RESUMEN
The anaphase-promoting complex/cyclosome (APC/C), a multi-subunit ubiquitin ligase essential for cell cycle control, is regulated by reversible phosphorylation. APC/C phosphorylation by cyclin-dependent kinase 1 (Cdk1) promotes Cdc20 co-activator loading in mitosis to form active APC/C-Cdc20. However, detailed phospho-regulation of APC/C dynamics through other kinases and phosphatases is still poorly understood. Here, we show that an interplay between polo-like kinase (Plx1) and PP2A-B56 phosphatase on a flexible loop domain of the subunit Apc1 (Apc1-loop500 ) controls APC/C activity and mitotic progression. Plx1 directly binds to the Apc1-loop500 in a phosphorylation-dependent manner and promotes the formation of APC/C-Cdc20 via Apc3 phosphorylation. Upon phosphorylation of loop residue T532, PP2A-B56 is recruited to the Apc1-loop500 and differentially promotes dissociation of Plx1 and PP2A-B56 through dephosphorylation of Plx1-binding sites. Stable Plx1 binding, which prevents PP2A-B56 recruitment, prematurely activates the APC/C and delays APC/C dephosphorylation during mitotic exit. Furthermore, the phosphorylation status of the Apc1-loop500 is controlled by distant Apc3-loop phosphorylation. Our study suggests that phosphorylation-dependent feedback regulation through flexible loop domains within a macromolecular complex coordinates the activity and dynamics of the APC/C during the cell cycle.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Mitosis/fisiología , Proteína Fosfatasa 2/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transducción de Señal , Humanos , Fosforilación , Unión Proteica , Quinasa Tipo Polo 1RESUMEN
Cell cycle progression and genome stability are regulated by a ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C). Cyclin-dependent kinase 1 (Cdk1) has long been implicated in APC/C activation; however, the molecular mechanisms of governing this process in vivo are largely unknown. Recently, a Cdk1-dependent phosphorylation relay within Apc3-Apc1 subunits has been shown to alleviate Apc1-mediated auto-inhibition by which a mitotic APC/C co-activator Cdc20 binds to and activates the APC/C. However, the underlying mechanism for dephosphorylation of Cdc20 and APC/C remains elusive. Here, we show that a disordered loop domain of Apc1 (Apc1-loop500 ) directly binds the B56 regulatory subunit of protein phosphatase 2A (PP2A) and stimulates Cdc20 loading to the APC/C. Using the APC/C reconstitution system in Xenopus egg extracts, we demonstrate that mutations in Apc1-loop500 that abolish B56 binding decrease Cdc20 loading and APC/C-dependent ubiquitylation. Conversely, a non-phosphorylatable mutant Cdc20 can efficiently bind the APC/C even when PP2A-B56 binding is impeded. Furthermore, PP2A-B56 preferentially dephosphorylates Cdc20 over the Apc1 inhibitory domain. These results indicate that Apc1-loop500 plays a role in dephosphorylating Cdc20, promoting APC/C-Cdc20 complex formation in mitosis.
Asunto(s)
Ciclosoma-Complejo Promotor de la Anafase , Proteínas Cdc20 , Mitosis , Proteína Fosfatasa 2 , Ubiquitina , Ciclosoma-Complejo Promotor de la Anafase/genética , Animales , Proteínas Cdc20/genética , Proteínas de Ciclo Celular/genética , Mitosis/genética , Proteína Fosfatasa 2/genética , XenopusRESUMEN
In Escherichia coli, the initiator protein ATP-DnaA promotes initiation of chromosome replication in a timely manner. After initiation, DnaA-bound ATP is hydrolyzed to yield ADP-DnaA, which is inactive in initiation. DnaA-reactivating sequences (DARS1 and DARS2) on the chromosome have predominant roles in catalysis of nucleotide exchange, producing ATP-DnaA from ADP-DnaA, which is prerequisite for timely initiation. Both DARS sequences have a core region containing a cluster of three DnaA-binding sites. DARS2 is more effective in vivo than DARS1, and timely activation of DARS2 depends on binding of two nucleoid-associated proteins, IHF and Fis. DARS2 is located centrally between the chromosomal replication origin oriC and the terminus region terC. We constructed mutants in which DARS2 was translocated to several chromosomal loci, including sites proximal to oriC and to terC. Replication initiation was inhibited in cells in which DARS2 was translocated to terC-proximal sites when the cells were grown at 42 °C, although overall binding efficiency of IHF and Fis to the translocated DARS2 was not affected. Inhibition was largely sustained even in cells lacking MatP, a DNA-binding protein responsible for terC-specific subchromosomal structure. These results suggest that functional regulation of DARS2 is correlated with its chromosomal location under certain conditions.
Asunto(s)
Proteínas Bacterianas/genética , Replicación del ADN/genética , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Mapeo Cromosómico , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Origen de RéplicaRESUMEN
In Escherichia coli, ATP-DnaA, unlike ADP-DnaA, can initiate chromosomal replication at oriC. The level of cellular ATP-DnaA fluctuates, peaking at around the time of replication initiation. However, it remains unknown how the ATP-DnaA level increases coordinately with the replication cycle. In this study, we show that two chromosomal intergenic regions, herein termed DnaA-reactivating sequence 1 (DARS1) and DnaA-reactivating sequence 2 (DARS2), directly promote regeneration of ATP-DnaA from ADP-DnaA by nucleotide exchange, resulting in the promotion of replication initiation in vitro and in vivo. Coordination of initiation with the cell cycle requires DARS activity and its regulation. Oversupply of DARSs results in increase in the ATP-DnaA level and enhancement of replication initiation, which can inhibit cell growth in an oriC-dependent manner. Deletion of DARSs results in decrease in the ATP-DnaA level and inhibition of replication initiation, which can cause synthetic lethality with a temperature-sensitive mutant dnaA and suppression of overinitiation by the lack of seqA or datA, negative regulators for initiation. DARSs bear a cluster of DnaA-binding sites. DnaA molecules form specific homomultimers on DARS1, which causes specific interactions among the protomers, reducing their affinity for ADP. Our findings reveal a novel regulatory pathway that promotes the initiation of chromosomal replication via DnaA reactivation.
Asunto(s)
Adenosina Difosfato/metabolismo , Proteínas Bacterianas/metabolismo , ADN Intergénico/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genoma Bacteriano/fisiología , Adenosina Trifosfatasas/metabolismo , ADN Helicasas/metabolismo , Enzimas Reparadoras del ADN/metabolismo , Replicación del ADN/genética , ADN Intergénico/genética , Proteínas de Drosophila/metabolismo , Endodesoxirribonucleasas/metabolismo , Proteínas de Escherichia coli/metabolismo , Genoma Bacteriano/genética , Nucleótidos/genética , Estabilidad Proteica , Estructura Terciaria de Proteína , Eliminación de SecuenciaRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) ubiquitin ligase is tightly regulated to ensure programmed proteolysis in cells. The activity of the APC/C is positively controlled by cyclin-dependent kinase (CDK), but a second level of control must also exist because phosphorylation inactivates Cdc20, a mitotic APC/C co-activator. How Cdc20 is dephosphorylated specifically, when CDK is high, has remained unexplained. Here, we show that phosphatases are crucial to activate the APC/C. Cdc20 is phosphorylated at six conserved residues (S50/T64/T68/T79/S114/S165) by CDK in Xenopus egg extracts. When all the threonine residues are phosphorylated, Cdc20 binding to and activation of the APC/C are inhibited. Their dephosphorylation is regulated depending on the sites and protein phosphatase 2A, active in mitosis, is essential to dephosphorylate the threonine residues and activate the APC/C. Consistently, most of the Cdc20 bound to the APC/C in anaphase evades phosphorylation at T79. Furthermore, we show that the 'activation domain' of Cdc20 associates with the Apc6 and Apc8 core subunits. Our data suggest that dephosphorylation of Cdc20 is required for its loading and activation of the APC/C ubiquitin ligase.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Fosfoproteínas Fosfatasas/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Proteínas de Xenopus/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Animales , Proteínas Cdc20 , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/fisiología , Células Cultivadas , Activación Enzimática , Femenino , Ratones , Ratones Endogámicos BALB C , Mitosis/genética , Mitosis/fisiología , Modelos Biológicos , Fosfoproteínas Fosfatasas/fisiología , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional/genética , Estructura Terciaria de Proteína/fisiología , Spodoptera , Complejos de Ubiquitina-Proteína Ligasa/química , Proteínas de Xenopus/química , Proteínas de Xenopus/fisiología , Xenopus laevisRESUMEN
In Escherichia coli, the ATP-bound form of DnaA (ATP-DnaA) promotes replication initiation. During replication, the bound ATP is hydrolyzed to ADP to yield the ADP-bound form (ADP-DnaA), which is inactive for initiation. The chromosomal site DARS2 facilitates the regeneration of ATP-DnaA by catalyzing nucleotide exchange between free ATP and ADP bound to DnaA. However, the regulatory mechanisms governing this exchange reaction are unclear. Here, using in vitro reconstituted experiments, we show that two nucleoid-associated proteins, IHF and Fis, bind site-specifically to DARS2 to activate coordinately the exchange reaction. The regenerated ATP-DnaA was fully active in replication initiation and underwent DnaA-ATP hydrolysis. ADP-DnaA formed heteromultimeric complexes with IHF and Fis on DARS2, and underwent nucleotide dissociation more efficiently than ATP-DnaA. Consistently, mutant analyses demonstrated that specific binding of IHF and Fis to DARS2 stimulates the formation of ATP-DnaA production, thereby promoting timely initiation. Moreover, we show that IHF-DARS2 binding is temporally regulated during the cell cycle, whereas Fis only binds to DARS2 in exponentially growing cells. These results elucidate the regulation of ATP-DnaA and replication initiation in coordination with the cell cycle and growth phase.
Asunto(s)
Proteínas Bacterianas/metabolismo , Cromosomas Bacterianos/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Factor Proteico para Inverción de Estimulación/metabolismo , Factores de Integración del Huésped/metabolismo , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Secuencia de Bases , Sitios de Unión , Ciclo Celular , Cromosomas Bacterianos/química , Secuencia Conservada , Escherichia coli/crecimiento & desarrollo , Escherichia coli/metabolismoRESUMEN
Histone transcription and deposition are tightly regulated with the DNA replication cycle to maintain genetic integrity. Ams2 is a GATA-containing transcription factor responsible for core histone gene expression and for CENP-A loading at centromeres in fission yeast. Ams2 levels are cell cycle-regulated, and after the S phase Ams2 is degraded by the SCF(pof3) ubiquitin ligase; however, the regulation of Ams2 in G(1) or meiosis is poorly understood. Here we show that another ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C) targets Ams2 for destruction in G(1). Ubiquitylation and destruction of Ams2 is dependent upon a coactivator Cdh1/Ste9 and the KEN box in the C terminus of Ams2. We also find that stabilization of Ams2 sensitizes cells to the anti-microtubule drug thiabendazole and the histone deacetylase inhibitor tricostatin A when a histone deacetylase gene hst4 is deleted, suggesting that histone acetylation together with Ams2 stability ensures the coupling of mitosis to DNA replication. Furthermore, in meiosis, the failure of the APC/C-mediated destruction of Ams2 is deleterious, and pre-meiotic DNA replication is barely completed. These data suggest that Ams2 destruction via both the APC/C and the SCF ubiquitin ligases underlies the coordination of histone expression and DNA replication.
Asunto(s)
Replicación del ADN , ADN de Hongos/biosíntesis , Fase G1 , Factores de Transcripción GATA/metabolismo , Regulación Fúngica de la Expresión Génica/fisiología , Histonas/genética , Proteolisis , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Complejos de Ubiquitina-Proteína Ligasa/fisiología , Acetilación , Ciclosoma-Complejo Promotor de la Anafase , ADN de Hongos/genética , Histonas/metabolismo , Meiosis , Schizosaccharomyces/citología , Transcripción Genética , UbiquitinaciónRESUMEN
The anaphase-promoting complex/cyclosome (APC/C) is a critical and tightly regulated E3 ligase that orchestrates the cellular life cycle by controlling the degradation of cell cycle regulators. An intriguing feature of this complex is an autoinhibition mechanism: an intrinsically disordered loop domain, Apc1-300L, blocks Cdc20 coactivator binding, yet phosphorylation of Apc1-300L counteracts this autoinhibition. Many such disordered loops within APC/C remain unexplored. Our systematic analysis of loop-deficient APC/C mutants uncovered a pivotal role for Apc8's C-terminal loop (Apc8-L) in mitotic activation. Apc8-L directly recruits the CDK adaptor protein, Xe-p9/Cks2, positioning the Xe-p9-CDK-CycB complex near Apc1-300L. This stimulates the phosphorylation and removal of Apc1-300L, prompting the formation of active APC/CCdc20. Strikingly, without both Apc8-L and Apc3-L, the APC/C is rendered inactive during mitosis, highlighting Apc8-L's synergistic role with other loops and kinases. This study broadens our understanding of the intricate dynamics in APC/C regulation and provides insights on the regulation of macromolecular complexes.
Asunto(s)
Mitosis , Animales , Femenino , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc8 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdc20/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Fosforilación , Unión Proteica , Dominios Proteicos , Xenopus laevisRESUMEN
Cell cycle control relies on a delicate balance of phosphorylation with CDK1 and phosphatases like PP1 and PP2A-B55. Yet, identifying the primary substrate responsible for cell cycle oscillations remains a challenge. We uncover the pivotal role of phospho-regulation in the anaphase-promoting complex/cyclosome (APC/C), particularly through the Apc1-loop300 domain (Apc1-300L), orchestrated by CDK1 and PP2A-B55. Premature activation of PP2A-B55 during mitosis, induced by Greatwall kinase depletion, leads to Apc1-300L dephosphorylation, stalling APC/C activity and delaying Cyclin B degradation. This effect can be counteracted using the B55-specific inhibitor pEnsa or by removing Apc1-300L. We also show Cdc20's dynamic APC/C interaction across cell cycle stages, but dephosphorylation of Apc1-300L specifically inhibits further Cdc20 recruitment. Our study underscores APC/C's central role in cell cycle oscillation, identifying it as a primary substrate regulated by the CDK-PP2A partnership.
Asunto(s)
Proteína Quinasa CDC2 , Ciclo Celular , Proteína Fosfatasa 2 , Animales , Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteína Quinasa CDC2/metabolismo , Proteínas Cdc20/metabolismo , Mitosis , Fosforilación , Proteína Fosfatasa 2/metabolismo , Células Sf9 , XenopusRESUMEN
Chromosome replication in Escherichia coli is limited by the supply of DnaA associated with ATP. Cells deficient in RIDA (Regulatory Inactivation of DnaA) due to a deletion of the hda gene accumulate suppressor mutations (hsm) to counteract the overinitiation caused by an elevated DnaA(ATP) level. Eight spontaneous hda suppressor mutations were identified by whole-genome sequencing, and three of these were analysed further. Two mutations (hsm-2 and hsm-4) mapped in the dnaA gene and led to a reduced ability to initiate replication from oriC. One mutation (hsm-1) mapped to the seqA promoter and increased the SeqA protein level in the cell. hsm-1 cells had prolonged origin sequestration, reduced DnaA protein level and reduced DnaA-Reactivating Sequence (DARS)-mediated rejuvenation of DnaA(ADP) to DnaA(ATP) , all of which could contribute to the suppression of RIDA deficiency. Despite of these defects hsm-1 cells were quite similar to wild type with respect to cell cycle parameters. We speculate that since SeqA binding sites might overlap with DnaA binding sites spread throughout the chromosome, excess SeqA could interfere with DnaA titration and thereby increase free DnaA level. Thus, in spite of reduction in total DnaA, the amount of DnaA molecules available for initiation may not be reduced.
Asunto(s)
Proteínas Bacterianas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Supresión Genética , Adenosina Difosfato/análisis , Adenosina Trifosfato/análisis , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Cromosomas Bacterianos , ADN Bacteriano/genética , Proteínas de Unión al ADN/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , MutaciónRESUMEN
Initiation of chromosome replication in Escherichia coli is limited by the initiator protein DnaA associated with ATP. Within the replication origin, binding sites for DnaA associated with ATP or ADP (R boxes) and the DnaA(ATP) specific sites (I-boxes, tau-boxes and 6-mer sites) are found. We analysed chromosome replication of cells carrying mutations in conserved regions of oriC. Cells carrying mutations in DnaA-boxes I2, I3, R2, R3 and R5 as well as FIS and IHF binding sites resembled wild-type cells with respect to origin concentration. Initiation of replication in these mutants occurred in synchrony or with slight asynchrony only. Furthermore, lack of Hda stimulated initiation in all these mutants. The DnaA(ATP) containing complex that leads to initiation can therefore be formed in the absence of several of the origin DnaA binding sites including both DnaA(ATP) specific I-boxes. However, competition between I-box mutant and wild-type origins, revealed a positive role of I-boxes on initiation. On the other hand, mutations affecting DnaA-box R4 were found to be compromised for initiation and could not be augmented by an increase in cellular DnaA(ATP)/DnaA(ADP) ratio. Compared with the sites tested here, R4 therefore seems to contribute to initiation most critically.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Cromosomas Bacterianos , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Mutagénesis Sitio-Dirigida , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Análisis de Secuencia de ADNRESUMEN
The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the beta clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25 degrees C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25 degrees C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42 degrees C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25 degrees C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25 degrees C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Proteínas Bacterianas/metabolismo , División Celular , Replicación del ADN , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/citología , Adenosina Trifosfatasas/genética , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Frío , Citosol/química , Proteínas de Unión al ADN/genética , Nucleótidos de Desoxiadenina/análisis , Nucleótidos de Desoxicitosina/análisis , AdnB Helicasas/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrollo , Proteínas de Escherichia coli/genética , Dosificación de Gen , Perfilación de la Expresión Génica , Calor , Mutagénesis Insercional , Mutación , Ribonucleósido Difosfato Reductasa/genética , Ribonucleótido Reductasas/genéticaRESUMEN
Error-free genome duplication and segregation are ensured through the timely activation of ubiquitylation enzymes. The anaphase-promoting complex or cyclosome (APC/C), a multisubunit E3 ubiquitin ligase, is regulated by phosphorylation. However, the mechanism remains elusive. Using systematic reconstitution and analysis of vertebrate APC/Cs under physiological conditions, we show how cyclin-dependent kinase 1 (CDK1) activates the APC/C through coordinated phosphorylation between Apc3 and Apc1. Phosphorylation of the loop domains by CDK1 in complex with p9/Cks2 (a CDK regulatory subunit) controlled loading of coactivator Cdc20 onto APC/C. A phosphomimetic mutation introduced into Apc1 allowed Cdc20 to increase APC/C activity in interphase. These results define a previously unrecognized subunit-subunit communication over a distance and the functional consequences of CDK phosphorylation. Cdc20 is a potential therapeutic target, and our findings may facilitate the development of specific inhibitors.
Asunto(s)
Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase/metabolismo , Proteínas Cdh1/metabolismo , Anafase , Animales , Subunidad Apc1 del Ciclosoma-Complejo Promotor de la Anafase/genética , Subunidad Apc3 del Ciclosoma-Complejo Promotor de la Anafase/genética , Proteínas Cdc20/metabolismo , Activación Enzimática , Humanos , Mutación , Fosforilación , XenopusRESUMEN
A multi-subunit ubiquitin ligase, the anaphase-promoting complex/cyclosome (APC/C), regulates critical cellular processes including the cell cycle. To accomplish its diverse functions, APC/C activity must be precisely regulated in time and space. The interphase APC/C activator Fizzy-related (Fzr or Cdh1) is localized at centrosomes in animal cells. However, neither the mechanism of its localization nor its importance is clear. Here we identify the centrosome component Spd2 as a major partner of Fzr in Drosophila. The localization of Fzr to the centriole during interphase depends on direct interaction with Spd2. By generating Spd2 mutants unable to bind Fzr, we show that centrosomal localization of Fzr is essential for optimal APC/C activation towards its centrosomal substrate Aurora A. Finally, we show that Spd2 is also a novel APC/C(Fzr) substrate. Our study is the first to demonstrate the critical importance of distinct subcellular pools of APC/C activators in the spatiotemporal control of APC/C activity.
Asunto(s)
Proteínas Cdh1/metabolismo , Centrosoma/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/fisiología , Animales , Aurora Quinasa A/metabolismo , Proteínas Cdh1/genética , Proteínas de Drosophila/genética , Femenino , Interfase/fisiología , Mitosis/fisiología , Mutación , Unión Proteica/genética , Factores de TiempoRESUMEN
The Anaphase-promoting complex/cyclosome (APC/C) cofactor Cdh1 modulates cell proliferation by targeting multiple cell-cycle regulators for ubiquitin-dependent degradation. Lack of Cdh1 results in structural and numerical chromosome aberrations, a hallmark of genomic instability. By using a proteomic approach in Cdh1-null cells and mouse tissues, we have identified kinesin Eg5 and topoisomerase 2α as Cdh1 targets involved in the maintenance of genomic stability. These proteins are ubiquitinated and degraded through specific KEN and D boxes in a Cdh1-dependent manner. Whereas Cdh1-null cells display partial resistance to Eg5 inhibitors such as monastrol, lack of Cdh1 results in a dramatic sensitivity to Top2α poisons as a consequence of increased levels of trapped Top2α-DNA complexes. Chemical inhibition of the APC/C in cancer cells results in increased sensitivity to Top2α poisons. This work identifies in vivo targets of the mammalian APC/C-Cdh1 complex and reveals synthetic lethal interactions of relevance in anticancer treatments.
Asunto(s)
Proteínas Cdh1/metabolismo , Proteoma/metabolismo , Pirimidinas/farmacología , Tionas/farmacología , Inhibidores de Topoisomerasa II/farmacología , Animales , Antígenos de Neoplasias/química , Antígenos de Neoplasias/metabolismo , Sitios de Unión , Proteínas Cdh1/genética , ADN-Topoisomerasas de Tipo II/química , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas de Unión al ADN/antagonistas & inhibidores , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Inestabilidad Genómica , Células HEK293 , Células HeLa , Humanos , Cinesinas/química , Cinesinas/metabolismo , Ratones , Unión Proteica , Ubiquitinación , XenopusRESUMEN
Chromosomal replication must be limited to once and only once per cell cycle. This is accomplished by multiple regulatory pathways that govern initiator proteins and replication origins. A principal feature of DNA replication is the coupling of the replication reaction to negative-feedback regulation. Some of the factors that are important in this process have been discovered, including the clamp (DNA polymerase III subunit-beta (DnaN)), the datA locus, SeqA, DnaA homologue protein (Hda) and YabA, as well as factors that are involved at other stages of the regulatory mechanism, such as DnaA initiator-associating protein (DiaA), the DnaA-reactivating sequence (DARS) loci and Soj. Here, we describe the regulation of DnaA, one of the central proteins involved in bacterial DNA replication, by these factors in Escherichia coli, Bacillus subtilis and Caulobacter crescentus.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/fisiología , Caulobacter crescentus/fisiología , Replicación del ADN , Proteínas de Unión al ADN/fisiología , Escherichia coli/fisiología , Animales , Eucariontes/fisiología , Retroalimentación FisiológicaRESUMEN
Escherichia coli DiaA is a DnaA-binding protein that is required for the timely initiation of chromosomal replication during the cell cycle. In this study, we determined the crystal structure of DiaA at 1.8 A resolution. DiaA forms a homotetramer consisting of a symmetrical pair of homodimers. Mutational analysis revealed that the DnaA-binding activity and formation of homotetramers are required for the stimulation of initiation by DiaA. DiaA tetramers can bind multiple DnaA molecules simultaneously. DiaA stimulated the assembly of multiple DnaA molecules on oriC, conformational changes in ATP-DnaA-specific initiation complexes, and unwinding of oriC duplex DNA. The mutant DiaA proteins are defective in these stimulations. DiaA associated also with ADP-DnaA, and stimulated the assembly of inactive ADP-DnaA-oriC complexes. Specific residues in the putative phosphosugar-binding motif of DiaA were required for the stimulation of initiation and formation of ATP-DnaA-specific-oriC complexes. Our data indicate that DiaA regulates initiation by a novel mechanism, in which DiaA tetramers most likely bind to multiple DnaA molecules and stimulate the assembly of specific ATP-DnaA-oriC complexes. These results suggest an essential role for DiaA in the promotion of replication initiation in a cell cycle coordinated manner.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Secuencia de Bases , Proteínas Portadoras/química , Proteínas Portadoras/genética , Ciclo Celular , Replicación del ADN , ADN Bacteriano/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/citología , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Sustancias Macromoleculares , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Mutación , Complejo de Reconocimiento del Origen/genética , Complejo de Reconocimiento del Origen/metabolismo , Unión Proteica , Estructura Cuaternaria de Proteína , Homología de Secuencia de Aminoácido , Factores Generales de Transcripción/química , Factores Generales de Transcripción/genética , Factores Generales de Transcripción/metabolismoRESUMEN
In Escherichia coli, ATP-DnaA, but not ADP-DnaA, forms an initiation complex that undergoes site-specific duplex DNA unwinding, open complex formation. However, it remains unclear how highly the ATP-dependent activation of the initiation factor is conserved in evolution. The hyperthermophile Thermotoga maritima is one of the most ancient eubacteria in evolution. Here, we show that the DnaA homolog (tmaDnaA) of this bacterium forms open complexes with the predicted origin region (tma-oriC) in vitro. TmaDnaA has a strong and specific affinity for ATP/ADP as well as for 12-mer repeating sequences within the tma-oriC. Unlike ADP-tmaDnaA, ATP-tmaDnaA is highly cooperative in DNA binding and forms open complexes in a manner that depends on temperature and the superhelical tension of the tma-oriC-bearing plasmid. The minimal tma-oriC required for unwinding is a 149-bp region containing five repeats of the 12-mer sequence and two AT-rich 9-mer repeats. TmaDnaA-binding to the 12-mer motif provokes DNA bending. The 9-mer region is the duplex-unwinding site. The tmaDnaA-binding and unwinding motifs of tma-oriC share sequence homology with corresponding archaeal and eukaryotic sequences. These findings suggest that the ATP-dependent molecular switch of the initiator and the mechanisms in the replication initiation complex are highly conserved in eubacterial evolution.
Asunto(s)
Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Origen de Réplica/fisiología , Thermotoga maritima/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Secuencia Conservada , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/aislamiento & purificación , Escherichia coli/genética , Escherichia coli/metabolismo , Técnicas In Vitro , Sustancias Macromoleculares/metabolismo , Datos de Secuencia Molecular , Temperatura , Thermotoga maritima/genéticaRESUMEN
Escherichia coli DnaA, an AAA+ superfamily protein, initiates chromosomal replication in an ATP-binding-dependent manner. Although DnaA has conserved Walker A/B motifs, it binds adenine nucleotides 10- to 100-fold more tightly than do many other AAA+ proteins. This study shows that the DnaA Asp-269 residue, located in the sensor 1 motif, plays a specific role in supporting high-affinity ATP/ADP binding. The affinity of the DnaA D269A mutant for ATP/ADP is at least 10- to 100-fold reduced compared with that of the wild-type and DnaA R270A proteins. In contrast, the abilities of DnaA D269A to bind a typical DnaA box, unwind oriC duplex in the presence of elevated concentrations of ATP, load DnaB onto DNA and support minichromosomal replication in a reconstituted system are retained. Whereas the acidic Asp residue is highly conserved among eubacterial DnaA homologues, the corresponding residue in many other AAA+ proteins is Asn/Thr and in some AAA+ proteins these neutral residues are essential for ATP hydrolysis but not ATP binding. As the intrinsic ATPase activity of DnaA is extremely weak, this study reveals a novel and specific function for the sensor 1 motif in tight ATP/ADP binding, one that depends on the alternate key residue Asp.
Asunto(s)
Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Ácido Aspártico/química , Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Secuencias de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/crecimiento & desarrolloRESUMEN
The minimization of a genome is necessary to identify experimentally the minimal gene set that contains only those genes that are essential and sufficient to sustain a functioning cell. Recent developments in genetic techniques have made it possible to generate bacteria with a markedly reduced genome. We developed a simple system for formation of markerless chromosomal deletions, and constructed and characterized a series of large-scale chromosomal deletion mutants of Escherichia coli that lack between 2.4 and 29.7% of the parental chromosome. Combining deletion mutations changes cell length and width, and the mutant cells with larger deletions were even longer and wider than the parental cells. The nucleoid organization of the mutants is also changed: the nucleoids occur as multiple small nucleoids and are localized peripherally near the envelope. Inhibition of translation causes them to condense into one or two packed nucleoids, suggesting that the coupling of transcription and translation of membrane proteins peripherally localizes chromosomes. Because these phenotypes are similar to those of spherical cells, those may be a consequence of the morphological change. Based on the nucleoid localization observed with these mutants, we discuss the cellular nucleoid dynamics.