RESUMEN
DNA2 is an essential nuclease-helicase implicated in DNA repair, lagging-strand DNA synthesis, and the recovery of stalled DNA replication forks (RFs). In Saccharomyces cerevisiae, dna2Δ inviability is reversed by deletion of the conserved helicase PIF1 and/or DNA damage checkpoint-mediator RAD9. It has been suggested that Pif1 drives the formation of long 5'-flaps during Okazaki fragment maturation, and that the essential function of Dna2 is to remove these intermediates. In the absence of Dna2, 5'-flaps are thought to accumulate on the lagging strand, resulting in DNA damage-checkpoint arrest and cell death. In line with Dna2's role in RF recovery, we find that the loss of Dna2 results in severe chromosome under-replication downstream of endogenous and exogenous RF-stalling. Importantly, unfaithful chromosome replication in Dna2-mutant cells is exacerbated by Pif1, which triggers the DNA damage checkpoint along a pathway involving Pif1's ability to promote homologous recombination-coupled replication. We propose that Dna2 fulfils its essential function by promoting RF recovery, facilitating replication completion while suppressing excessive RF restart by recombination-dependent replication (RDR) and checkpoint activation. The critical nature of Dna2's role in controlling the fate of stalled RFs provides a framework to rationalize the involvement of DNA2 in Seckel syndrome and cancer.
Asunto(s)
ADN Helicasas/metabolismo , Replicación del ADN , Enfermedades Genéticas Congénitas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Puntos de Control del Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN , Daño del ADN , ADN Helicasas/genética , Humanos , Mutación , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
PURPOSE: Cholinergic signals can be important modulators of cellular signaling in cancer. We recently have shown that knockdown of nicotinic acetylcholine receptor subunit alpha 5, CHRNA5, diminishes the proliferative potential of breast cancer cells. However, modulation of CHRNA5 expression in the context of estrogen signaling and its prognostic implications in breast cancer remained unexplored. METHODS: Meta-analyses of large breast cancer microarray cohorts were used to evaluate the association of CHRNA5 expression with estrogen (E2) treatment, estrogen receptor (ER) status and patient prognosis. The results were validated through RT-qPCR analyses of multiple E2 treated cell lines, CHRNA5 depleted MCF7 cells and across a breast cancer patient cDNA panel. We also calculated a predicted secondary (PS) score representing direct/indirect induction of gene expression by E2 based on a public dataset (GSE8597). Co-expression analysis was performed using a weighted gene co-expression network analysis (WGCNA) pipeline. Multiple other publicly available datasets such as CCLE, COSMIC and TCGA were also analyzed. RESULTS: Herein we found that CHRNA5 expression was induced by E2 in a dose- and time-dependent manner in breast cancer cell lines. ER- breast tumors exhibited higher CHRNA5 expression levels than ER+ tumors. Independent meta-analysis for survival outcome revealed that higher CHRNA5 expression was associated with a worse prognosis in untreated breast cancer patients. Furthermore, CHRNA5 and its co-expressed gene network emerged as secondarily induced targets of E2 stimulation. These targets were largely downregulated by exposure to CHRNA5 siRNA in MCF7 cells while the response of primary ESR1 targets was dependent on the direction of the PS-score. Moreover, primary and secondary target genes were uncoupled and clustered distinctly based on multiple public datasets. CONCLUSION: Our findings strongly associate increased expression of CHRNA5 and its co-expression network with secondary E2 signaling and a worse prognosis in breast cancer.
Asunto(s)
Neoplasias de la Mama/metabolismo , Estrógenos/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Nicotínicos/metabolismo , Transducción de Señal , Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Línea Celular Tumoral , Receptor alfa de Estrógeno/metabolismo , Estrógenos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Redes Reguladoras de Genes , Humanos , Proteínas del Tejido Nervioso/genética , Pronóstico , ARN Interferente Pequeño/metabolismo , Receptores de Estrógenos/metabolismo , Receptores Nicotínicos/genética , Transducción de Señal/efectos de los fármacos , Factores de Transcripción/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genéticaRESUMEN
Cells have evolved mechanisms to protect, restart and repair perturbed replication forks, allowing full genome duplication, even under replication stress. Interrogating the interplay between nuclease-helicase Dna2 and Holliday junction (HJ) resolvase Yen1, we find the Dna2 helicase activity acts parallel to homologous recombination (HR) in promoting DNA replication and chromosome detachment at mitosis after replication fork stalling. Yen1, but not the HJ resolvases Slx1-Slx4 and Mus81-Mms4, safeguards chromosome segregation by removing replication intermediates that escape Dna2. Post-replicative DNA damage checkpoint activation in Dna2 helicase-defective cells causes terminal G2/M arrest by precluding Yen1-dependent repair, whose activation requires progression into anaphase. These findings explain the exquisite replication stress sensitivity of Dna2 helicase-defective cells, and identify a non-canonical role for Yen1 in the processing of replication intermediates that is distinct from HJ resolution. The involvement of Dna2 helicase activity in completing replication may have implications for DNA2-associated pathologies, including cancer and Seckel syndrome.
Asunto(s)
ADN Helicasas/genética , Replicación del ADN , Regulación Fúngica de la Expresión Génica , Resolvasas de Unión Holliday/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Segregación Cromosómica , Cromosomas Fúngicos/química , Cromosomas Fúngicos/metabolismo , ADN Helicasas/metabolismo , ADN Cruciforme/genética , ADN Cruciforme/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Endodesoxirribonucleasas/genética , Endodesoxirribonucleasas/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Endonucleasas de ADN Solapado/genética , Endonucleasas de ADN Solapado/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Resolvasas de Unión Holliday/metabolismo , Recombinación Homóloga , Mitosis , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
PBY1 continues to be linked with DNA repair through functional genomics studies in yeast. Using the yeast knockout (YKO) strain collection, high-throughput genetic interaction screens have identified a large set of negative interactions between PBY1 and genes involved in genome stability. In drug sensitivity screens, the YKO collection pby1Δ strain exhibits a sensitivity profile typical for genes involved in DNA replication and repair. We show that these findings are not related to loss of Pby1. On the basis of genetic interaction profile similarity, we pinpoint disruption of Holliday junction resolvase Mus81-Mms4 as the mutation responsible for DNA repair phenotypes currently ascribed to pby1. The finding that Pby1 is not a DNA repair factor reconciles discrepancies in the data available for PBY1, and indirectly supports a role for Pby1 in mRNA metabolism. Data that has been collected using the YKO collection pby1Δ strain confirms and expands the chemical-genetic interactome of MUS81-MMS4.