RESUMO
Mutations in the nuclear trypsin-like serine protease FAM111A cause Kenny-Caffey syndrome (KCS2) with hypoparathyroidism and skeletal dysplasia or perinatally lethal osteocraniostenosis (OCS). In addition, FAM111A was identified as a restriction factor for certain host range mutants of the SV40 polyomavirus and VACV orthopoxvirus. However, because FAM111A function is poorly characterized, its roles in restricting viral replication and the etiology of KCS2 and OCS remain undefined. We find that FAM111A KCS2 and OCS patient mutants are hyperactive and cytotoxic, inducing apoptosis-like phenotypes such as disruption of nuclear structure and pore distribution, in a protease-dependent manner. Moreover, wild-type FAM111A activity causes similar nuclear phenotypes, including the loss of nuclear barrier function, when SV40 host range mutants attempt to replicate in restrictive cells. Interestingly, pan-caspase inhibitors do not block these FAM111A-induced phenotypes, implying it acts independently or upstream of caspases. In this regard, we identify nucleoporins and the associated GANP transcription/replication factor as FAM111A interactors and candidate targets. Overall, we reveal a potentially unifying mechanism through which deregulated FAM111A activity restricts viral replication and causes KCS2 and OCS.
Assuntos
Doenças do Desenvolvimento Ósseo , Núcleo Celular/patologia , Anormalidades Craniofaciais , Hiperostose Cortical Congênita , Hipoparatireoidismo , Receptores Virais , Humanos , Vírus 40 dos Símios , Replicação ViralRESUMO
The posttranslational modifiers SUMO and ubiquitin critically regulate the DNA damage response (DDR). Important crosstalk between these modifiers at DNA lesions is mediated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitinates SUMO chains to generate SUMO-ubiquitin hybrids. These SUMO-ubiquitin hybrids attract DDR proteins able to bind both modifiers, and/or are degraded at the proteasome. Despite these insights, specific roles for SUMO chains and STUbL in the DDR remain poorly defined. Notably, fission yeast defective in SUMO chain formation exhibit near wild-type resistance to genotoxins and moreover, have a greatly reduced dependency on STUbL activity for DNA repair. Based on these and other data, we propose that a critical role of STUbL is to antagonize DDR-inhibitory SUMO chain formation at DNA lesions. In this regard, we identify a SUMO-binding Swi2/Snf2 translocase called Rrp2 (ScUls1) as a mediator of the DDR defects in STUbL mutant cells. Therefore, in support of our proposal, SUMO chains attract activities that can antagonize STUbL and other DNA repair factors. Finally, we find that Taz1TRF1/TRF2-deficiency triggers extensive telomeric poly-SUMOylation. In this setting STUbL, together with its cofactor Cdc48p97, actually promotes genomic instability caused by the aberrant processing of taz1Δ telomeres by DNA repair factors. In summary, depending on the nature of the initiating DNA lesion, STUbL activity can either be beneficial or harmful.
Assuntos
Instabilidade Genômica , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Genoma Fúngico , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Ubiquitina-Proteína Ligases/genética , Proteína com ValosinaRESUMO
Duplication of the genome poses one of the most significant threats to genetic integrity, cellular fitness, and organismal health. Therefore, numerous mechanisms have evolved that maintain replication fork stability in the face of DNA damage and allow faithful genome duplication. The fission yeast BRCT-domain-containing protein Brc1, and its budding yeast orthologue Rtt107, has emerged as a "hub" factor that integrates multiple replication fork protection mechanisms. Notably, the cofactors and pathways through which Brc1, Rtt107, and their human orthologue (PTIP) act have appeared largely distinct. This either represents true evolutionary functional divergence, or perhaps an incomplete genetic and biochemical analysis of each protein. In this regard, we recently showed that like Rtt107, Brc1 supports key functions of the Smc5-Smc6 complex, including its recruitment into DNA repair foci, chromatin association, and SUMO ligase activity. Furthermore, fission yeast cells lacking the Nse5-Nse6 genome stability factor were found to exhibit defects in Smc5-Smc6 function, similar to but more severe than those in cells lacking Brc1. Here, we place these findings in context with the known functions of Brc1, Rtt107, and Smc5-Smc6, present data suggesting a role for acetylation in Smc5-Smc6 chromatin loading, and discuss wider implications for genome stability.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Instabilidade Genômica , Ligases/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas de Ciclo Celular/genética , Dano ao DNA , Replicação do DNA , Ligases/genética , Proteína SUMO-1/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Posttranslational modifications (PTMs) provide dynamic regulation of the cellular proteome, which is critical for both normal cell growth and for orchestrating rapid responses to environmental stresses, e.g. genotoxins. Key PTMs include ubiquitin, the Small Ubiquitin-like MOdifier SUMO, and phosphorylation. Recently, SUMO-targeted ubiquitin ligases (STUbLs) were found to integrate signaling through the SUMO and ubiquitin pathways. In general, STUbLs are recruited to target proteins decorated with poly-SUMO chains to ubiquitinate them and drive either their extraction from protein complexes, and/or their degradation at the proteasome. In fission yeast, reducing or preventing the formation of SUMO chains can circumvent the essential and DNA damage response functions of STUbL. This result indicates that whilst some STUbL "targets" have been identified, the crucial function of STUbL is to antagonize SUMO chain formation. Herein, by screening for additional STUbL suppressors, we reveal crosstalk between the serine/threonine phosphatase PP2A-Pab1B55 and the SUMO pathway. A hypomorphic Pab1B55 mutant not only suppresses STUbL dysfunction, but also mitigates the phenotypes associated with deletion of the SUMO protease Ulp2, or mutation of the STUbL cofactor Rad60. Together, our results reveal a novel role for PP2A-Pab1B55 in modulating SUMO pathway output, acting in parallel to known critical regulators of SUMOylation homeostasis. Given the broad evolutionary functional conservation of the PP2A and SUMO pathways, our results could be relevant to the ongoing attempts to therapeutically target these factors.
Assuntos
Proteína Fosfatase 2/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ciclo Celular , Proliferação de Células , Proteínas Cromossômicas não Histona/metabolismo , Dano ao DNA , Reparo do DNA , Deleção de Genes , Dosagem de Genes , Genoma Fúngico , Genótipo , Proteínas de Fluorescência Verde/metabolismo , Mutação , Fenótipo , Proteínas de Ligação a Poli(A)/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Processamento de Proteína Pós-Traducional , Análise de Sequência de DNA , SumoilaçãoRESUMO
Covalent modification of the proteome by SUMO is critical for genetic stability and cell growth. Equally crucial to these processes is the removal of SUMO from its targets by the Ulp1 (HuSENP1/2) family of SUMO proteases. Ulp1 activity is normally spatially restricted, because it is localized to the nuclear periphery via interactions with the nuclear pore. Delocalization of Ulp1 causes DNA damage and cell cycle defects, phenotypes thought to be caused by inappropriate desumoylation of nucleoplasmic targets that are normally spatially protected from Ulp1. Here, we define a novel consequence of Ulp1 deregulation, with a major impact on SUMO pathway function. In fission yeast lacking Nup132 (Sc/HuNUP133), Ulp1 is delocalized and can no longer antagonize sumoylation of the PIAS family SUMO E3 ligase, Pli1. Consequently, SUMO chain-modified Pli1 is targeted for proteasomal degradation by the concerted action of a SUMO-targeted ubiquitin ligase (STUbL) and Cdc48-Ufd1-Npl4. Pli1 degradation causes the profound SUMO pathway defects and associated centromere dysfunction in cells lacking Nup132. Thus, perhaps counterintuitively, Ulp1-mediated desumoylation can promote SUMO modification by stabilizing a SUMO E3 ligase.
Assuntos
Cisteína Endopeptidases/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sumoilação/fisiologia , Cisteína Endopeptidases/genética , Endopeptidases/genética , Endopeptidases/metabolismo , Humanos , Proteínas Inibidoras de STAT Ativados/genética , Proteínas Inibidoras de STAT Ativados/metabolismo , Proteína SUMO-1/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The post-translational modification of DNA repair and checkpoint proteins by ubiquitin and small ubiquitin-like modifier (SUMO) critically orchestrates the DNA damage response (DDR). The ubiquitin ligase RNF4 integrates signaling by SUMO and ubiquitin, through its selective recognition and ubiquitination of SUMO-modified proteins. Here, we define a key new determinant for target discrimination by RNF4, in addition to interaction with SUMO. We identify a nucleosome-targeting motif within the RNF4 RING domain that can bind DNA and thereby enables RNF4 to selectively ubiquitinate nucleosomal histones. Furthermore, RNF4 nucleosome-targeting is crucially required for the repair of TRF2-depleted dysfunctional telomeres by 53BP1-mediated non-homologous end joining.
Assuntos
Reparo do DNA , Proteínas Nucleares/metabolismo , Proteínas Nucleares/ultraestrutura , Nucleossomos/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/ultraestrutura , Motivos de Aminoácidos , Animais , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , Dano ao DNA , Proteínas de Ligação a DNA/metabolismo , Técnicas de Inativação de Genes , Camundongos , Proteínas Nucleares/genética , Processamento de Proteína Pós-Traducional , Estrutura Terciária de Proteína , Tamoxifeno/análogos & derivados , Tamoxifeno/farmacologia , Telômero/efeitos dos fármacos , Telômero/genética , Proteína 2 de Ligação a Repetições Teloméricas/genética , Fatores de Transcrição/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53 , Ubiquitina/metabolismo , Ubiquitina-Proteína Ligases , UbiquitinaçãoRESUMO
Faithful chromosome segregation in meiosis is crucial to form viable, healthy offspring and in most species, it requires programmed recombination between homologous chromosomes. In fission yeast, meiotic recombination is initiated by Rec12 (Spo11 homolog) and generates single Holliday junction (HJ) intermediates, which are resolved by the Mus81-Eme1 endonuclease to generate crossovers and thereby allow proper chromosome segregation. Although Mus81 contains the active site for HJ resolution, the regulation of Mus81-Eme1 is unclear. In cells lacking Nse5-Nse6 of the Smc5-Smc6 genome stability complex, we observe persistent meiotic recombination intermediates (DNA joint molecules) resembling HJs that accumulate in mus81Δ cells. Elimination of Rec12 nearly completely rescues the meiotic defects of nse6Δ and mus81Δ single mutants and partially rescues nse6Δ mus81Δ double mutants, indicating that these factors act after DNA double-strand break formation. Likewise, expression of the bacterial HJ resolvase RusA partially rescues the defects of nse6Δ, mus81Δ and nse6Δ mus81Δ mitotic cells, as well as the meiotic defects of nse6Δ and mus81Δ cells. Partial rescue likely reflects the accumulation of structures other than HJs, such as hemicatenanes, and an additional role for Nse5-Nse6 most prominent during mitotic growth. Our results indicate a regulatory role for the Smc5-Smc6 complex in HJ resolution via Mus81-Eme1.
Assuntos
Proteínas Cromossômicas não Histona/fisiologia , Meiose/genética , Reparo de DNA por Recombinação , Proteínas de Schizosaccharomyces pombe/fisiologia , Proteínas de Ciclo Celular/fisiologia , Proteínas Cromossômicas não Histona/genética , Troca Genética , Quebras de DNA de Cadeia Dupla , Proteínas de Escherichia coli/metabolismo , Deleção de Genes , Resolvases de Junção Holliday/metabolismo , Mitose/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
Through as yet undefined proteins and pathways, the SUMO-targeted ubiquitin ligase (STUbL) suppresses genomic instability by ubiquitinating SUMO conjugated proteins and driving their proteasomal destruction. Here, we identify a critical function for fission yeast STUbL in suppressing spontaneous and chemically induced topoisomerase I (Top1)-mediated DNA damage. Strikingly, cells with reduced STUbL activity are dependent on tyrosyl-DNA phosphodiesterase 1 (Tdp1). This is notable, as cells lacking Tdp1 are largely aphenotypic in the vegetative cell cycle due to the existence of alternative pathways for the removal of covalent Top1-DNA adducts (Top1cc). We further identify Rad60, a SUMO mimetic and STUbL-interacting protein, and the SUMO E3 ligase Nse2 as critical Top1cc repair factors in cells lacking Tdp1. Detection of Top1ccs using chromatin immunoprecipitation and quantitative PCR shows that they are elevated in cells lacking Tdp1 and STUbL, Rad60, or Nse2 SUMO ligase activity. These unrepaired Top1ccs are shown to cause DNA damage, hyper-recombination, and checkpoint-mediated cell cycle arrest. We further determine that Tdp1 and the nucleotide excision repair endonuclease Rad16-Swi10 initiate the major Top1cc repair pathways of fission yeast. Tdp1-based repair is the predominant activity outside S phase, likely acting on transcription-coupled Top1cc. Epistasis analyses suggest that STUbL, Rad60, and Nse2 facilitate the Rad16-Swi10 pathway, parallel to Tdp1. Collectively, these results reveal a unified role for STUbL, Rad60, and Nse2 in protecting genome stability against spontaneous Top1-mediated DNA damage.
Assuntos
Proteínas Cromossômicas não Histona , Dano ao DNA/genética , DNA Topoisomerases Tipo I , Instabilidade Genômica , Proteínas de Schizosaccharomyces pombe , Schizosaccharomyces , Ubiquitina-Proteína Ligases , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Adutos de DNA/genética , Adutos de DNA/metabolismo , Reparo do DNA , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Diester Fosfórico Hidrolases/genética , Diester Fosfórico Hidrolases/metabolismo , Proteína SUMO-1/genética , Proteína SUMO-1/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Transdução de Sinais , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Protein modification by SUMO and ubiquitin critically impacts genome stability via effectors that "read" their signals using SUMO interaction motifs or ubiquitin binding domains, respectively. A novel mixed SUMO and ubiquitin signal is generated by the SUMO-targeted ubiquitin ligase (STUbL), which ubiquitylates SUMO conjugates. Herein, we determine that the "ubiquitin-selective" segregase Cdc48-Ufd1-Npl4 also binds SUMO via a SUMO interaction motif in Ufd1 and can thus act as a selective receptor for STUbL targets. Indeed, we define key cooperative DNA repair functions for Cdc48-Ufd1-Npl4 and STUbL, thereby revealing a new signaling mechanism involving dual recruitment by SUMO and ubiquitin for Cdc48-Ufd1-Npl4 functions in maintaining genome stability.
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Instabilidade Genômica/fisiologia , Proteína SUMO-1/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina/metabolismo , Ubiquitinação/fisiologia , Adenosina Trifosfatases/genética , Motivos de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Reparo do DNA/fisiologia , DNA Fúngico/genética , DNA Fúngico/metabolismo , Ligação Proteica , Proteína SUMO-1/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genética , Transdução de Sinais/fisiologia , Ubiquitina/genética , Ubiquitina-Proteína Ligases/genética , Proteína com ValosinaRESUMO
Ubiquitin and ubiquitin-like proteins (Ubls) share a beta-GRASP fold and have key roles in cellular growth and suppression of genome instability. Despite their common fold, SUMO and ubiquitin are classically portrayed as distinct, and they can have antagonistic roles. Recently, a new family of proteins, the small ubiquitin-related modifier (SUMO)-targeted ubiquitin ligases (STUbLs), which directly connect sumoylation and ubiquitylation, has been discovered. Uniquely, STUbLs use SUMO-interaction motifs (SIMs) to recognize their sumoylated targets. STUbLs are global regulators of protein sumoylation levels, and cells lacking STUbLs display genomic instability and hypersensitivity to genotoxic stress. The human STUbL, RNF4, is implicated in several diseases including cancer, highlighting the importance of characterizing the cellular functions of STUbLs.
Assuntos
Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/fisiologia , Ubiquitina/fisiologia , Sequência de Aminoácidos , Humanos , Modelos Biológicos , Dados de Sequência Molecular , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Complexo de Endopeptidases do Proteassoma/fisiologia , Domínios e Motivos de Interação entre Proteínas , Saccharomycetales/fisiologia , Alinhamento de Sequência , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
SUMOylation of proteins is a cyclic process that requires both conjugation and deconjugation of SUMO moieties. Besides modification by a single SUMO, SUMO chains have also been observed, yet the dynamics of SUMO conjugation/deconjugation remain poorly understood. Using a non-deconjugatable form of SUMO we demonstrate the underappreciated existence of SUMO chains in vivo, we highlight the importance of SUMO deconjugation, and we demonstrate the highly dynamic nature of the SUMO system. We show that SUMO-specific proteases (SENPs) play a crucial role in the dynamics of SUMO chains in vivo by constant deconjugation. Preventing deSUMOylation in Schizosaccharomyces pombe results in slow growth and a sensitivity to replication stress, highlighting the biological requirement for deSUMOylation dynamics. Furthermore, we present the mechanism of SUMO chain deconjugation by SENPs, which occurs via a stochastic mechanism, resulting in cleavage anywhere within a chain. Our results offer mechanistic insights into the workings of deSUMOylating proteases and highlight their importance in the homeostasis of (poly)SUMO-modified substrates.
Assuntos
Peptídeo Hidrolases/metabolismo , Proteína SUMO-1/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Schizosaccharomyces/metabolismo , Sumoilação/fisiologia , Células HEK293 , Células HeLa , Humanos , Peptídeo Hidrolases/genética , Proteína SUMO-1/genética , Schizosaccharomyces/genética , Proteínas de Schizosaccharomyces pombe/genéticaRESUMO
The Smc5/6 holocomplex executes key functions in genome maintenance that include ensuring the faithful segregation of chromosomes at mitosis and facilitating critical DNA repair pathways. Smc5/6 is essential for viability and therefore, dissecting its chromosome segregation and DNA repair roles has been challenging. We have identified distinct epigenetic and post-translational modifications that delineate roles for fission yeast Smc5/6 in centromere function, versus replication fork-associated DNA repair. We monitored Smc5/6 subnuclear and genomic localization in response to different replicative stresses, using fluorescence microscopy and chromatin immunoprecipitation (ChIP)-on-chip methods. Following hydroxyurea treatment, and during an unperturbed S phase, Smc5/6 is transiently enriched at the heterochromatic outer repeats of centromeres in an H3-K9 methylation-dependent manner. In contrast, methyl methanesulphonate treatment induces the accumulation of Smc5/6 at subtelomeres, in an Nse2 SUMO ligase-dependent, but H3-K9 methylation-independent manner. Finally, we determine that Smc5/6 loads at all genomic tDNAs, a phenomenon that requires intact consensus TFIIIC-binding sites in the tDNAs.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Heterocromatina/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/metabolismo , Telômero/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Núcleo Celular/metabolismo , Núcleo Celular/ultraestrutura , Proteínas Cromossômicas não Histona/genética , Replicação do DNA , DNA Fúngico/química , DNA Fúngico/metabolismo , Hidroxiureia/metabolismo , Metanossulfonato de Metila/metabolismo , Mutagênicos/metabolismo , Inibidores da Síntese de Ácido Nucleico/metabolismo , Schizosaccharomyces/citologia , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genéticaRESUMO
The human SMC5/6 complex is a conserved guardian of genome stability and an emerging component of antiviral responses. These disparate functions likely require distinct mechanisms of SMC5/6 regulation. In yeast, Smc5/6 is regulated by its Nse5/6 subunits, but such regulatory subunits for human SMC5/6 are poorly defined. Here, we identify a novel SMC5/6 subunit called SIMC1 that contains SUMO interacting motifs (SIMs) and an Nse5-like domain. We isolated SIMC1 from the proteomic environment of SMC5/6 within polyomavirus large T antigen (LT)-induced subnuclear compartments. SIMC1 uses its SIMs and Nse5-like domain to localize SMC5/6 to polyomavirus replication centers (PyVRCs) at SUMO-rich PML nuclear bodies. SIMC1's Nse5-like domain binds to the putative Nse6 orthologue SLF2 to form an anti-parallel helical dimer resembling the yeast Nse5/6 structure. SIMC1-SLF2 structure-based mutagenesis defines a conserved surface region containing the N-terminus of SIMC1's helical domain that regulates SMC5/6 localization to PyVRCs. Furthermore, SLF1, which recruits SMC5/6 to DNA lesions via its BRCT and ARD motifs, binds SLF2 analogously to SIMC1 and forms a separate Nse5/6-like complex. Thus, two Nse5/6-like complexes with distinct recruitment domains control human SMC5/6 localization.
Assuntos
Proteínas de Ciclo Celular , Proteínas Cromossômicas não Histona , Humanos , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Proteômica , Compartimentos de Replicação ViralRESUMO
Stabilization and processing of stalled replication forks is critical for cell survival and genomic integrity. We characterize a novel DNA repair heterodimer of Nse5 and Nse6, which are nonessential nuclear proteins critical for chromosome segregation in fission yeast. The Nse5/6 dimer facilitates DNA repair as part of the Smc5-Smc6 holocomplex (Smc5/6), the basic architecture of which we define. Nse5-Nse6 [corrected] (Nse5 and Nse6) [corrected] mutants display a high level of spontaneous DNA damage and mitotic catastrophe in the absence of the master checkpoint regulator Rad3 (hATR). Nse5/6 mutants are required for the response to genotoxic agents that block the progression of replication forks, acting in a pathway that allows the tolerance of irreparable UV lesions. Interestingly, the UV sensitivity of Nse5/6 [corrected] is suppressed by concomitant deletion of the homologous recombination repair factor, Rhp51 (Rad51). Further, the viability of Nse5/6 mutants depends on Mus81 and Rqh1, factors that resolve or prevent the formation of Holliday junctions. Consistently, the UV sensitivity of cells lacking Nse5/6 can be partially suppressed by overexpressing the bacterial resolvase RusA. We propose a role for Nse5/6 mutants in suppressing recombination that results in Holliday junction formation or in Holliday junction resolution.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA/fisiologia , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA/fisiologia , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dimerização , Endonucleases/genética , Endonucleases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Genoma Fúngico/genética , Instabilidade Genômica , Resolvases de Junção Holliday/genética , Resolvases de Junção Holliday/metabolismo , Complexos Multiproteicos , Mutação , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Recombinação Genética , Schizosaccharomyces/genética , Schizosaccharomyces/efeitos da radiação , Proteínas de Schizosaccharomyces pombe/genética , Raios UltravioletaRESUMO
As genetic instability drives disease or loss of cell fitness, cellular safeguards have evolved to protect the genome, especially during sensitive cell cycle phases, such as DNA replication. Fission yeast Brc1 has emerged as a key factor in promoting cell survival when replication forks are stalled or collapsed. Brc1 is a multi-BRCT protein that is structurally related to the budding yeast Rtt107 and human PTIP DNA damage response factors, but functional similarities appear limited. Brc1 is a dosage suppressor of a mutation in the essential Smc5-Smc6 genome stability complex and is thought to act in a bypass pathway. In this study, we reveal an unexpectedly intimate connection between Brc1 and Smc5-Smc6 function. Brc1 is required for the accumulation of the Smc5-Smc6 genome stability complex in foci during replication stress and for activation of the intrinsic SUMO ligase activity of the complex by collapsed replication forks. Moreover, we show that the chromatin association and SUMO ligase activity of Smc5-Smc6 require the Nse5-Nse6 heterodimer, explaining how this nonessential cofactor critically supports the DNA repair roles of Smc5-Smc6. We also found that Brc1 interacts with Nse5-Nse6, as well as gamma-H2A, so it can tether Smc5-Smc6 at replicative DNA lesions to promote survival.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Schizosaccharomyces pombe/metabolismo , Proteínas de Ciclo Celular/genética , Dano ao DNA , Reparo do DNA , Replicação do DNA , Instabilidade Genômica , Mutação , Recombinação Genética/genética , Proteína SUMO-1/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/genética , SumoilaçãoRESUMO
Genome integrity is protected by Cds1 (Chk2), a checkpoint kinase that stabilizes arrested replication forks. How Cds1 accomplishes this task is unknown. We report that Cds1 interacts with Rad60, a protein required for recombinational repair in fission yeast. Cds1 activation triggers Rad60 phosphorylation and nuclear delocalization. A Rad60 mutant that inhibits regulation by Cds1 renders cells specifically sensitive to replication fork arrest. Genetic and biochemical studies indicate that Rad60 functions codependently with Smc5 and Smc6, subunits of an SMC (structural maintenance of chromosomes) complex required for recombinational repair. These studies indicate that regulation of Rad60 is an important part of the replication checkpoint response controlled by Cds1. We propose that control of Rad60 regulates recombination events at stalled forks.
Assuntos
Proteínas Cromossômicas não Histona/metabolismo , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Recombinação Genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Sequência de Aminoácidos , Núcleo Celular/metabolismo , Sobrevivência Celular , Quinase do Ponto de Checagem 2 , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Immunoblotting , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Fosforilação , Ligação Proteica , SaccharomycetalesRESUMO
The structural maintenance of chromosomes (SMC) family of proteins play key roles in the organization, packaging, and repair of chromosomes. Cohesin (Smc1+3) holds replicated sister chromatids together until mitosis, condensin (Smc2+4) acts in chromosome condensation, and Smc5+6 performs currently enigmatic roles in DNA repair and chromatin structure. The SMC heterodimers must associate with non-SMC subunits to perform their functions. Using both biochemical and genetic methods, we have isolated a novel subunit of the Smc5+6 complex, Nse3. Nse3 is an essential nuclear protein that is required for normal mitotic chromosome segregation and cellular resistance to a number of genotoxic agents. Epistasis with Rhp51 (Rad51) suggests that like Smc5+6, Nse3 functions in the homologous recombination based repair of DNA damage. We previously identified two non-SMC subunits of Smc5+6 called Nse1 and Nse2. Analysis of nse1-1, nse2-1, and nse3-1 mutants demonstrates that they are crucial for meiosis. The Nse1 mutant displays meiotic DNA segregation and homologous recombination defects. Spore viability is reduced by nse2-1 and nse3-1, without affecting interhomolog recombination. Finally, genetic interactions shared by the nse mutants suggest that the Smc5+6 complex is important for replication fork stability.
Assuntos
Meiose , Proteínas Nucleares/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Proteínas de Schizosaccharomyces pombe/fisiologia , Schizosaccharomyces/fisiologia , Sequência de Aminoácidos , Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/metabolismo , Sobrevivência Celular , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos/ultraestrutura , Reparo do DNA , Raios gama , Deleção de Genes , Immunoblotting , Imunoprecipitação , Mitose , Modelos Biológicos , Dados de Sequência Molecular , Mutação , Proteínas Nucleares/genética , Peptídeos/química , Ligação Proteica , Recombinação Genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Homologia de Sequência de Aminoácidos , Técnicas do Sistema de Duplo-Híbrido , Raios UltravioletaRESUMO
Mus81 is a highly conserved endonuclease with homology to the XPF subunit of the XPF-ERCC1 complex. In yeast Mus81 associates with a second subunit, Eme1 or Mms4, which is essential for endonuclease activity in vitro and for in vivo function. Human Mus81 binds to a homolog of fission yeast Eme1 in vitro and in vivo. We show that recombinant Mus81-Eme1 cleaves replication forks, 3' flap substrates, and Holliday junctions in vitro. By use of differentially tagged versions of Mus81 and Eme1, we find that Mus81 associates with Mus81 and that Eme1 associates with Eme1. Thus, complexes containing two or more Mus81-Eme1 units could function to coordinate substrate cleavage in vivo. Down-regulation of Mus81 by RNA interference reduces mitotic recombination in human somatic cells. The recombination defect is rescued by expression of a bacterial Holliday junction resolvase. These data provide direct evidence for a role of Mus81-Eme1 in mitotic recombination in higher eukaryotes and support the hypothesis that Mus81-Eme1 resolves Holliday junctions in vivo.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Endonucleases/genética , Endonucleases/metabolismo , Mitose/genética , Interferência de RNA , Recombinação Genética/genética , Proteínas de Schizosaccharomyces pombe/genética , Sequência de Aminoácidos , Nucléolo Celular/genética , Nucléolo Celular/metabolismo , Sobrevivência Celular/fisiologia , Clonagem Molecular , Dano ao DNA/fisiologia , Proteínas de Ligação a DNA/genética , Células HeLa , Resolvases de Junção Holliday/metabolismo , Humanos , Dados de Sequência Molecular , Ligação Proteica , Proteínas de Saccharomyces cerevisiae , Proteínas de Schizosaccharomyces pombe/metabolismoRESUMO
Covalent protein modification by sumoylation (i.e., addition of small ubiquitin-like modifiers [SUMOs]) regulates a broad spectrum of critical functions in eukaryotic cells; however, usually ≤1% of a given protein is modified as a result of the highly dynamic nature of sumoylation. As such, capturing and identifying sumoylated proteins are both important in biological studies and very challenging tasks. Here we report a tailored purification protocol that includes rapid and complete cell disruption, coupled to highly stringent isolation of sumoylated proteins. Proteins purified using this protocol are compatible with common downstream applications such as western and mass spectrometry analyses. This protocol will work equally well to study other key covalent modifiers such as ubiquitin and Ned8.
Assuntos
Proteínas Fúngicas/química , Proteínas Fúngicas/isolamento & purificação , Biologia Molecular/métodos , Proteína SUMO-1/análise , Schizosaccharomyces/química , Schizosaccharomyces/metabolismo , SumoilaçãoRESUMO
The tandem affinity purification (TAP) method uses an epitope that contains two different affinity purification tags separated by a site-specific protease site to isolate a protein rapidly and easily. Proteins purified via the TAP tag are eluted under mild conditions, allowing them to be used for structural and biochemical analyses. The original TAP tag contains a calmodulin-binding peptide and the IgG-binding domain from protein A separated by a tobacco etch virus (TEV) protease cleavage site. After capturing the Protein A epitope on an IgG resin, bound proteins are released by incubation with the TEV protease and then isolated on a calmodulin matrix in the presence of calcium; elution from this resin is achieved by chelating calcium with EGTA. However, because the robustness of the calmodulin-binding step in this procedure is highly variable, we replaced the calmodulin-binding peptide with three copies of the FLAG epitope, (3× FLAG)-TEV-Protein A, which can be isolated using an anti-FLAG resin. Elution from this matrix is achieved in the presence of an excess of a 3× FLAG peptide. In addition to allowing proteins to be released under mild conditions, elution by the 3× FLAG peptide adds an extra layer of specificity to the TAP procedure, because it liberates only FLAG-tagged proteins.