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1.
Mol Cell ; 81(3): 426-441.e8, 2021 02 04.
Artículo en Inglés | MEDLINE | ID: mdl-33545059

RESUMEN

Eukaryotic genomes replicate via spatially and temporally regulated origin firing. Cyclin-dependent kinase (CDK) and Dbf4-dependent kinase (DDK) promote origin firing, whereas the S phase checkpoint limits firing to prevent nucleotide and RPA exhaustion. We used chemical genetics to interrogate human DDK with maximum precision, dissect its relationship with the S phase checkpoint, and identify DDK substrates. We show that DDK inhibition (DDKi) leads to graded suppression of origin firing and fork arrest. S phase checkpoint inhibition rescued origin firing in DDKi cells and DDK-depleted Xenopus egg extracts. DDKi also impairs RPA loading, nascent-strand protection, and fork restart. Via quantitative phosphoproteomics, we identify the BRCA1-associated (BRCA1-A) complex subunit MERIT40 and the cohesin accessory subunit PDS5B as DDK effectors in fork protection and restart. Phosphorylation neutralizes autoinhibition mediated by intrinsically disordered regions in both substrates. Our results reveal mechanisms through which DDK controls the duplication of large vertebrate genomes.


Asunto(s)
Replicación del ADN , Origen de Réplica , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Fosforilación , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Puntos de Control de la Fase S del Ciclo Celular , Especificidad por Sustrato , Factores de Tiempo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xenopus laevis
2.
Nucleic Acids Res ; 52(6): 3146-3163, 2024 Apr 12.
Artículo en Inglés | MEDLINE | ID: mdl-38349040

RESUMEN

Sensing and processing of DNA double-strand breaks (DSBs) are vital to genome stability. DSBs are primarily detected by the ATM checkpoint pathway, where the Mre11-Rad50-Nbs1 (MRN) complex serves as the DSB sensor. Subsequent DSB end resection activates the ATR checkpoint pathway, where replication protein A, MRN, and the Rad9-Hus1-Rad1 (9-1-1) clamp serve as the DNA structure sensors. ATR activation depends also on Topbp1, which is loaded onto DNA through multiple mechanisms. While different DNA structures elicit specific ATR-activation subpathways, the regulation and mechanisms of the ATR-activation subpathways are not fully understood. Using DNA substrates that mimic extensively resected DSBs, we show here that MRN and 9-1-1 redundantly stimulate Dna2-dependent long-range end resection and ATR activation in Xenopus egg extracts. MRN serves as the loading platform for ATM, which, in turn, stimulates Dna2- and Topbp1-loading. Nevertheless, MRN promotes Dna2-mediated end processing largely independently of ATM. 9-1-1 is dispensable for bulk Dna2 loading, and Topbp1 loading is interdependent with 9-1-1. ATR facilitates Mre11 phosphorylation and ATM dissociation. These data uncover that long-range end resection activates two redundant pathways that facilitate ATR checkpoint signaling and DNA processing in a vertebrate system.


Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN , Proteínas de Xenopus , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , ADN/genética , ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Enzimas Reparadoras del ADN/metabolismo , Proteína Homóloga de MRE11/genética , Proteína Homóloga de MRE11/metabolismo , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismo , Xenopus laevis/genética , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Activación Enzimática/genética , Fosforilación/genética
3.
Genes Dev ; 32(11-12): 806-821, 2018 06 01.
Artículo en Inglés | MEDLINE | ID: mdl-29899141

RESUMEN

Post-replicative correction of replication errors by the mismatch repair (MMR) system is critical for suppression of mutations. Although the MMR system may need to handle nucleosomes at the site of chromatin replication, how MMR occurs in the chromatin environment remains unclear. Here, we show that nucleosomes are excluded from a >1-kb region surrounding a mismatched base pair in Xenopus egg extracts. The exclusion was dependent on the Msh2-Msh6 mismatch recognition complex but not the Mlh1-containing MutL homologs and counteracts both the HIRA- and CAF-1 (chromatin assembly factor 1)-mediated chromatin assembly pathways. We further found that the Smarcad1 chromatin remodeling ATPase is recruited to mismatch-carrying DNA in an Msh2-dependent but Mlh1-independent manner to assist nucleosome exclusion and that Smarcad1 facilitates the repair of mismatches when nucleosomes are preassembled on DNA. In budding yeast, deletion of FUN30, the homolog of Smarcad1, showed a synergistic increase of spontaneous mutations in combination with MSH6 or MSH3 deletion but no significant increase with MSH2 deletion. Genetic analyses also suggested that the function of Fun30 in MMR is to counteract CAF-1. Our study uncovers that the eukaryotic MMR system has an ability to exclude local nucleosomes and identifies Smarcad1/Fun30 as an accessory factor for the MMR reaction.


Asunto(s)
Disparidad de Par Base/fisiología , ADN Helicasas/metabolismo , Reparación de la Incompatibilidad de ADN/genética , Proteína 2 Homóloga a MutS/metabolismo , Nucleosomas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Animales , Disparidad de Par Base/genética , Ensamble y Desensamble de Cromatina/genética , ADN/genética , ADN/metabolismo , ADN Helicasas/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Xenopus laevis
4.
J Biol Chem ; 300(1): 105588, 2024 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-38141767

RESUMEN

Proliferating cell nuclear antigen (PCNA) is a homo-trimeric clamp complex that serves as the molecular hub for various DNA transactions, including DNA synthesis and post-replicative mismatch repair. Its timely loading and unloading are critical for genome stability. PCNA loading is catalyzed by Replication factor C (RFC) and the Ctf18 RFC-like complex (Ctf18-RLC), and its unloading is catalyzed by Atad5/Elg1-RLC. However, RFC, Ctf18-RLC, and even some subcomplexes of their shared subunits are capable of unloading PCNA in vitro, leaving an ambiguity in the division of labor in eukaryotic clamp dynamics. By using a system that specifically detects PCNA unloading, we show here that Atad5-RLC, which accounts for only approximately 3% of RFC/RLCs, nevertheless provides the major PCNA unloading activity in Xenopus egg extracts. RFC and Ctf18-RLC each account for approximately 40% of RFC/RLCs, while immunodepletion of neither Rfc1 nor Ctf18 detectably affects the rate of PCNA unloading in our system. PCNA unloading is dependent on the ATP-binding motif of Atad5, independent of nicks on DNA and chromatin assembly, and inhibited effectively by PCNA-interacting peptides. These results support a model in which Atad5-RLC preferentially unloads DNA-bound PCNA molecules that are free from their interactors.


Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Proteínas de Unión al ADN , Antígeno Nuclear de Célula en Proliferación , Animales , ADN , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Xenopus laevis/metabolismo , Oocitos , ATPasas Asociadas con Actividades Celulares Diversas/genética , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo
5.
EMBO J ; 37(15)2018 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-29997179

RESUMEN

DNA replication initiates at many discrete loci on eukaryotic chromosomes, and individual replication origins are regulated under a spatiotemporal program. However, the underlying mechanisms of this regulation remain largely unknown. In the fission yeast Schizosaccharomyces pombe, the telomere-binding protein Taz1, ortholog of human TRF1/TRF2, regulates a subset of late replication origins by binding to the telomere-like sequence near the origins. Here, we showed using a lacO/LacI-GFP system that Taz1-dependent late origins were predominantly localized at the nuclear periphery throughout interphase, and were localized adjacent to the telomeres in the G1/S phase. The peripheral localization that depended on the nuclear membrane protein Bqt4 was not necessary for telomeric association and replication-timing control of the replication origins. Interestingly, the shelterin components Rap1 and Poz1 were required for replication-timing control and telomeric association of Taz1-dependent late origins, and this requirement was bypassed by a minishelterin Tpz1-Taz1 fusion protein. Our results suggest that Taz1 suppresses replication initiation through shelterin-mediated telomeric association of the origins at the onset of S phase.


Asunto(s)
Origen de Réplica/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Proteínas de Unión a Telómeros/metabolismo , Telómero/metabolismo , Proteína 2 de Unión a Repeticiones Teloméricas/metabolismo , Replicación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Fase G1/genética , Proteínas de la Membrana/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Fase S/genética , Proteínas de Schizosaccharomyces pombe/genética , Complejo Shelterina , Proteínas de Unión a Telómeros/genética
6.
Genes Dev ; 26(18): 2050-62, 2012 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-22987637

RESUMEN

In eukaryotes, the replication of chromosome DNA is coordinated by a replication timing program that temporally regulates the firing of individual replication origins. However, the molecular mechanism underlying the program remains elusive. Here, we report that the telomere-binding protein Taz1 plays a crucial role in the control of replication timing in fission yeast. A DNA element located proximal to a late origin in the chromosome arm represses initiation from the origin in early S phase. Systematic deletion and substitution experiments demonstrated that two tandem telomeric repeats are essential for this repression. The telomeric repeats recruit Taz1, a counterpart of human TRF1 and TRF2, to the locus. Genome-wide analysis revealed that Taz1 regulates about half of chromosomal late origins, including those in subtelomeres. The Taz1-mediated mechanism prevents Dbf4-dependent kinase (DDK)-dependent Sld3 loading onto the origins. Our results demonstrate that the replication timing program in fission yeast uses the internal telomeric repeats and binding of Taz1.


Asunto(s)
Replicación del ADN/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/fisiología , Proteínas de Unión a Telómeros/metabolismo , Secuencia de Bases , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas de Unión al ADN/metabolismo , Datos de Secuencia Molecular , Unión Proteica , Transporte de Proteínas , Origen de Réplica/fisiología , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Unión a Telómeros/genética
7.
Nucleic Acids Res ; 45(19): 11222-11235, 2017 Nov 02.
Artículo en Inglés | MEDLINE | ID: mdl-28977643

RESUMEN

Centromeres that are essential for faithful segregation of chromosomes consist of unique DNA repeats in many eukaryotes. Although recombination is under-represented around centromeres during meiosis, little is known about recombination between centromere repeats in mitotic cells. Here, we compared spontaneous recombination that occurs between ade6B/ade6X inverted repeats integrated at centromere 1 (cen1) or at a non-centromeric ura4 locus in fission yeast. Remarkably, distinct mechanisms of homologous recombination (HR) were observed in centromere and non-centromere regions. Rad51-dependent HR that requires Rad51, Rad54 and Rad52 was predominant in the centromere, whereas Rad51-independent HR that requires Rad52 also occurred in the arm region. Crossovers between inverted repeats (i.e. inversions) were under-represented in the centromere as compared to the arm region. While heterochromatin was dispensable, Mhf1/CENP-S, Mhf2/CENP-X histone-fold proteins and Fml1/FANCM helicase were required to suppress crossovers. Furthermore, Mhf1 and Fml1 were found to prevent gross chromosomal rearrangements mediated by centromere repeats. These data for the first time uncovered the regulation of mitotic recombination between DNA repeats in centromeres and its physiological role in maintaining genome integrity.


Asunto(s)
Centrómero/genética , ADN de Hongos/genética , Recombinación Homóloga , Mitosis/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , ADN Helicasas/genética , ADN Helicasas/metabolismo , ADN de Hongos/metabolismo , Genoma Fúngico/genética , Modelos Genéticos , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo , Proteína Recombinante y Reparadora de ADN Rad52/genética , Proteína Recombinante y Reparadora de ADN Rad52/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
8.
Nucleic Acids Res ; 44(22): 10744-10757, 2016 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-27697832

RESUMEN

Centromeres consist of DNA repeats in many eukaryotes. Non-allelic homologous recombination (HR) between them can result in gross chromosomal rearrangements (GCRs). In fission yeast, Rad51 suppresses isochromosome formation that occurs between inverted repeats in the centromere. However, how the HR enzyme prevents homology-mediated GCRs remains unclear. Here, we provide evidence that Rad51 with the aid of the Swi/Snf-type motor protein Rad54 promotes non-crossover recombination between centromere repeats to prevent isochromosome formation. Mutations in Rad51 and Rad54 epistatically increased the rates of isochromosome formation and chromosome loss. In sharp contrast, these mutations decreased gene conversion between inverted repeats in the centromere. Remarkably, analysis of recombinant DNAs revealed that rad51 and rad54 increase the proportion of crossovers. In the absence of Rad51, deletion of the structure-specific endonuclease Mus81 decreased both crossovers and isochromosomes, while the cdc27/pol32-D1 mutation, which impairs break-induced replication, did not. We propose that Rad51 and Rad54 promote non-crossover recombination between centromere repeats on the same chromatid, thereby suppressing crossover between non-allelic repeats on sister chromatids that leads to chromosomal rearrangements. Furthermore, we found that Rad51 and Rad54 are required for gene silencing in centromeres, suggesting that HR also plays a role in the structure and function of centromeres.


Asunto(s)
ADN Helicasas/fisiología , Recombinasa Rad51/fisiología , Proteínas de Schizosaccharomyces pombe/fisiología , Schizosaccharomyces/genética , Centrómero , Cromátides , Cromosomas Fúngicos , Intercambio Genético , ADN de Hongos/genética , Reparación del ADN por Recombinación , Secuencias Repetitivas de Ácidos Nucleicos , Schizosaccharomyces/metabolismo
9.
Genes Dev ; 24(24): 2723-31, 2010 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-21159813

RESUMEN

The ring-shaped cohesin complex links sister chromatids and plays crucial roles in homologous recombination and mitotic chromosome segregation. In cycling cells, cohesin's ability to generate cohesive linkages is restricted to S phase and depends on loading and establishment factors that are intimately connected to DNA replication. Here we review how cohesin is regulated by the replication machinery, as well as recent evidence that cohesin itself influences how chromosomes are replicated.


Asunto(s)
Proteínas de Ciclo Celular/fisiología , Proteínas Cromosómicas no Histona/fisiología , Replicación del ADN , Proteínas de Ciclo Celular/metabolismo , Cromátides/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas , Cohesinas
10.
EMBO J ; 31(9): 2182-94, 2012 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-22433840

RESUMEN

The CMG complex composed of Mcm2-7, Cdc45 and GINS is postulated to be the eukaryotic replicative DNA helicase, whose activation requires sequential recruitment of replication proteins onto Mcm2-7. Current models suggest that Mcm10 is involved in assembly of the CMG complex, and in tethering of DNA polymerase α at replication forks. Here, we report that Mcm10 is required for origin DNA unwinding after association of the CMG components with replication origins in fission yeast. A combination of promoter shut-off and the auxin-inducible protein degradation (off-aid) system efficiently depleted cellular Mcm10 to <0.5% of the wild-type level. Depletion of Mcm10 did not affect origin loading of Mcm2-7, Cdc45 or GINS, but impaired recruitment of RPA and DNA polymerases. Mutations in a conserved zinc finger of Mcm10 abolished RPA loading after recruitment of Mcm10. These results show that Mcm10, together with the CMG components, plays a novel essential role in origin DNA unwinding through its zinc-finger function.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN de Hongos/metabolismo , Proteínas Fúngicas/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Levaduras
11.
J Biol Chem ; 289(33): 23043-23055, 2014 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-24947512

RESUMEN

The E3 ubiquitin ligase CRL4(Cdt2) targets proteins for destruction in S phase and after DNA damage by coupling ubiquitylation to DNA-bound proliferating cell nuclear antigen (PCNA). Coupling to PCNA involves a PCNA-interacting peptide (PIP) degron motif in the substrate that recruits CRL4(Cdt2) while binding to PCNA. In vertebrates, CRL4(Cdt2) promotes degradation of proteins whose presence in S phase is deleterious, including Cdt1, Set8, and p21. Here, we show that CRL4(Cdt2) targets thymine DNA glycosylase (TDG), a base excision repair enzyme that is involved in DNA demethylation. TDG contains a conserved and nearly perfect match to the PIP degron consensus. TDG is ubiquitylated and destroyed in a PCNA-, Cdt2-, and PIP degron-dependent manner during DNA repair in Xenopus egg extract. The protein can also be destroyed during DNA replication in this system. During Xenopus development, TDG first accumulates during gastrulation, and its expression is down-regulated by CRL4(Cdt2). Our results expand the group of vertebrate CRL4(Cdt2) substrates to include a bona fide DNA repair enzyme.


Asunto(s)
Metilación de ADN/fisiología , Gástrula/enzimología , Timina ADN Glicosilasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/fisiología , Proteínas de Xenopus/metabolismo , Animales , Gástrula/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Regulación Enzimológica de la Expresión Génica/fisiología , Complejos de Ubiquitina-Proteína Ligasa , Ubiquitina-Proteína Ligasas/genética , Proteínas de Xenopus/genética , Xenopus laevis
13.
Proc Natl Acad Sci U S A ; 109(24): 9366-71, 2012 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-22628566

RESUMEN

The loading of cohesin onto chromatin requires the heterodimeric complex sister chromatid cohesion (Scc)2 and Scc4 (Scc2/4), which is highly conserved in all species. Here, we describe the purification of the human (h)-Scc2/4 and show that it interacts with h-cohesin and the heterodimeric Smc1-Smc3 complex but not with the Smc1 or Smc3 subunit alone. We demonstrate that both h-Scc2/4 and h-cohesin are loaded onto dsDNA containing the prereplication complex (pre-RC) generated in vitro by Xenopus high-speed soluble extracts. The addition of geminin, which blocks pre-RC formation, prevents the loading of Scc2/4 and cohesin. Xenopus extracts depleted of endogenous Scc2/4 with specific antibodies, although able to form pre-RCs, did not support cohesin loading unless supplemented with purified h-Scc2/4. The results presented here indicate that the Xenopus or h-Scc2/4 complex supports the loading of Xenopus and/or h-cohesin onto pre-RCs formed by Xenopus high-speed extracts. We show that cohesin loaded onto pre-RCs either by h-Scc2/4 and/or the Xenopus complex was dissociated from chromatin by low salt extraction, similar to cohesin loaded onto chromatin in G(1) by HeLa cells in vivo. Replication of cohesin-loaded DNA, both in vitro and in vivo, markedly increased the stability of cohesin associated with DNA. Collectively, these in vitro findings partly recapitulate the in vivo pathway by which sister chromatids are linked together, leading to cohesion.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/metabolismo , Animales , Ciclo Celular , Cromatina/metabolismo , Dimerización , Humanos , Xenopus , Cohesinas
14.
Elife ; 122023 02 03.
Artículo en Inglés | MEDLINE | ID: mdl-36734974

RESUMEN

UHRF1-dependent ubiquitin signaling plays an integral role in the regulation of maintenance DNA methylation. UHRF1 catalyzes transient dual mono-ubiquitylation of PAF15 (PAF15Ub2), which regulates the localization and activation of DNMT1 at DNA methylation sites during DNA replication. Although the initiation of UHRF1-mediated PAF15 ubiquitin signaling has been relatively well characterized, the mechanisms underlying its termination and how they are coordinated with the completion of maintenance DNA methylation have not yet been clarified. This study shows that deubiquitylation by USP7 and unloading by ATAD5 (ELG1 in yeast) are pivotal processes for the removal of PAF15 from chromatin. On replicating chromatin, USP7 specifically interacts with PAF15Ub2 in a complex with DNMT1. USP7 depletion or inhibition of the interaction between USP7 and PAF15 results in abnormal accumulation of PAF15Ub2 on chromatin. Furthermore, we also find that the non-ubiquitylated form of PAF15 (PAF15Ub0) is removed from chromatin in an ATAD5-dependent manner. PAF15Ub2 was retained at high levels on chromatin when the catalytic activity of DNMT1 was inhibited, suggesting that the completion of maintenance DNA methylation is essential for the termination of UHRF1-mediated ubiquitin signaling. This finding provides a molecular understanding of how the maintenance DNA methylation machinery is disassembled at the end of the S phase.


Asunto(s)
Ubiquitina-Proteína Ligasas , Ubiquitina , Ubiquitina/metabolismo , Peptidasa Específica de Ubiquitina 7/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Unión Proteica , Cromatina , ADN (Citosina-5-)-Metiltransferasa 1/genética , Metilación de ADN
15.
J Biol Chem ; 286(48): 41701-41710, 2011 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-21971174

RESUMEN

Mcm2-7 complexes are loaded onto chromatin with the aid of Cdt1 and Cdc18/Cdc6 and form prereplicative complexes (pre-RCs) at multiple sites on each chromosome. Pre-RCs are essential for DNA replication and surviving replication stress. However, the mechanism by which pre-RCs contribute to surviving replication stress is largely unknown. Here, we isolated the fission yeast mcm6-S1 mutant that was hypersensitive to methyl methanesulfonate (MMS) and camptothecin (CPT), both of which cause forks to collapse. The mcm6-S1 mutation impaired the interaction with Cdt1 and decreased the binding of minichromosome maintenance (MCM) proteins to replication origins. Overexpression of Cdt1 restored MCM binding and suppressed the sensitivity to MMS and CPT, suggesting that the Cdt1-Mcm6 interaction is important for the assembly of pre-RCs and the repair of collapsed forks. MMS-induced Chk1 phosphorylation and Rad22/Rad52 focus formation occurred normally, whereas cells containing Rhp54/Rad54 foci, which are involved in DNA strand exchange and dissociation of the joint molecules, were increased. Remarkably, G(1) phase extension through deletion of an S phase cyclin, Cig2, as well as Cdt1 overexpression restored pre-RC assembly and suppressed Rhp54 accumulation. A cdc18 mutation also caused hypersensitivity to MMS and CPT and accumulation of Rhp54 foci. These data suggest that an abundance of pre-RCs facilitates a late step in the recombinational repair of collapsed forks in the following S phase.


Asunto(s)
ADN de Hongos/biosíntesis , Fase G1/fisiología , Complejos Multiproteicos/metabolismo , Fase S/fisiología , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , ADN de Hongos/genética , Regulación Fúngica de la Expresión Génica/fisiología , Complejos Multiproteicos/genética , Mutación , Fosforilación , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética
16.
EMBO J ; 27(22): 3036-46, 2008 Nov 19.
Artículo en Inglés | MEDLINE | ID: mdl-18923422

RESUMEN

Centromere that plays a pivotal role in chromosome segregation is composed of repetitive elements in many eukaryotes. Although chromosomal regions containing repeats are the hotspots of rearrangements, little is known about the stability of centromere repeats. Here, by using a minichromosome that has a complete set of centromere sequences, we have developed a fission yeast system to detect gross chromosomal rearrangements (GCRs) that occur spontaneously. Southern and comprehensive genome hybridization analyses of rearranged chromosomes show two types of GCRs: translocation between homologous chromosomes and formation of isochromosomes in which a chromosome arm is replaced by a copy of the other. Remarkably, all the examined isochromosomes contain the breakpoint in centromere repeats, showing that isochromosomes are produced by centromere rearrangement. Mutations in the Rad3 checkpoint kinase increase both types of GCRs. In contrast, the deletion of Rad51 recombinase preferentially elevates isochromosome formation. Chromatin immunoprecipitation analysis shows that Rad51 localizes at centromere around S phase. These data suggest that Rad51 suppresses rearrangements of centromere repeats that result in isochromosome formation.


Asunto(s)
Centrómero/metabolismo , Aberraciones Cromosómicas , Cromosomas Fúngicos/metabolismo , Recombinasa Rad51/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/genética , Ciclo Celular/fisiología , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrómero/genética , Quinasa de Punto de Control 2 , Segregación Cromosómica , ADN de Hongos/genética , ADN de Hongos/metabolismo , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Recombinasa Rad51/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética
17.
BMC Cell Biol ; 12: 8, 2011 Feb 11.
Artículo en Inglés | MEDLINE | ID: mdl-21314938

RESUMEN

BACKGROUND: Inducible inactivation of a protein is a powerful approach for analysis of its function within cells. Fission yeast is a useful model for studying the fundamental mechanisms such as chromosome maintenance and cell cycle. However, previously published strategies for protein-depletion are successful only for some proteins in some specific conditions and still do not achieve efficient depletion to cause acute phenotypes such as immediate cell cycle arrest. The aim of this work was to construct a useful and powerful protein-depletion system in Shizosaccaromyces pombe. RESULTS: We constructed an auxin-inducible degron (AID) system, which utilizes auxin-dependent poly-ubiquitination of Aux/IAA proteins by SCFTIR1 in plants, in fission yeast. Although expression of a plant F-box protein, TIR1, decreased Mcm4-aid, a component of the MCM complex essential for DNA replication tagged with Aux/IAA peptide, depletion did not result in an evident growth defect. We successfully improved degradation efficiency of Mcm4-aid by fusion of TIR1 with fission yeast Skp1, a conserved F-box-interacting component of SCF (improved-AID system; i-AID), and the cells showed severe defect in growth. The i-AID system induced degradation of Mcm4-aid in the chromatin-bound MCM complex as well as those in soluble fractions. The i-AID system in conjunction with transcription repression (off-AID system), we achieved more efficient depletion of other proteins including Pol1 and Cdc45, causing early S phase arrest. CONCLUSION: Improvement of the AID system allowed us to construct conditional null mutants of S. pombe. We propose that the off-AID system is the powerful method for in vivo protein-depletion in fission yeast.


Asunto(s)
Ciclo Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Proteínas Recombinantes de Fusión/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromatina/genética , Cromatina/metabolismo , Replicación del ADN/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas F-Box/genética , Proteínas F-Box/metabolismo , Expresión Génica/genética , Ácidos Indolacéticos/farmacología , Componente 4 del Complejo de Mantenimiento de Minicromosoma , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas del Complejo de Iniciación de Transcripción Pol1/genética , Proteínas del Complejo de Iniciación de Transcripción Pol1/metabolismo , Poliubiquitina/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Ligasas SKP Cullina F-box/genética , Proteínas Ligasas SKP Cullina F-box/metabolismo , Schizosaccharomyces/citología , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Activación Transcripcional , Transformación Genética
18.
Nat Cell Biol ; 6(10): 991-6, 2004 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-15448702

RESUMEN

Cohesin is a multi-subunit, ring-shaped protein complex that holds sister chromatids together from the time of their synthesis in S phase until they are segregated in anaphase. In yeast, the loading of cohesin onto chromosomes requires the Scc2 protein. In vertebrates, cohesins first bind to chromosomes as cells exit mitosis, but the mechanism is unknown. Concurrent with cohesin binding, pre-replication complexes (pre-RCs) are assembled at origins of DNA replication through the sequential loading of the initiation factors ORC, Cdc6, Cdt1 and MCM2-7 (the 'licensing' reaction). In S phase, the protein kinase Cdk2 activates pre-RCs, causing origin unwinding and DNA replication. Here, we use Xenopus egg extracts to show that the recruitment of cohesins to chromosomes requires fully licensed chromatin and is dependent on ORC, Cdc6, Cdt1 and MCM2-7, but is independent of Cdk2. We further show that Xenopus Scc2 is required for cohesin loading and that binding of XScc2 to chromatin is MCM2-7 dependent. Our results define a novel pre-RC-dependent pathway for cohesin recruitment to chromosomes in a vertebrate model system.


Asunto(s)
Cromatina/metabolismo , Proteínas Cromosómicas no Histona/química , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Nucleares/metabolismo , Origen de Réplica , Proteínas de Xenopus/química , Proteínas de Xenopus/metabolismo , Animales , Proteínas de Ciclo Celular/metabolismo , Extractos Celulares , Proteínas Cromosómicas no Histona/aislamiento & purificación , Replicación del ADN , Electroforesis en Gel Bidimensional , Escherichia coli/genética , Proteínas Fúngicas , Masculino , Espectrometría de Masas , Datos de Secuencia Molecular , Oocitos/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Espermatozoides/química , Xenopus , Proteínas de Xenopus/aislamiento & purificación , Cohesinas
19.
Trends Biochem Sci ; 30(8): 437-44, 2005 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-16002295

RESUMEN

In eukaryotes, numerous lines of evidence have coalesced into a convincing case that the MCM2-7 complex - a heterohexameric ATPase - is the replicative DNA helicase. However, almost nothing is known about how this enzyme functions in a cellular context. Some models for the mechanism of the MCM2-7 helicase envision that it translocates along single-stranded DNA (ssDNA), whereas, more recently, it is has been suggested that it pumps double-stranded DNA (dsDNA) through its central channel. In particular, one model in which a double hexamer of MCM2-7 pumps dsDNA towards the hexamer interface and extrudes ssDNA laterally as a result of torsional strain is gaining popularity. Here, we discuss existing models and propose a new variation in which a single hexamer is the functional unit of the helicase. Duplex DNA is pumped into MCM2-7 and, as it emerges from the complex, a rigid protein that we term the 'ploughshare' splits the duplex.


Asunto(s)
Proteínas de Ciclo Celular/metabolismo , ADN Helicasas/metabolismo , Proteínas Nucleares/metabolismo , Animales , ADN/genética , Replicación del ADN , Humanos , Unión Proteica
20.
Commun Biol ; 2: 17, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-30652128

RESUMEN

Heterochromatin, characterized by histone H3 lysine 9 (H3K9) methylation, assembles on repetitive regions including centromeres. Although centromeric heterochromatin is important for correct segregation of chromosomes, its exact role in maintaining centromere integrity remains elusive. Here, we found in fission yeast that heterochromatin suppresses gross chromosomal rearrangements (GCRs) at centromeres. Mutations in Clr4/Suv39 methyltransferase increased the formation of isochromosomes, whose breakpoints were located in centromere repeats. H3K9A and H3K9R mutations also increased GCRs, suggesting that Clr4 suppresses centromeric GCRs via H3K9 methylation. HP1 homologs Swi6 and Chp2 and the RNAi component Chp1 were the chromodomain proteins essential for full suppression of GCRs. Remarkably, mutations in RNA polymerase II (RNAPII) or Tfs1/TFIIS, the transcription factor that facilitates restart of RNAPII after backtracking, specifically bypassed the requirement of Clr4 for suppressing GCRs. These results demonstrate that heterochromatin suppresses GCRs by repressing Tfs1-dependent transcription of centromere repeats.


Asunto(s)
Centrómero/metabolismo , Heterocromatina/metabolismo , Isocromosomas/genética , Schizosaccharomyces/genética , Transcripción Genética/genética , Factores de Elongación Transcripcional/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , Histonas/genética , Metilación , Plásmidos/genética , Interferencia de ARN , ARN Polimerasa II/genética , Proteínas Represoras/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo
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