RESUMEN
Obstructions to replication fork progression, referred to collectively as DNA replication stress, challenge genome stability. In Saccharomyces cerevisiae, cells lacking RTT107 or SLX4 show genome instability and sensitivity to DNA replication stress and are defective in the completion of DNA replication during recovery from replication stress. We demonstrate that Slx4 is recruited to chromatin behind stressed replication forks, in a region that is spatially distinct from that occupied by the replication machinery. Slx4 complex formation is nucleated by Mec1 phosphorylation of histone H2A, which is recognized by the constitutive Slx4 binding partner Rtt107. Slx4 is essential for recruiting the Mec1 activator Dpb11 behind stressed replication forks, and Slx4 complexes are important for full activity of Mec1. We propose that Slx4 complexes promote robust checkpoint signaling by Mec1 by stably recruiting Dpb11 within a discrete domain behind the replication fork, during DNA replication stress.
Asunto(s)
Replicación del ADN , ADN de Hongos/metabolismo , Endodesoxirribonucleasas/metabolismo , Multimerización de Proteína , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/fisiología , Proteínas de Ciclo Celular , Histonas , Péptidos y Proteínas de Señalización Intracelular , Proteínas Nucleares , Unión Proteica , Proteínas Serina-Treonina Quinasas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMEN
Condition-dependent genetic interactions can reveal functional relationships between genes that are not evident under standard culture conditions. State-of-the-art yeast genetic interaction mapping, which relies on robotic manipulation of arrays of double-mutant strains, does not scale readily to multi-condition studies. Here, we describe barcode fusion genetics to map genetic interactions (BFG-GI), by which double-mutant strains generated via en masse "party" mating can also be monitored en masse for growth to detect genetic interactions. By using site-specific recombination to fuse two DNA barcodes, each representing a specific gene deletion, BFG-GI enables multiplexed quantitative tracking of double mutants via next-generation sequencing. We applied BFG-GI to a matrix of DNA repair genes under nine different conditions, including methyl methanesulfonate (MMS), 4-nitroquinoline 1-oxide (4NQO), bleomycin, zeocin, and three other DNA-damaging environments. BFG-GI recapitulated known genetic interactions and yielded new condition-dependent genetic interactions. We validated and further explored a subnetwork of condition-dependent genetic interactions involving MAG1, SLX4, and genes encoding the Shu complex, and inferred that loss of the Shu complex leads to an increase in the activation of the checkpoint protein kinase Rad53.
Asunto(s)
Mapeo Cromosómico , Código de Barras del ADN Taxonómico , Daño del ADN , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Reparación del ADN , Epistasis Genética , Eliminación de Gen , Sitios Genéticos , Secuenciación de Nucleótidos de Alto Rendimiento , Metilmetanosulfonato , Modelos Teóricos , Regiones Promotoras Genéticas , Reproducibilidad de los ResultadosRESUMEN
The DNA damage checkpoint pathway is activated in response to DNA lesions and replication stress to preserve genome integrity. However, hyper-activation of this surveillance system is detrimental to the cell, because it might prevent cell cycle re-start after repair, which may also lead to senescence. Here we show that the scaffold proteins Slx4 and Rtt107 limit checkpoint signalling at a persistent double-strand DNA break (DSB) and at uncapped telomeres. We found that Slx4 is recruited within a few kilobases of an irreparable DSB, through the interaction with Rtt107 and the multi-BRCT domain scaffold Dpb11. In the absence of Slx4 or Rtt107, Rad9 binding near the irreparable DSB is increased, leading to robust checkpoint signalling and slower nucleolytic degradation of the 5' strand. Importantly, in slx4Δ sae2Δ double mutant cells these phenotypes are exacerbated, causing a severe Rad9-dependent defect in DSB repair. Our study sheds new light on the molecular mechanism that coordinates the processing and repair of DSBs with DNA damage checkpoint signalling, preserving genome integrity.
Asunto(s)
Roturas del ADN de Doble Cadena , Endodesoxirribonucleasas/metabolismo , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Camptotecina/farmacología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endonucleasas/genética , Endonucleasas/metabolismo , Metilmetanosulfonato/farmacología , Mutación , Proteínas Nucleares/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transducción de Señal , Telómero/genética , Telómero/metabolismo , Inhibidores de Topoisomerasa I/farmacologíaRESUMEN
To systematically explore complex genetic interactions, we constructed ~200,000 yeast triple mutants and scored negative trigenic interactions. We selected double-mutant query genes across a broad spectrum of biological processes, spanning a range of quantitative features of the global digenic interaction network and tested for a genetic interaction with a third mutation. Trigenic interactions often occurred among functionally related genes, and essential genes were hubs on the trigenic network. Despite their functional enrichment, trigenic interactions tended to link genes in distant bioprocesses and displayed a weaker magnitude than digenic interactions. We estimate that the global trigenic interaction network is ~100 times as large as the global digenic network, highlighting the potential for complex genetic interactions to affect the biology of inheritance, including the genotype-to-phenotype relationship.
Asunto(s)
Redes Reguladoras de Genes , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Mutación , Análisis de Secuencia por Matrices de OligonucleótidosRESUMEN
In response to replication stress, signaling mediated by DNA damage checkpoint kinases protects genome integrity. However, following repair or bypass of DNA lesions, checkpoint signaling needs to be terminated for continued cell cycle progression and proliferation. In budding yeast, the PP4 phosphatase has been shown to play a key role in preventing hyperactivation of the checkpoint kinase Rad53. In addition, we recently uncovered a phosphatase-independent mechanism for downregulating Rad53 in which the DNA repair scaffold Slx4 decreases engagement of the checkpoint adaptor Rad9 at DNA lesions. Here we reveal that proper termination of checkpoint signaling following the bypass of replication blocks imposed by alkylated DNA adducts requires the concerted action of these two fundamentally distinct mechanisms of checkpoint downregulation. Cells lacking both SLX4 and the PP4-subunit PPH3 display a synergistic increase in Rad53 signaling and are exquisitely sensitive to the DNA alkylating agent methyl methanesulfonate, which induces replication blocks and extensive formation of chromosomal linkages due to template switching mechanisms required for fork bypass. Rad53 hypersignaling in these cells seems to converge to a strong repression of Mus81-Mms4, the endonuclease complex responsible for resolving chromosomal linkages, thus explaining the selective sensitivity of slx4Δ pph3Δ cells to alkylation damage. Our results support a model in which Slx4 acts locally to downregulate Rad53 activation following fork bypass, while PP4 acts on pools of active Rad53 that have diffused from the site of lesions. We propose that the proper spatial coordination of the Slx4 scaffold and PP4 action is crucial to allow timely activation of Mus81-Mms4 and, therefore, proper chromosome segregation.