RESUMEN
Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Cohesion establishment requires acetylation of conserved cohesin lysine residues by Eco1 acetyltransferase. Here, we explore how cohesin acetylation is linked to DNA replication. Biochemical reconstitution of replication-coupled cohesin acetylation reveals that transient DNA structures, which form during DNA replication, control the acetylation reaction. As polymerases complete lagging strand replication, strand displacement synthesis produces DNA flaps that are trimmed to result in nicked double-stranded DNA. Both flaps and nicks stimulate cohesin acetylation, while subsequent nick ligation to complete Okazaki fragment maturation terminates the acetylation reaction. A flapped or nicked DNA substrate constitutes a transient molecular clue that directs cohesin acetylation to a window behind the replication fork, next to where cohesin likely entraps both sister chromatids. Our results provide an explanation for how DNA replication is linked to sister chromatid cohesion establishment.
Asunto(s)
Cromátides , Proteínas de Saccharomyces cerevisiae , Cromátides/metabolismo , Proteínas Nucleares/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Replicación del ADN , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN , Acetiltransferasas/genética , Acetiltransferasas/metabolismoRESUMEN
Retrons are toxin-antitoxin systems protecting bacteria against bacteriophages via abortive infection. The Retron-Eco1 antitoxin is formed by a reverse transcriptase (RT) and a non-coding RNA (ncRNA)/multi-copy single-stranded DNA (msDNA) hybrid that neutralizes an uncharacterized toxic effector. Yet, the molecular mechanisms underlying phage defense remain unknown. Here, we show that the N-glycosidase effector, which belongs to the STIR superfamily, hydrolyzes NAD+ during infection. Cryoelectron microscopy (cryo-EM) analysis shows that the msDNA stabilizes a filament that cages the effector in a low-activity state in which ADPr, a NAD+ hydrolysis product, is covalently linked to the catalytic E106 residue. Mutations shortening the msDNA induce filament disassembly and the effector's toxicity, underscoring the msDNA role in immunity. Furthermore, we discovered a phage-encoded Retron-Eco1 inhibitor (U56) that binds ADPr, highlighting the intricate interplay between retron systems and phage evolution. Our work outlines the structural basis of Retron-Eco1 defense, uncovering ADPr's pivotal role in immunity.
Asunto(s)
Bacteriófagos , Microscopía por Crioelectrón , NAD , NAD/metabolismo , Bacteriófagos/genética , Bacteriófagos/metabolismo , Bacteriófagos/inmunología , Hidrólisis , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , ADN de Cadena Simple/inmunología , Sistemas Toxina-Antitoxina/genética , Escherichia coli/virología , Escherichia coli/genética , Escherichia coli/inmunología , Escherichia coli/metabolismoRESUMEN
The evolutionarily conserved cohesin complex mediates sister chromatid cohesion and facilitates mitotic chromosome condensation, DNA repair, and transcription regulation. These biological functions require cohesin's two ATPases, formed by the Smc1p and Smc3p subunits. Cohesin's ATPase activity is stimulated by the Scc2p auxiliary factor. This stimulation is inhibited by Eco1p acetylation of Smc3p at an interface with Scc2p. It was unclear how cohesin's ATPase activity is stimulated by Scc2p or how acetylation inhibits Scc2p, given that the acetylation site is distal to cohesin's ATPase active sites. Here, we identify mutations in budding yeast that suppressed the in vivo defects caused by Smc3p acetyl-mimic and acetyl-defective mutations. We provide compelling evidence that Scc2p activation of cohesin ATPase depends on an interface between Scc2p and a region of Smc1p proximal to cohesin's Smc3p ATPase active site. Furthermore, substitutions at this interface increase or decrease ATPase activity to overcome ATPase modulation by acetyl-mimic and acetyl-null mutations. Using these observations and an existing cryo-EM structure, we propose a model for regulating cohesin ATPase activity. We suggest that Scc2p binding to Smc1p causes the adjacent Smc1p residues and ATP to shift, stimulating Smc3p's ATPase. This stimulatory shift is inhibited through acetylation of the distal Scc2p-Smc3p interface.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Adenosina Trifosfatasas/metabolismo , Acetilación , Cromátides/metabolismo , Proteínas de Ciclo Celular/metabolismo , CohesinasRESUMEN
Cohesin is an ATPase that drives chromosome organization through the generation of intramolecular loops and sister chromatid cohesion. Cohesin's ATPase is stimulated by Scc2 binding but attenuated by acetylation of its Smc3 subunit. In this issue of Genes & Development, Boardman and colleagues (pp. 277-290) take a genetic approach to generate a mechanistic model for the opposing regulation of cohesin's ATPase by Scc2 and Smc3 acetylation. Their findings provide in vivo insight into how this important genome organizer functions in vivo.
Asunto(s)
Adenosina Trifosfatasas , Proteínas de Saccharomyces cerevisiae , Adenosina Trifosfatasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromatina , Cromátides/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Concomitant with DNA replication, the chromosomal cohesin complex establishes cohesion between newly replicated sister chromatids. Several replication-fork-associated "cohesion establishment factors," including the multifunctional Ctf18-RFC complex, aid this process in as yet unknown ways. Here, we show that Ctf18-RFC's role in sister chromatid cohesion correlates with PCNA loading but is separable from its role in the replication checkpoint. Ctf18-RFC loads PCNA with a slight preference for the leading strand, which is dispensable for DNA replication. Conversely, the canonical Rfc1-RFC complex preferentially loads PCNA onto the lagging strand, which is crucial for DNA replication but dispensable for sister chromatid cohesion. The downstream effector of Ctf18-RFC is cohesin acetylation, which we place toward a late step during replication maturation. Our results suggest that Ctf18-RFC enriches and balances PCNA levels at the replication fork, beyond the needs of DNA replication, to promote establishment of sister chromatid cohesion and possibly other post-replicative processes.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Cromátides/fisiología , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/fisiología , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Saccharomyces cerevisiae/crecimiento & desarrollo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Cohesin, a member of the SMC complex family, holds sister chromatids together but also shapes chromosomes by promoting the formation of long-range intra-chromatid loops, a process proposed to be mediated by DNA loop extrusion. Here we describe the roles of three cohesin partners, Pds5, Wpl1, and Eco1, in loop formation along either unreplicated or mitotic Saccharomyces cerevisiae chromosomes. Pds5 limits the size of DNA loops via two different pathways: the canonical Wpl1-mediated releasing activity and an Eco1-dependent mechanism. In the absence of Pds5, the main barrier to DNA loop expansion appears to be the centromere. Our data also show that Eco1 acetyl-transferase inhibits the translocase activity that powers loop formation and contributes to the positioning of loops through a mechanism that is distinguishable from its role in cohesion establishment. This study reveals that the mechanisms regulating cohesin-dependent chromatin loops are conserved among eukaryotes while promoting different functions.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/química , Proteínas Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferasas/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Cromosomas Fúngicos/genética , Cromosomas Fúngicos/metabolismo , Mitosis , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
During DNA replication, proliferating cell nuclear antigen (PCNA) adopts a ring-shaped structure to promote processive DNA synthesis, acting as a sliding clamp for polymerases. Known posttranslational modifications function at the outer surface of the PCNA ring to favor DNA damage bypass. Here, we demonstrate that acetylation of lysine residues at the inner surface of PCNA is induced by DNA lesions. We show that cohesin acetyltransferase Eco1 targets lysine 20 at the sliding surface of the PCNA ring in vitro and in vivo in response to DNA damage. Mimicking constitutive acetylation stimulates homologous recombination and robustly suppresses the DNA damage sensitivity of mutations in damage tolerance pathways. In comparison to the unmodified trimer, structural differences are observed at the interface between protomers in the crystal structure of the PCNA-K20ac ring. Thus, acetylation regulates PCNA sliding on DNA in the presence of DNA damage, favoring homologous recombination linked to sister-chromatid cohesion.
Asunto(s)
Acetiltransferasas/metabolismo , Cromátides , Cromosomas Fúngicos , Daño del ADN , Inestabilidad Genómica , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Procesamiento Proteico-Postraduccional , Reparación del ADN por Recombinación , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Acetilación , Acetiltransferasas/química , Acetiltransferasas/genética , ADN Polimerasa III/genética , ADN Polimerasa III/metabolismo , Genotipo , Humanos , Lisina , Modelos Moleculares , Mutación , Proteínas Nucleares/química , Proteínas Nucleares/genética , Fenotipo , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Conformación Proteica , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Relación Estructura-ActividadRESUMEN
Cohesin is a DNA-associated protein complex that forms a tripartite ring controlling sister chromatid cohesion, chromosome segregation and organization, DNA replication, and gene expression. Sister chromatid cohesion is established by the protein acetyltransferase Eco1, which acetylates two conserved lysine residues on the cohesin subunit Smc3 and thereby ensures correct chromatid separation in yeast (Saccharomyces cerevisiae) and other eukaryotes. However, the consequence of Eco1-catalyzed cohesin acetylation is unknown, and the exact nature of the cohesive state of chromatids remains controversial. Here, we show that self-interactions of the cohesin subunits Scc1/Rad21 and Scc3 occur in a DNA replication-coupled manner in both yeast and human cells. Using cross-linking MS-based and in vivo disulfide cross-linking analyses of purified cohesin, we show that a subpopulation of cohesin may exist as dimers. Importantly, upon temperature-sensitive and auxin-induced degron-mediated Eco1 depletion, the cohesin-cohesin interactions became significantly compromised, whereas deleting either the deacetylase Hos1 or the Eco1 antagonist Wpl1/Rad61 increased cohesin dimer levels by â¼20%. These results indicate that cohesin dimerizes in the S phase and monomerizes in mitosis, processes that are controlled by Eco1, Wpl1, and Hos1 in the sister chromatid cohesion-dissolution cycle. These findings suggest that cohesin dimerization is controlled by the cohesion cycle and support the notion that a double-ring cohesin model operates in sister chromatid cohesion.
Asunto(s)
Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas Fúngicos/metabolismo , Proteínas Nucleares/metabolismo , Multimerización de Proteína/fisiología , Fase S/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetiltransferasas/genética , Proteínas de Ciclo Celular/genética , Cromátides/genética , Proteínas Cromosómicas no Histona/genética , Cromosomas Fúngicos/genética , Histona Demetilasas/genética , Histona Demetilasas/metabolismo , Humanos , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Mistranslation occurs when an amino acid not specified by the standard genetic code is incorporated during translation. Since the ribosome does not read the amino acid, tRNA variants aminoacylated with a non-cognate amino acid or containing a non-cognate anticodon dramatically increase the frequency of mistranslation. In a systematic genetic analysis, we identified a suppression interaction between tRNASerUGG, G26A, which mistranslates proline codons by inserting serine, and eco1-1, a temperature sensitive allele of the gene encoding an acetyltransferase required for sister chromatid cohesion. The suppression was partial, with a tRNA that inserts alanine at proline codons and not apparent for a tRNA that inserts serine at arginine codons. Sequencing of the eco1-1 allele revealed a mutation that would convert the highly conserved serine 213 within ß7 of the GCN5-related N-acetyltransferase core to proline. Mutation of P213 in eco1-1 back to the wild-type serine restored the function of the enzyme at elevated temperatures. Our results indicate the utility of mistranslating tRNA variants to identify functionally relevant mutations and identify eco1 as a reporter for mistranslation. We propose that mistranslation could be used as a tool to treat genetic disease.
Asunto(s)
Acetiltransferasas/genética , Alelos , Mutación , Proteínas Nucleares/genética , Prolina/genética , ARN de Transferencia/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Serina/genéticaRESUMEN
BACKGROUND: Roberts syndrome (RBS) is a rare genetic disorder characterized by craniofacial abnormalities, limb malformation, and often severe mental retardation. RBS arises from mutations in ESCO2 that encodes an acetyltransferase and modifies the cohesin subunit SMC3. Mutations in SCC2/NIPBL (encodes a cohesin loader), SMC3 or other cohesin genes (SMC1, RAD21/MCD1) give rise to a related developmental malady termed Cornelia de Lange syndrome (CdLS). RBS and CdLS exhibit overlapping phenotypes, but RBS is thought to arise through mitotic failure and limited progenitor cell proliferation while CdLS arises through transcriptional dysregulation. Here, we use the zebrafish regenerating fin model to test the mechanism through which RBS-type phenotypes arise. RESULTS: esco2 is up-regulated during fin regeneration and specifically within the blastema. esco2 knockdown adversely affects both tissue and bone growth in regenerating fins-consistent with a role in skeletal morphogenesis. esco2-knockdown significantly diminishes cx43/gja1 expression which encodes the gap junction connexin subunit required for cell-cell communication. cx43 mutations cause the short fin (sof(b123) ) phenotype in zebrafish and oculodentodigital dysplasia (ODDD) in humans. Importantly, miR-133-dependent cx43 overexpression rescues esco2-dependent growth defects. CONCLUSIONS: These results conceptually link ODDD to cohesinopathies and provide evidence that ESCO2 may play a transcriptional role critical for human development.
Asunto(s)
Acetiltransferasas/genética , Aletas de Animales/fisiología , Huesos/fisiología , Conexina 43/genética , Regeneración/genética , Proteínas de Pez Cebra/genética , Pez Cebra/fisiología , Acetiltransferasas/metabolismo , Animales , Apoptosis/genética , Proliferación Celular/genética , Conexina 43/metabolismo , Regulación de la Expresión Génica , Regulación hacia Arriba , Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The proper transmission of DNA in dividing cells is crucial for the survival of eukaryotic organisms. During cell division, faithful segregation of replicated chromosomes requires their tight attachment, known as sister chromatid cohesion, until anaphase. Sister chromatid cohesion is established during S-phase in a process requiring an acetyltransferase that in yeast is known as Establishment of cohesion 1 (Eco1). Inactivation of Eco1 typically disrupts chromosome segregation and homologous recombination-dependent DNA repair in dividing cells, ultimately resulting in lethality. We report here the isolation and detailed characterization of two homozygous T-DNA insertion mutants for the Arabidopsis thaliana Eco1 homolog, CHROMOSOME TRANSMISSION FIDELITY 7/ESTABLISHMENT OF COHESION 1 (CTF7/ECO1), called ctf7-1 and ctf7-2. Mutants exhibited dwarfism, poor anther development and sterility. Analysis of somatic tissues by flow cytometry, scanning electron microscopy and quantitative real-time PCR identified defects in DNA repair and cell division, including an increase in the area of leaf epidermal cells, an increase in DNA content and the upregulation of genes involved in DNA repair including BRCA1 and PARP2. No significant change was observed in the expression of genes that influence entry into the endocycle. Analysis of meiocytes identified changes in chromosome morphology and defective segregation; the abundance of chromosomal-bound cohesion subunits was also reduced. Transcript levels for several meiotic genes, including the recombinase genes DMC1 and RAD51C and the S-phase licensing factor CDC45 were elevated in mutant anthers. Taken together our results demonstrate that Arabidopsis CTF7/ECO1 plays important roles in the preservation of genome integrity and meiosis.
Asunto(s)
Acetiltransferasas/genética , Proteínas de Arabidopsis/genética , Arabidopsis/genética , Cromátides/fisiología , Genoma de Planta , Meiosis , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica/genética , Reparación del ADN/genética , Meiosis/genética , Mitosis/genética , Mutagénesis Insercional , Polen/genética , Polen/crecimiento & desarrollo , CohesinasRESUMEN
Cohesins promote proper chromosome segregation, gene transcription, genomic architecture, DNA condensation, and DNA damage repair. Mutations in either cohesin subunits or regulatory genes can give rise to severe developmental abnormalities (such as Robert Syndrome and Cornelia de Lange Syndrome) and also are highly correlated with cancer. Despite this, little is known about cohesin regulation. Eco1 (ESCO2/EFO2 in humans) and Rad61 (WAPL in humans) represent two such regulators but perform opposing roles. Eco1 acetylation of cohesin during S phase, for instance, stabilizes cohesin-DNA binding to promote sister chromatid cohesion. On the other hand, Rad61 promotes the dissociation of cohesin from DNA. While Eco1 is essential, ECO1 and RAD61 co-deletion results in yeast cell viability, but only within a limited temperature range. Here, we report that eco1 rad61 cell lethality is due to reduced levels of the cohesin subunit Mcd1. Results from a suppressor screen further reveals that FDO1 deletion rescues the temperature sensitive (ts) growth defects exhibited by eco1 rad61 double mutant cells by increasing Mcd1 levels. Regulation of MCD1 expression, however, appears more complex. Elevated expression of MBP1, which encodes a subunit of the MBF transcription complex, also rescues eco1 rad61 cell growth defects. Elevated expression of SWI6, however, which encodes the Mbp1-binding partner of MBF, exacerbates eco1 rad61 cell growth and also abrogates the Mpb1-dependent rescue. Finally, we identify two additional transcription factors, Fkh1 and Fkh2, that impact MCD1 expression. In combination, these findings provide new insights into the nuanced and multi-faceted transcriptional pathways that impact MCD1 expression.
RESUMEN
Cohesin is a central architectural element of chromosomes that regulates numerous DNA-based events. The complex holds sister chromatids together until anaphase onset and organizes individual chromosomal DNAs into loops and self-associating domains. Purified cohesin diffuses along DNA in an ATP-independent manner but can be propelled by transcribing RNA polymerase. In conjunction with a cofactor, the complex also extrudes DNA loops in an ATP-dependent manner. In this study we examine transcription-driven translocation of cohesin under various conditions in yeast. To this end, obstacles of increasing size were tethered to DNA to act as roadblocks to complexes mobilized by an inducible gene. The obstacles were built from a GFP-lacI core fused to one or more mCherries. A chimera with four mCherries blocked cohesin passage in late G1. During M phase, the threshold barrier depended on the state of cohesion: non-cohesive complexes were also blocked by four mCherries whereas cohesive complexes were blocked by as few as three mCherries. Furthermore cohesive complexes that were stalled at obstacles, in turn, blocked the passage of non-cohesive complexes. That synthetic barriers capture mobilized cohesin demonstrates that transcription-driven complexes translocate processively in vivo. Together, this study reveals unexplored limitations to cohesin movement on chromosomes.
Asunto(s)
Proteínas Cromosómicas no Histona , Proteínas de Saccharomyces cerevisiae , Proteínas Cromosómicas no Histona/genética , Saccharomyces cerevisiae/genética , Proteínas de Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Cromátides , ADN , Adenosina Trifosfato , CohesinasRESUMEN
Roberts syndrome (RBS) is a multispectrum developmental disorder characterized by severe limb, craniofacial, and organ abnormalities and often intellectual disabilities. The genetic basis of RBS is rooted in loss-of-function mutations in the essential N-acetyltransferase ESCO2 which is conserved from yeast (Eco1/Ctf7) to humans. ESCO2/Eco1 regulate many cellular processes that impact chromatin structure, chromosome transmission, gene expression, and repair of the genome. The etiology of RBS remains contentious with current models that include transcriptional dysregulation or mitotic failure. Here, we report evidence that supports an emerging model rooted in defective DNA damage responses. First, the results reveal that redox stress is elevated in both eco1 and cohesion factor Saccharomyces cerevisiae mutant cells. Second, we provide evidence that Eco1 and cohesion factors are required for the repair of oxidative DNA damage such that ECO1 and cohesin gene mutations result in reduced cell viability and hyperactivation of DNA damage checkpoints that occur in response to oxidative stress. Moreover, we show that mutation of ECO1 is solely sufficient to induce endogenous redox stress and sensitizes mutant cells to exogenous genotoxic challenges. Remarkably, antioxidant treatment desensitizes eco1 mutant cells to a range of DNA damaging agents, raising the possibility that modulating the cellular redox state may represent an important avenue of treatment for RBS and tumors that bear ESCO2 mutations.
Asunto(s)
Ectromelia , Hipertelorismo , Proteínas de Saccharomyces cerevisiae , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromátides , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Anomalías Craneofaciales , Ectromelia/genética , Ectromelia/metabolismo , Ectromelia/patología , Humanos , Hipertelorismo/genética , Hipertelorismo/metabolismo , Hipertelorismo/patología , Proteínas Nucleares/genética , Oxidación-Reducción , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The intersection through which two fundamental processes meet provides a unique vantage point from which to view cellular regulation. On the one hand, DNA replication is at the heart of cell division, generating duplicate chromosomes that allow each daughter cell to inherit a complete copy of the parental genome. Among other factors, the PCNA (proliferating cell nuclear antigen) sliding clamp ensures processive DNA replication during S phase and is essential for cell viability. On the other hand, the process of chromosome segregation during M phase-an act that occurs long after DNA replication-is equally fundamental to a successful cell division. Eco1/Ctf7 ensures that chromosomes faithfully segregate during mitosis, but functions during DNA replication to activate cohesins and thereby establish cohesion between sister chromatids. To achieve this, Eco1 binds PCNA and numerous other DNA replication fork factors that include MCM helicase, Chl1 helicase, and the Rtt101-Mms1-Mms22 E3 ubiquitin ligase. Here, we review the multi-faceted coordination between cohesion establishment and DNA replication. SUMMARY STATEMENT: New findings provide important insights into the mechanisms through which DNA replication and the establishment of sister chromatid cohesion are coupled.
Asunto(s)
Cromátides , Proteínas de Saccharomyces cerevisiae , Acetiltransferasas/genética , Cromátides/genética , Segregación Cromosómica , Replicación del ADN/genética , Proteínas Nucleares/genética , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Eco1/Ctf7 is a highly conserved acetyltransferase that activates cohesin complexes and is critical for sister chromatid cohesion, chromosome condensation, DNA damage repair, nucleolar integrity, and gene transcription. Mutations in the human homolog of ECO1 (ESCO2/EFO2), or in genes that encode cohesin subunits, result in severe developmental abnormalities and intellectual disabilities referred to as Roberts syndrome and Cornelia de Lange syndrome, respectively. In yeast, deletion of ECO1 results in cell inviability. Codeletion of RAD61 (WAPL in humans), however, produces viable yeast cells. These eco1 rad61 double mutants, however, exhibit a severe temperature-sensitive growth defect, suggesting that Eco1 or cohesins respond to hyperthermic stress through a mechanism that occurs independent of Rad61. Here, we report that deletion of the G1 cyclin CLN2 rescues the temperature-sensitive lethality otherwise exhibited by eco1 rad61 mutant cells, such that the triple mutant cells exhibit robust growth over a broad range of temperatures. While Cln1, Cln2, and Cln3 are functionally redundant G1 cyclins, neither CLN1 nor CLN3 deletions rescue the temperature-sensitive growth defects otherwise exhibited by eco1 rad61 double mutants. We further provide evidence that CLN2 deletion rescues hyperthermic growth defects independent of START and impacts the state of chromosome condensation. These findings reveal novel roles for Cln2 that are unique among the G1 cyclin family and appear critical for cohesin regulation during hyperthermic stress.
Asunto(s)
Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Acetiltransferasas/genética , Proteínas de Ciclo Celular/genética , Cromátides , Proteínas Cromosómicas no Histona/genética , Segregación Cromosómica , Ciclinas/genética , Humanos , Linfocitos Nulos , Glicoproteínas de Membrana/genética , Chaperonas Moleculares/genética , Proteínas Nucleares/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
Double-strand breaks that are induced postreplication trigger establishment of damage-induced cohesion in Saccharomyces cerevisiae, locally at the break site and genome-wide on undamaged chromosomes. The translesion synthesis polymerase, polymerase η, is required for generation of damage-induced cohesion genome-wide. However, its precise role and regulation in this process is unclear. Here, we investigated the possibility that the cyclin-dependent kinase Cdc28 and the acetyltransferase Eco1 modulate polymerase η activity. Through in vitro phosphorylation and structure modeling, we showed that polymerase η is an attractive substrate for Cdc28 Mutation of the putative Cdc28-phosphorylation site Ser14 to Ala not only affected polymerase η protein level, but also prevented generation of damage-induced cohesion in vivo We also demonstrated that Eco1 acetylated polymerase η in vitro Certain nonacetylatable polymerase η mutants showed reduced protein level, deficient nuclear accumulation, and increased ultraviolet irradiation sensitivity. In addition, we found that both Eco1 and subunits of the cohesin network are required for cell survival after ultraviolet irradiation. Our findings support functionally important Cdc28-mediated phosphorylation, as well as post-translational modifications of multiple lysine residues that modulate polymerase η activity, and provide new insights into understanding the regulation of polymerase η for damage-induced cohesion.
Asunto(s)
Reparación del ADN , ADN Polimerasa Dirigida por ADN/metabolismo , Procesamiento Proteico-Postraduccional , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/genética , Proteína Quinasa CDC28 de Saccharomyces cerevisiae/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Cellular genomes undergo various structural changes that include cis tethering (the tethering together of two loci within a single DNA molecule), which promotes chromosome condensation and transcriptional activation, and trans tethering (the tethering together of two DNA molecules), which promotes sister chromatid cohesion and DNA repair. The protein complex termed cohesin promotes both cis and trans forms of DNA tethering, but the extent to which these cohesin functions occur in temporally or spatially defined contexts remains largely unknown. Prior studies indicate that DNA polymerase sliding clamp PCNA recruits cohesin acetyltransferase Eco1, suggesting that sister chromatid cohesion is established in the context of the DNA replication fork. In support of this model, elevated levels of PCNA rescue the temperature growth and cohesion defects exhibited by eco1 mutant cells. Here, we test whether Eco1-dependent chromatin condensation is also promoted in the context of this DNA replication fork component. Our results reveal that overexpressed PCNA does not promote DNA condensation in eco1 mutant cells, even though Smc3 acetylation levels are increased. We further provide evidence that replication fork-associated E3 ligase impacts on Eco1 are more complex that previously described. In combination, the data suggests that Eco1 acetylates Smc3 and thus promotes sister chromatid cohesion in context of the DNA replication fork, whereas a distinct cohesin population participates in chromatin condensation outside the context of the DNA replication fork.
Asunto(s)
Cromátides , Ensamble y Desensamble de Cromatina , Cromosomas Fúngicos , Replicación del ADN , ADN de Hongos/biosíntesis , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Acetilación , Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN de Hongos/genética , Mutación , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , CohesinasRESUMEN
Robust progression through the cell-division cycle depends on the precisely ordered phosphorylation of hundreds of different proteins by cyclin-dependent kinases (CDKs) and other kinases. The order of CDK substrate phosphorylation depends on rising CDK activity, coupled with variations in substrate affinities for different CDK-cyclin complexes and the opposing phosphatases [1-4]. Here, we address the ordering of substrate phosphorylation by a second major cell-cycle kinase, Cdc7-Dbf4 or Dbf4-dependent kinase (DDK). The primary function of DDK is to initiate DNA replication by phosphorylating the Mcm2-7 replicative helicase [5-7]. DDK also phosphorylates the cohesin acetyltransferase Eco1 [8]. Sequential phosphorylations of Eco1 by CDK, DDK, and Mck1 create a phosphodegron that is recognized by the ubiquitin ligase SCFCdc4. DDK, despite being activated in early S phase, does not phosphorylate Eco1 to trigger its degradation until late S phase [8]. DDK associates with docking sites on loaded Mcm double hexamers at unfired replication origins [9, 10]. We hypothesized that these docking interactions sequester limiting amounts of DDK, delaying Eco1 phosphorylation by DDK until replication is complete. Consistent with this hypothesis, we find that overproduction of DDK leads to premature Eco1 degradation. Eco1 degradation also occurs prematurely if Mcm complex loading at origins is prevented by depletion of Cdc6, and Eco1 is stabilized if loaded Mcm complexes are prevented from firing by a Cdc45 mutant. We propose that the timing of Eco1 phosphorylation, and potentially that of other DDK substrates, is determined in part by sequestration of DDK at unfired replication origins during S phase.
Asunto(s)
Acetiltransferasas/genética , Proteínas de Ciclo Celular/genética , Proteínas Nucleares/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Acetiltransferasas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Replicación del ADN , Proteínas Nucleares/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteolisis , Origen de Réplica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
Multicellular organisms such as higher plants require timely regulation of DNA replication and cell division to grow and develop. Recent work in Arabidopsis has shown that chromosome segregation during meiosis and mitosis depends on the activity of several genes that in yeast are involved in the establishment of chromosomal cohesion. In this process, proteins of the structural maintenance of chromosomes (SMC) family tether chromosomes and establish inter- and intrachromosomal connections. In Arabidopsis, recruitment of SMC proteins and establishment of cohesion during key stages of the cell cycle depend on the activity of chromosome transmission fidelity 7/establishment of cohesion 1 (CTF7/ECO1). Here we show that loss of CTF7/ECO1 activity alters the status of cytosine methylation in both intergenic regions and transposon loci. An increase in expression was also observed for transposon copia28, which suggests a link between CTF7/ECO1 activity, DNA methylation and gene silencing. More work is needed to determine the mechanistic relationships that intervene in this process.