RESUMEN
The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which employs a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in Saccharomyces cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Histonas/metabolismo , Proteínas Nucleares/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Cromatina/genética , Chaperonas de Histonas/genética , ADN , Chaperonas Moleculares/metabolismoRESUMEN
The 43rd Asilomar Chromatin, Chromosomes, and Epigenetics Conference was held in an entirely online format from 9 to 11 December 2021. The conference enabled presenters at various career stages to share promising new findings, and presentations covered modern chromatin research across an array of model systems. Topics ranged from the fundamental principles of nuclear organization and transcription regulation to key mechanisms underlying human disease. The meeting featured five keynote speakers from diverse backgrounds and was organized by Juan Ausió, University of Victoria (British Columbia, Canada), James Davie, University of Manitoba (Manitoba, Canada), Philippe T. Georgel, Marshall University (West Virginia, USA), Michael Goldman, San Francisco State University (California, USA), LeAnn Howe, The University of British Columbia (British Columbia, Canada), Jennifer A. Mitchell, University of Toronto (Ontario, Canada), and Sally G. Pasion, San Francisco State University (California, USA).
Asunto(s)
Cromatina , Epigenómica , Canadá , Cromatina/genética , Cromosomas/genética , Epigénesis Genética , HumanosRESUMEN
Cells respond to DNA damage by activating cell cycle checkpoints, arresting cell division or DNA replication while damage is repaired. In Saccharomyces cerevisiae, activation of the checkpoint kinase Rad53 leads to cell cycle arrest, with Rad53 deactivation required for proper resumption of the cell cycle. Rtt107 is a S. cerevisiae protein that acts as a scaffold in the response to DNA damage, and rtt107Δ mutants exhibit prolonged activation of Rad53 when subjected to replication stress. This phenotype has been attributed to checkpoint dampening, wherein an Rtt107-Slx4-Dpb11 interaction limits formation of a Rad9-Dpb11 complex that promotes Rad53 activation. However, we found that the rtt107Δ mutant contains higher levels of DNA damage during replication stress, presenting an alternative possible cause of Rad53 hyperactivation. We therefore sought to address the relevance of checkpoint dampening to the Rad53 hyperactivation phenotype of the rtt107Δ mutant by using a rad9-ST462,474AA allele that specifically disrupts Rad9-Dpb11 interaction. Incorporation of the rad9-ST462,474AA allele slightly suppressed the rtt107Δ mutant's DNA damage sensitivity phenotypes, while having little effect on Rad53 hyperactivation. This indicated that in the context of acute replication stress, Rad53 hyperactivation in the rtt107Δ mutant did not primarily result from Rad9-Dpb11 interaction. A H2A-S129A mutation, which generally reduces Rad9-mediated Rad53 activation, led to more robust suppression of rtt107Δ mutant phenotypes. Suppression of rtt107Δ mutant DNA damage sensitivity by the H2A-S129A or the rad9-ST462,474AA alleles required intact DNA damage tolerance pathways, indicating a reliance of the rtt107Δ mutant on tolerance pathways for reasons other than misregulation of Rad53 activity. Collectively, this work proposed a revised model of Rad53 hyperactivation after acute replicative stress in the rtt107Δ mutant, in which this phenotype was primarily a consequence of excess DNA damage.
Asunto(s)
Puntos de Control del Ciclo Celular/genética , Daño del ADN , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Puntos de Control del Ciclo Celular/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Metilmetanosulfonato/farmacología , Mutación , Proteínas Nucleares/genética , Fenotipo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Cells are constantly exposed to assaults that cause DNA damage, which must be detected and repaired to prevent genome instability. The DNA damage response is mediated by key kinases that activate various signaling pathways. In Saccharomyces cerevisiae, one of these kinases is Mec1, which phosphorylates numerous targets, including H2A and the DNA damage protein Rtt107. In addition to being phosphorylated, Rtt107 contains six BRCA1 C-terminal (BRCT) domains, which typically recognize phospho-peptides. Thus Rtt107 represented an opportunity to study complementary aspects of the phosphorylation cascades within one protein. Here we sought to describe the functional roles of the multiple BRCT domains in Rtt107. Rtt107 BRCT5/6 facilitated recruitment to sites of DNA lesions via its interaction with phosphorylated H2A. Rtt107 BRCT3/4 also contributed to Rtt107 recruitment, but BRCT3/4 was not sufficient for recruitment when BRCT5/6 was absent. Intriguingly, both mutations that affected Rtt107 recruitment also abrogated its phosphorylation. Pointing to its modular nature, replacing Rtt107 BRCT5/6 with the BRCT domains from the checkpoint protein Rad9 was able to sustain Rtt107 function. Although Rtt107 physically interacts with both the endonuclease Slx4 and the DNA replication and repair protein Dpb11, only Slx4 was dependent on Rtt107 for its recruitment to DNA lesions. Fusing Rtt107 BRCT5/6 to Slx4, which presumably allows artificial recruitment of Slx4 to DNA lesions, alleviated some phenotypes of rtt107Δ mutants, indicating the functional importance of Slx4 recruitment. Together this data revealed a key function of the Rtt107 BRCT domains for targeting of both itself and its interaction partners to DNA lesions.