RESUMEN
Cellular homeostasis requires the coordination of several machineries concurrently engaged in the DNA. Wide-spread transcription can interfere with other processes, and transcription-replication conflicts (TRCs) threaten genome stability. The conserved Sen1 helicase not only terminates non-coding transcription but also interacts with the replisome and reportedly resolves genotoxic R-loops. Sen1 prevents genomic instability, but how this relates to its molecular functions remains unclear. We generated high-resolution, genome-wide maps of transcription-dependent conflicts and R-loops using a Sen1 mutant that has lost interaction with the replisome but is termination proficient. We show that, under physiological conditions, Sen1 removes RNA polymerase II at TRCs within genes and the rDNA and at sites of transcription-transcription conflicts, thus qualifying as a "key regulator of conflicts." We demonstrate that genomic stability is affected by Sen1 mutation only when in addition to its role at the replisome, the termination of non-coding transcription or R-loop removal are additionally compromised.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN/genética , Inestabilidad Genómica , ARN Helicasas/genética , ARN Helicasas/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción GenéticaRESUMEN
The disease-associated nuclease-helicase DNA2 has been implicated in DNA end-resection during DNA double-strand break repair, Okazaki fragment processing, and the recovery of stalled DNA replication forks (RFs). Its role in Okazaki fragment processing has been proposed to explain why DNA2 is indispensable for cell survival across organisms. Unexpectedly, we found that DNA2 has an essential role in suppressing homologous recombination (HR)-dependent replication restart at stalled RFs. In the absence of DNA2-mediated RF recovery, excessive HR-restart of stalled RFs results in toxic levels of abortive recombination intermediates that lead to DNA damage-checkpoint activation and terminal cell-cycle arrest. While HR proteins protect and restart stalled RFs to promote faithful genome replication, these findings show how HR-dependent replication restart is actively constrained by DNA2 to ensure cell survival. These new insights disambiguate the effects of DNA2 dysfunction on cell survival, and provide a framework to rationalize the association of DNA2 with cancer and the primordial dwarfism disorder Seckel syndrome based on its role in RF recovery.
Asunto(s)
ADN Helicasas/genética , Reparación del ADN/genética , Replicación del ADN/genética , Recombinación Homóloga/genética , Supervivencia Celular/genética , ADN/genética , Enanismo/genética , Genoma Humano/genética , Humanos , Neoplasias/genéticaRESUMEN
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
DNA replication and RNA transcription compete for the same substrate during S phase. Cells have evolved several mechanisms to minimize such conflicts. Here, we identify the mechanism by which the transcription termination helicase Sen1 associates with replisomes. We show that the N terminus of Sen1 is both sufficient and necessary for replisome association and that it binds to the replisome via the components Ctf4 and Mrc1. We generated a separation of function mutant, sen1-3, which abolishes replisome binding without affecting transcription termination. We observe that the sen1-3 mutants show increased genome instability and recombination levels. Moreover, sen1-3 is synthetically defective with mutations in genes involved in RNA metabolism and the S phase checkpoint. RNH1 overexpression suppresses defects in the former, but not the latter. These findings illustrate how Sen1 plays a key function at replication forks during DNA replication to promote fork progression and chromosome stability.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , ARN Helicasas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Proteínas de Ciclo Celular/genética , ADN Helicasas/genética , Proteínas de Unión al ADN/genética , Genómica , Humanos , ARN Helicasas/genética , ARN de Hongos/genética , ARN de Hongos/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Transcripción GenéticaRESUMEN
Replication fork stalling and accumulation of single-stranded DNA trigger the S phase checkpoint, a signalling cascade that, in budding yeast, leads to the activation of the Rad53 kinase. Rad53 is essential in maintaining cell viability, but its targets of regulation are still partially unknown. Here we show that Rad53 drives the hyper-SUMOylation of Pol2, the catalytic subunit of DNA polymerase ε, principally following replication forks stalling induced by nucleotide depletion. Pol2 is the main target of SUMOylation within the replisome and its modification requires the SUMO-ligase Mms21, a subunit of the Smc5/6 complex. Moreover, the Smc5/6 complex co-purifies with Pol ε, independently of other replisome components. Finally, we map Pol2 SUMOylation to a single site within the N-terminal catalytic domain and identify a SUMO-interacting motif at the C-terminus of Pol2. These data suggest that the S phase checkpoint regulate Pol ε during replication stress through Pol2 SUMOylation and SUMO-binding ability.