, compared to previously used BND-cellulose columns.


Asunto(s)
Replicación del ADN , Células Eucariotas/fisiología , Microscopía Electrónica , Animales , Reactivos de Enlaces Cruzados , ADN de Hongos/ultraestructura , ADN de Cadena Simple/ultraestructura , Nucleosomas/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
5.
Nat Commun ; 8(1): 859, 2017 10 16.
Artículo en Inglés | MEDLINE | ID: mdl-29038466

RESUMEN

Besides its role in homologous recombination, the tumor suppressor BRCA2 protects stalled replication forks from nucleolytic degradation. Defective fork stability contributes to chemotherapeutic sensitivity of BRCA2-defective tumors by yet-elusive mechanisms. Using DNA fiber spreading and direct visualization of replication intermediates, we report that reversed replication forks are entry points for fork degradation in BRCA2-defective cells. Besides MRE11 and PTIP, we show that RAD52 promotes stalled fork degradation and chromosomal breakage in BRCA2-defective cells. Inactivation of these factors restores reversed fork frequency and chromosome integrity in BRCA2-defective cells. Conversely, impairing fork reversal prevents fork degradation, but increases chromosomal breakage, uncoupling fork protection, and chromosome stability. We propose that BRCA2 is dispensable for RAD51-mediated fork reversal, but assembles stable RAD51 nucleofilaments on regressed arms, to protect them from degradation. Our data uncover the physiopathological relevance of fork reversal and illuminate a complex interplay of homologous recombination factors in fork remodeling and stability.BRCA2 is involved in both homologous recombination (HR) and the protection of stalled replication forks from degradation. Here the authors reveal how HR factors cooperate in fork remodeling, showing that BRCA2 supports RAD51 loading on the regressed arms of reversed replication forks to protect them from degradation.


Asunto(s)
Proteína BRCA2/metabolismo , Proteínas Portadoras/metabolismo , Replicación del ADN , Recombinación Homóloga , Proteína Homóloga de MRE11/metabolismo , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Línea Celular Tumoral , Inestabilidad Cromosómica , Proteínas de Unión al ADN , Humanos
6.
Mol Cell ; 67(5): 882-890.e5, 2017 Sep 07.
Artículo en Inglés | MEDLINE | ID: mdl-28886337

RESUMEN

DNA damage tolerance during eukaryotic replication is orchestrated by PCNA ubiquitination. While monoubiquitination activates mutagenic translesion synthesis, polyubiquitination activates an error-free pathway, elusive in mammals, enabling damage bypass by template switching. Fork reversal is driven in vitro by multiple enzymes, including the DNA translocase ZRANB3, shown to bind polyubiquitinated PCNA. However, whether this interaction promotes fork remodeling and template switching in vivo was unknown. Here we show that damage-induced fork reversal in mammalian cells requires PCNA ubiquitination, UBC13, and K63-linked polyubiquitin chains, previously involved in error-free damage tolerance. Fork reversal in vivo also requires ZRANB3 translocase activity and its interaction with polyubiquitinated PCNA, pinpointing ZRANB3 as a key effector of error-free DNA damage tolerance. Mutations affecting fork reversal also induced unrestrained fork progression and chromosomal breakage, suggesting fork remodeling as a global fork slowing and protection mechanism. Targeting these fork protection systems represents a promising strategy to potentiate cancer chemotherapy.


Asunto(s)
Daño del ADN , ADN Helicasas/metabolismo , Replicación del ADN , ADN de Neoplasias/biosíntesis , Neoplasias/enzimología , Poliubiquitina/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Origen de Réplica , Animales , Sistemas CRISPR-Cas , ADN Helicasas/genética , ADN de Neoplasias/genética , ADN de Neoplasias/ultraestructura , Células HCT116 , Células HEK293 , Humanos , Cinética , Ratones , Mutación , Neoplasias/genética , Neoplasias/ultraestructura , Antígeno Nuclear de Célula en Proliferación/genética , Interferencia de ARN , Transfección , Enzimas Ubiquitina-Conjugadoras/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitinación
7.
EMBO J ; 35(23): 2584-2601, 2016 12 01.
Artículo en Inglés | MEDLINE | ID: mdl-27797818

RESUMEN

Homologous recombination (HR) is a key pathway that repairs DNA double-strand breaks (DSBs) and helps to restart stalled or collapsed replication forks. How HR supports replication upon genotoxic stress is not understood. Using in vivo and in vitro approaches, we show that the MMS22L-TONSL heterodimer localizes to replication forks under unperturbed conditions and its recruitment is increased during replication stress in human cells. MMS22L-TONSL associates with replication protein A (RPA)-coated ssDNA, and the MMS22L subunit directly interacts with the strand exchange protein RAD51. MMS22L is required for proper RAD51 assembly at DNA damage sites in vivo, and HR-mediated repair of stalled forks is abrogated in cells expressing a MMS22L mutant deficient in RAD51 interaction. Similar to the recombination mediator BRCA2, recombinant MMS22L-TONSL limits the assembly of RAD51 on dsDNA, which stimulates RAD51-ssDNA nucleoprotein filament formation and RAD51-dependent strand exchange activity in vitro Thus, by specifically regulating RAD51 activity at uncoupled replication forks, MMS22L-TONSL stabilizes perturbed replication forks by promoting replication fork reversal and stimulating their HR-mediated restart in vivo.


Asunto(s)
Proteínas de Unión al ADN/metabolismo , FN-kappa B/metabolismo , Proteínas Nucleares/metabolismo , Recombinasa Rad51/metabolismo , Recombinación Genética , Daño del ADN , Reparación del ADN , Replicación del ADN , Células HeLa , Humanos , Mapeo de Interacción de Proteínas , Multimerización de Proteína
8.
Nat Cell Biol ; 18(7): 777-89, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27323328

RESUMEN

The cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21) is a cell-cycle checkpoint effector and inducer of senescence, regulated by p53. Yet, evidence suggests that p21 could also be oncogenic, through a mechanism that has so far remained obscure. We report that a subset of atypical cancerous cells strongly expressing p21 showed proliferation features. This occurred predominantly in p53-mutant human cancers, suggesting p53-independent upregulation of p21 selectively in more aggressive tumour cells. Multifaceted phenotypic and genomic analyses of p21-inducible, p53-null, cancerous and near-normal cellular models showed that after an initial senescence-like phase, a subpopulation of p21-expressing proliferating cells emerged, featuring increased genomic instability, aggressiveness and chemoresistance. Mechanistically, sustained p21 accumulation inhibited mainly the CRL4-CDT2 ubiquitin ligase, leading to deregulated origin licensing and replication stress. Collectively, our data reveal the tumour-promoting ability of p21 through deregulation of DNA replication licensing machinery-an unorthodox role to be considered in cancer treatment, since p21 responds to various stimuli including some chemotherapy drugs.


Asunto(s)
Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Replicación del ADN/genética , Inestabilidad Genómica/genética , Células Cultivadas , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Ciclinas/genética , Ciclinas/metabolismo , Humanos , Neoplasias/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo
9.
Cell Rep ; 15(2): 300-9, 2016 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-27050524

RESUMEN

During transcription and DNA replication, the DNA template is overwound ahead of RNA and DNA polymerases and relaxed by DNA topoisomerases. Inhibitors of topoisomerases are potent anti-cancer agents. Camptothecin traps topoisomerase I on DNA and exerts preferential cytotoxicity toward cancer cells by way of its interference with the progression of replication forks. Starting with an unbiased proteomic analysis, we find that the chromatin remodeling complex BAZ1B-SMARCA5 accumulates near replication forks in camptothecin-exposed cells. We report that BAZ1B associates with topoisomerase I and facilitates its access to replication forks. Single-molecule analyses of replication structures show that BAZ1B contributes to replication interference by camptothecin. A lack of BAZ1B confers increased cellular tolerance of camptothecin. These findings reveal BAZ1B as a key facilitator of topoisomerase I function during DNA replication that affects the response of cancer cells to topoisomerase I inhibitors.


Asunto(s)
Ensamble y Desensamble de Cromatina , Replicación del ADN , ADN-Topoisomerasas de Tipo I/metabolismo , ADN/metabolismo , Proteómica/métodos , Adenosina Trifosfatasas/metabolismo , Camptotecina/farmacología , Ensamble y Desensamble de Cromatina/efectos de los fármacos , Proteínas Cromosómicas no Histona/metabolismo , Replicación del ADN/efectos de los fármacos , Células HeLa , Humanos , Masculino , Inhibidores de Topoisomerasa I/farmacología , Factores de Transcripción/metabolismo , Transcripción Genética/efectos de los fármacos
10.
Nat Commun ; 7: 10660, 2016 Feb 15.
Artículo en Inglés | MEDLINE | ID: mdl-26876348

RESUMEN

Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.


Asunto(s)
Daño del ADN , Replicación del ADN , ADN de Cadena Simple/metabolismo , Proteínas de Unión al ADN/metabolismo , Puntos de Control de la Fase G1 del Ciclo Celular , Fase G1 , Células Madre Embrionarias de Ratones/metabolismo , Recombinasa Rad51/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Blastocisto/metabolismo , Western Blotting , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Electroforesis en Gel de Campo Pulsado , Citometría de Flujo , Histonas/metabolismo , Ratones , Microscopía Confocal , Microscopía Electrónica , Microscopía Fluorescente , Mitosis , Mórula/metabolismo , Fosforilación , Poli(ADP-Ribosa) Polimerasas/metabolismo , Proteína de Replicación A/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53
11.
J Cell Biol ; 208(5): 563-79, 2015 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-25733714

RESUMEN

Replication fork reversal protects forks from breakage after poisoning of Topoisomerase 1. We here investigated fork progression and chromosomal breakage in human cells in response to a panel of sublethal genotoxic treatments, using other topoisomerase poisons, DNA synthesis inhibitors, interstrand cross-linking inducers, and base-damaging agents. We used electron microscopy to visualize fork architecture under these conditions and analyzed the association of specific molecular features with checkpoint activation. Our data identify replication fork uncoupling and reversal as global responses to genotoxic treatments. Both events are frequent even after mild treatments that do not affect fork integrity, nor activate checkpoints. Fork reversal was found to be dependent on the central homologous recombination factor RAD51, which is consistently present at replication forks independently of their breakage, and to be antagonized by poly (ADP-ribose) polymerase/RECQ1-regulated restart. Our work establishes remodeling of uncoupled forks as a pivotal RAD51-regulated response to genotoxic stress in human cells and as a promising target to potentiate cancer chemotherapy.


Asunto(s)
Daño del ADN , Replicación del ADN , Recombinasa Rad51/metabolismo , Línea Celular Tumoral , Células HEK293 , Humanos , Poli(ADP-Ribosa) Polimerasas/genética , Poli(ADP-Ribosa) Polimerasas/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Inhibidores de Topoisomerasa/toxicidad
12.
J Cell Biol ; 208(5): 545-62, 2015 Mar 02.
Artículo en Inglés | MEDLINE | ID: mdl-25733713

RESUMEN

Accurate processing of stalled or damaged DNA replication forks is paramount to genomic integrity and recent work points to replication fork reversal and restart as a central mechanism to ensuring high-fidelity DNA replication. Here, we identify a novel DNA2- and WRN-dependent mechanism of reversed replication fork processing and restart after prolonged genotoxic stress. The human DNA2 nuclease and WRN ATPase activities functionally interact to degrade reversed replication forks with a 5'-to-3' polarity and promote replication restart, thus preventing aberrant processing of unresolved replication intermediates. Unexpectedly, EXO1, MRE11, and CtIP are not involved in the same mechanism of reversed fork processing, whereas human RECQ1 limits DNA2 activity by preventing extensive nascent strand degradation. RAD51 depletion antagonizes this mechanism, presumably by preventing reversed fork formation. These studies define a new mechanism for maintaining genome integrity tightly controlled by specific nucleolytic activities and central homologous recombination factors.


Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN/fisiología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , ADN Helicasas/genética , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Humanos , Proteína Homóloga de MRE11 , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , RecQ Helicasas/genética , RecQ Helicasas/metabolismo , Helicasa del Síndrome de Werner
13.
Cell Rep ; 10(10): 1749-1757, 2015 Mar 17.
Artículo en Inglés | MEDLINE | ID: mdl-25772361

RESUMEN

DNA replication fork perturbation is a major challenge to the maintenance of genome integrity. It has been suggested that processing of stalled forks might involve fork regression, in which the fork reverses and the two nascent DNA strands anneal. Here, we show that FBH1 catalyzes regression of a model replication fork in vitro and promotes fork regression in vivo in response to replication perturbation. Cells respond to fork stalling by activating checkpoint responses requiring signaling through stress-activated protein kinases. Importantly, we show that FBH1, through its helicase activity, is required for early phosphorylation of ATM substrates such as CHK2 and CtIP as well as hyperphosphorylation of RPA. These phosphorylations occur prior to apparent DNA double-strand break formation. Furthermore, FBH1-dependent signaling promotes checkpoint control and preserves genome integrity. We propose a model whereby FBH1 promotes early checkpoint signaling by remodeling of stalled DNA replication forks.

14.
Genes Dev ; 27(23): 2537-42, 2013 Dec 01.
Artículo en Inglés | MEDLINE | ID: mdl-24298053

RESUMEN

Deregulated origin licensing and rereplication promote genome instability and tumorigenesis by largely elusive mechanisms. Investigating the consequences of Early mitotic inhibitor 1 (Emi1) depletion in human cells, previously associated with rereplication, we show by DNA fiber labeling that origin reactivation occurs rapidly, well before accumulation of cells with >4N DNA, and is associated with checkpoint-blind ssDNA gaps and replication fork reversal. Massive RPA chromatin loading, formation of small chromosomal fragments, and checkpoint activation occur only later, once cells complete bulk DNA replication. We propose that deregulated origin firing leads to undetected discontinuities on newly replicated DNA, which ultimately cause breakage of rereplicating forks.


Asunto(s)
Rotura Cromosómica , Replicación del ADN/genética , Origen de Réplica/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular , ADN/biosíntesis , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Humanos , ARN Interferente Pequeño/metabolismo , Moldes Genéticos
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