RESUMEN
The systematic identification of tumour vulnerabilities through perturbational experiments on cancer models, including genome editing and drug screens, is playing a crucial role in combating cancer. This collective effort is known as the Cancer Dependency Map (DepMap). The 1st European Cancer Dependency Map Symposium (EuroDepMap), held in Milan last May, featured talks, a roundtable discussion, and a poster session, showcasing the latest discoveries and future challenges related to the DepMap. The symposium aimed to facilitate interactions among participants across Europe, encourage idea exchange with leading experts, and present their work and future projects. Importantly, it sparked discussions on future endeavours, such as screening more complex cancer models and accounting for tumour evolution.
Asunto(s)
Neoplasias , Humanos , Neoplasias/genética , Europa (Continente)RESUMEN
DNA double-strand break (DSB) repair by homologous recombination (HR) requires 3' single-stranded DNA (ssDNA) generation by 5' DNA-end resection. During meiosis, yeast Sae2 cooperates with the nuclease Mre11 to remove covalently bound Spo11 from DSB termini, allowing resection and HR to ensue. Mitotic roles of Sae2 and Mre11 nuclease have remained enigmatic, however, since cells lacking these display modest resection defects but marked DNA damage hypersensitivities. By combining classic genetic suppressor screening with high-throughput DNA sequencing, we identify Mre11 mutations that strongly suppress DNA damage sensitivities of sae2∆ cells. By assessing the impacts of these mutations at the cellular, biochemical and structural levels, we propose that, in addition to promoting resection, a crucial role for Sae2 and Mre11 nuclease activity in mitotic DSB repair is to facilitate the removal of Mre11 from ssDNA associated with DSB ends. Thus, without Sae2 or Mre11 nuclease activity, Mre11 bound to partly processed DSBs impairs strand invasion and HR.
Asunto(s)
Reparación del ADN/fisiología , ADN de Hongos/metabolismo , ADN de Cadena Simple/metabolismo , Endonucleasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , ADN de Hongos/genética , ADN de Cadena Simple/genética , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas/genética , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
The Mre11-Rad50-Nbs1 (MRN) complex tethers, processes and signals DNA double-strand breaks, promoting genomic stability. To understand the functional architecture of MRN, we determined the crystal structures of the Schizosaccharomyces pombe Mre11 dimeric catalytic domain alone and in complex with a fragment of Nbs1. Two Nbs1 subunits stretch around the outside of the nuclease domains of Mre11, with one subunit additionally bridging and locking the Mre11 dimer via a highly conserved asymmetrical binding motif. Our results show that Mre11 forms a flexible dimer and suggest that Nbs1 not only is a checkpoint adaptor but also functionally influences Mre11-Rad50. Clinical mutations in Mre11 are located along the Nbs1-interaction sites and weaken the Mre11-Nbs1 interaction. However, they differentially affect DNA repair and telomere maintenance in Saccharomyces cerevisiae, potentially providing insight into their different human disease pathologies.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Daño del ADN , Mutación , Proteínas de Schizosaccharomyces pombe/metabolismo , Transducción de Señal , Sitios de Unión , Proteínas Cromosómicas no Histona/química , Dimerización , Humanos , Modelos Moleculares , Conformación Proteica , Proteínas de Schizosaccharomyces pombe/químicaRESUMEN
The DNA-damage checkpoint kinase Chk1 is essential in higher eukaryotes due to its role in maintaining genome stability in proliferating cells. CHK1 gene deletion is embryonically lethal, and Chk1 inhibition in replicating cells causes cell-cycle defects that eventually lead to perturbed replication and replication-fork collapse, thus generating endogenous DNA damage. What is the cause of replication-fork collapse when Chk1 is inactivated, however, remains poorly understood. Here, we show that generation of DNA double-strand breaks at replication forks when Chk1 activity is compromised relies on the DNA endonuclease complex Mus81/Eme1. Importantly, we show that Mus81/Eme1-dependent DNA damage--rather than a global increase in replication-fork stalling--is the cause of incomplete replication in Chk1-deficient cells. Consequently, Mus81/Eme1 depletion alleviates the S-phase progression defects associated with Chk1 deficiency, thereby increasing cell survival. Chk1-mediated protection of replication forks from Mus81/Eme1 even under otherwise unchallenged conditions is therefore vital to prevent uncontrolled fork collapse and ensure proper S-phase progression in human cells.
Asunto(s)
Daño del ADN , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Endonucleasas/metabolismo , Proteínas Quinasas/metabolismo , Western Blotting , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1) , Ensayo Cometa , ADN/química , ADN/genética , Roturas del ADN de Doble Cadena/efectos de los fármacos , Reparación del ADN , Replicación del ADN , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Activación Enzimática/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Humanos , Conformación de Ácido Nucleico , Proteínas Quinasas/genética , Interferencia de ARN , Fase S , Tiofenos/farmacología , Urea/análogos & derivados , Urea/farmacologíaRESUMEN
Meiotic recombination requires the formation of programmed Spo11-dependent DNA double strand breaks (DSBs). In Saccharomyces cerevisiae, the Sae2 protein and the Mre11-Rad50-Xrs2 complex are necessary to remove the covalently attached Spo11 protein from the DNA ends, which are then resected by so far unknown nucleases. Here, we demonstrate that phosphorylation of Sae2 Ser-267 by cyclin-dependent kinase 1 (Cdk1) is required to initiate meiotic DSB resection by allowing Spo11 removal from DSB ends. This finding suggests that Cdk1 activity is required for the processing of Spo11-induced DSBs, thus providing a mechanism for coordinating DSB resection with progression through meiotic prophase. Furthermore, the helicase Sgs1 and the nucleases Exo1 and Dna2 participate in lengthening the 5'-3' resection tracts during meiosis by controlling a step subsequent to Spo11 removal.
Asunto(s)
Quinasas Ciclina-Dependientes/metabolismo , Roturas del ADN de Doble Cadena , Daño del ADN , Meiosis , Saccharomyces cerevisiae/fisiología , Ciclo Celular , ADN Helicasas/metabolismo , ADN-Topoisomerasas de Tipo II/metabolismo , Endodesoxirribonucleasas , Endonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Fosforilación , RecQ Helicasas/metabolismo , Recombinación Genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
DNA double-strand breaks (DSBs) are highly hazardous for genome integrity, but meiotic cells deliberately introduce them into their genome in order to initiate homologous recombination, which ensures proper homologous chromosome segregation. To minimize the risk of deleterious effects, meiotic DSB formation, processing and repair are tightly regulated in order to occur only at the right time and place. Furthermore, a highly conserved signal-transduction pathway, called meiotic recombination checkpoint, coordinates DSB repair with meiotic progression and promotes meiotic recombination.
Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Meiosis , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Recombinación Genética/genética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
DNA double-strand breaks (DSBs) are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR). HR requires 5' DSB end degradation that occurs in the presence of cyclin-dependent kinase (CDK) activity. Here, we show that a lack of any of the NHEJ proteins Yku (Yku70-Yku80), Lif1 or DNA ligase IV (Dnl4) increases 5' DSB end degradation in G1 phase, with ykuDelta cells showing the strongest effect. This increase depends on MRX, the recruitment of which at DSBs is enhanced in ykuDelta G1 cells. DSB processing in G2 is not influenced by the absence of Yku, but it is delayed by Yku overproduction, which also decreases MRX loading on DSBs. Moreover, DSB resection in ykuDelta cells occurs independently of CDK activity, suggesting that it might be promoted by CDK-dependent inhibition of Yku.
Asunto(s)
Ciclo Celular/fisiología , Roturas del ADN de Doble Cadena , Proteínas de Unión al ADN/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Western Blotting , Ciclo Celular/genética , Inmunoprecipitación de Cromatina , Quinasas Ciclina-Dependientes/genética , Quinasas Ciclina-Dependientes/metabolismo , ADN Ligasa (ATP) , ADN Ligasas/genética , ADN Ligasas/metabolismo , Reparación del ADN , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Exodesoxirribonucleasas/metabolismo , Fase G1/genética , Fase G1/fisiología , Fase G2/genética , Fase G2/fisiología , Recombinación Genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
DNA double-strand breaks (DSBs) can arise at unpredictable locations after DNA damage or in a programmed manner during meiosis. DNA damage checkpoint response to accidental DSBs during mitosis requires the Rad53 effector kinase, whereas the meiosis-specific Mek1 kinase, together with Red1 and Hop1, mediates the recombination checkpoint in response to programmed meiotic DSBs. Here we provide evidence that exogenous DSBs lead to Rad53 phosphorylation during the meiotic cell cycle, whereas programmed meiotic DSBs do not. However, the latter can trigger phosphorylation of a protein fusion between Rad53 and the Mec1-interacting protein Ddc2, suggesting that the inability of Rad53 to transduce the meiosis-specific DSB signals might be due to its failure to access the meiotic recombination sites. Rad53 phosphorylation/activation is elicited when unrepaired meiosis-specific DSBs escape the recombination checkpoint. This activation requires homologous chromosome segregation and delays the second meiotic division. Altogether, these data indicate that Rad53 prevents sister chromatid segregation in the presence of unrepaired programmed meiotic DSBs, thus providing a salvage mechanism ensuring genetic integrity in the gametes even in the absence of the recombination checkpoint.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Roturas del ADN de Doble Cadena , Meiosis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Quinasa de Punto de Control 2 , Proteínas de Unión al ADN/genética , Endonucleasas , Péptidos y Proteínas de Señalización Intracelular , Fosforilación , Recombinación Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
DNA double-strand breaks (DSBs) are highly hazardous for genome integrity, because failure to repair them can lead to genome rearrangements or chromosome loss. They can arise at unpredictable locations as a consequence of DNA damage during both the mitotic and the meiotic cell cycle or in a programmed manner during meiosis. Cellular response to accidental or programmed DSBs involves highly conserved surveillance mechanisms, called DNA damage checkpoint and recombination checkpoint, which coordinate DSB repair with mitotic or meiotic cell cycle progression, respectively. Although these protective signal-transduction pathways share several upstream components, activation of the recombination checkpoint requires meiosis-specific proteins. These proteins are structural components of the meiotic chromosomes, indicating that the system monitoring programmed meiotic DSBs is an integral part of the chromosome structure formed during meiosis.
Asunto(s)
Rotura Cromosómica , Reparación del ADN , Meiosis/genética , Mitosis/genética , Animales , Humanos , Transducción de SeñalRESUMEN
DNA double-strand breaks (DSBs) are introduced into the genome to initiate meiotic recombination. Their accurate repair is monitored by the meiotic recombination checkpoint that prevents nuclear division until completion of meiotic DSB repair. We show that the Saccharomyces cerevisiae Sae2 protein, known to be involved in processing meiotic DSBs, is phosphorylated periodically during the meiotic cycle. Sae2 phosphorylation occurs at the onset of premeiotic S phase, is maximal at the time of meiotic DSB generation and decreases when DSBs are repaired by homologous recombination. Hyperactivation of the meiotic recombination checkpoint caused by the failure to repair DSBs results in accumulation and persistence of phosphorylated Sae2, indicating a possible link between checkpoint activation and meiosis-induced Sae2 phosphorylation. Accordingly, Sae2 phosphorylation depends on the checkpoint kinases Mec1 and Tel1, whose simultaneous deletion also impairs meiotic DSB repair. Moreover, replacing with alanines the Sae2 serine and threonine residues belonging to Mec1/Tel1-dependent putative phosphorylation sites impairs not only Sae2 phosphorylation during meiosis, but also meiotic DSB repair. Thus, checkpoint-mediated phosphorylation of Sae2 is important to support its meiotic recombination functions.