RESUMO
High-intensity transcription and replication supercoil DNA to levels that can impede or halt these processes. As a potent transcription amplifier and replication accelerator, the proto-oncogene MYC must manage this interfering torsional stress. By comparing gene expression with the recruitment of topoisomerases and MYC to promoters, we surmised a direct association of MYC with topoisomerase 1 (TOP1) and TOP2 that was confirmed in vitro and in cells. Beyond recruiting topoisomerases, MYC directly stimulates their activities. We identify a MYC-nucleated "topoisome" complex that unites TOP1 and TOP2 and increases their levels and activities at promoters, gene bodies, and enhancers. Whether TOP2A or TOP2B is included in the topoisome is dictated by the presence of MYC versus MYCN, respectively. Thus, in vitro and in cells, MYC assembles tools that simplify DNA topology and promote genome function under high output conditions.
Assuntos
DNA Topoisomerases Tipo II/metabolismo , Neoplasias/enzimologia , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transcrição Gênica , Animais , Replicação do DNA , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , DNA Topoisomerases Tipo II/genética , DNA de Neoplasias/biossíntese , DNA de Neoplasias/genética , DNA Super-Helicoidal/biossíntese , DNA Super-Helicoidal/genética , Ativação Enzimática , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Células K562 , Complexos Multienzimáticos , Neoplasias/genética , Neoplasias/patologia , Proteínas de Ligação a Poli-ADP-Ribose/genética , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-myc/genética , RatosRESUMO
As cells enter mitosis, chromatin compacts to facilitate chromosome segregation yet remains transcribed. Transcription supercoils DNA to levels that can impede further progression of RNA polymerase II (RNAPII) unless it is removed by DNA topoisomerase 1 (TOP1). Using ChIP-seq on mitotic cells, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity by RNAPII during elongation allowed RNAPII clearance from genes in prometaphase and enabled chromosomal segregation. Disruption of the TOP1-RNAPII interaction impaired RNAPII spiking at promoters and triggered defects in the post-mitotic transcription program. This program includes factors necessary for cell growth, and cells with impaired TOP1-RNAPII interaction are more sensitive to inhibitors of mTOR signaling. We conclude that TOP1 is necessary for assisting transcription during mitosis with consequences for growth and gene expression long after mitosis is completed. In this sense, TOP1 ensures that cellular memory is preserved in subsequent generations.
Assuntos
Proliferação de Células , Montagem e Desmontagem da Cromatina , Neoplasias Colorretais/enzimologia , DNA Topoisomerases Tipo I/metabolismo , Fase G1 , Mitose , RNA Polimerase II/metabolismo , Transcrição Gênica , Proliferação de Células/efeitos dos fármacos , Sequenciamento de Cromatina por Imunoprecipitação , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , DNA Topoisomerases Tipo I/genética , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica , Células HCT116 , Humanos , Inibidores de MTOR/farmacologia , Mitose/efeitos dos fármacos , RNA Polimerase II/genéticaRESUMO
Termination is a ubiquitous phase in every transcription cycle but is incompletely understood and a subject of debate. We used gene editing as a new approach to address its mechanism through engineered conditional depletion of the 5' â 3' exonuclease Xrn2 or the polyadenylation signal (PAS) endonuclease CPSF73 (cleavage and polyadenylation specificity factor 73). The ability to rapidly control Xrn2 reveals a clear and general role for it in cotranscriptional degradation of 3' flanking region RNA and transcriptional termination. This defect is characterized genome-wide at high resolution using mammalian native elongating transcript sequencing (mNET-seq). An Xrn2 effect on termination requires prior RNA cleavage, and we provide evidence for this by showing that catalytically inactive CPSF73 cannot restore termination to cells lacking functional CPSF73. Notably, Xrn2 plays no significant role in either Histone or small nuclear RNA (snRNA) gene termination even though both RNA classes undergo 3' end cleavage. In sum, efficient termination on most protein-coding genes involves CPSF73-mediated RNA cleavage and cotranscriptional degradation of polymerase-associated RNA by Xrn2. However, as CPSF73 loss caused more extensive readthrough transcription than Xrn2 elimination, it likely plays a more underpinning role in termination.
Assuntos
Fator de Especificidade de Clivagem e Poliadenilação/metabolismo , Exorribonucleases/fisiologia , RNA Polimerase II/metabolismo , Terminação da Transcrição Genética , Regiões 3' não Traduzidas , Linhagem Celular , Fator de Especificidade de Clivagem e Poliadenilação/antagonistas & inibidores , Fator de Especificidade de Clivagem e Poliadenilação/genética , Exorribonucleases/antagonistas & inibidores , Exorribonucleases/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Histonas , Humanos , Ácidos Indolacéticos/farmacologia , Mutação , RNA Nuclear Pequeno/genética , Análise de Sequência de RNARESUMO
The conditional depletion of a protein of interest (POI) is useful not only for loss-of-function studies, but also for the modulation of biological pathways. Technologies that work at the level of DNA, mRNA, and protein are available for temporal protein depletion. Compared with technologies targeting the pretranslation steps, direct protein depletion (or protein knockdown approaches) is advantageous in terms of specificity, reversibility, and time required for depletion, which can be achieved by fusing a POI with a protein domain called a degron that induces rapid proteolysis of the fusion protein. Conditional degrons can be activated or inhibited by temperature, small molecules, light, or the expression of another protein. The conditional degron-based technologies currently available are described and discussed.
Assuntos
Regulação da Expressão Gênica , Complexo de Endopeptidases do Proteassoma/metabolismo , Biossíntese de Proteínas , Proteômica/métodos , Animais , Sistemas CRISPR-Cas , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos da radiação , Humanos , Integrases/genética , Integrases/metabolismo , Luz , Morfolinos/genética , Morfolinos/metabolismo , Plantas/genética , Plantas/metabolismo , Biossíntese de Proteínas/efeitos dos fármacos , Biossíntese de Proteínas/efeitos da radiação , Domínios Proteicos , Engenharia de Proteínas , Proteólise/efeitos dos fármacos , Proteólise/efeitos da radiação , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologiaRESUMO
DNA replication fork progression can be disrupted at difficult to replicate loci in the human genome, which has the potential to challenge chromosome integrity. This replication fork disruption can lead to the dissociation of the replisome and the formation of DNA damage. To model the events stemming from replisome dissociation during DNA replication perturbation, we used a degron-based system for inducible proteolysis of a subunit of the replicative helicase. We show that MCM2-depleted cells activate a DNA damage response pathway and generate replication-associated DNA double-strand breaks (DSBs). Remarkably, these cells maintain some DNA synthesis in the absence of MCM2, and this requires the MCM8-9 complex, a paralog of the MCM2-7 replicative helicase. We show that MCM8-9 functions in a homologous recombination-based pathway downstream from RAD51, which is promoted by DSB induction. This RAD51/MCM8-9 axis is distinct from the recently described RAD52-dependent DNA synthesis pathway that operates in early mitosis at common fragile sites. We propose that stalled replication forks can be restarted in S phase via homologous recombination using MCM8-9 as an alternative replicative helicase.
Assuntos
Replicação do DNA/genética , DNA/biossíntese , Proteínas de Manutenção de Minicromossomo/metabolismo , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla , Ativação Enzimática/genética , Células HCT116 , Recombinação Homóloga/genética , Humanos , Componente 2 do Complexo de Manutenção de Minicromossomo/genética , Componente 2 do Complexo de Manutenção de Minicromossomo/metabolismo , Proteínas de Manutenção de Minicromossomo/genética , Mutação , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Fase S/genéticaRESUMO
Cowden syndrome (CS) is a rare autosomal dominant disorder associated with multiple hamartomatous and neoplastic lesions in various organs. Most CS patients have been found to have germline mutations in the PTEN tumor suppressor. In the present study, we investigated the causative gene of CS in a family of PTEN (phosphatase and tensin homolog deleted on chromosome 10) -negative CS patients. Whole exome sequencing analysis revealed AMBRA1 (Autophagy and Beclin 1 Regulator 1) as a novel candidate gene harboring two germline variants: p.Gln30Arg (Q30R) and p.Arg1195Ser (R1195S). AMBRA1 is a key regulator of the autophagy signaling network and a tumor suppressor. To functionally validate the role of AMBRA1 in the clinical manifestations of CS, we generated AMBRA1 depletion and Q30R mutation in hTERT-RPE1 (humanTelomerase Reverse Transcriptase-immortalized Retinal Pigmented Epithelial cells) using the CRISPR-Cas9 gene editing system. We observed that both AMBRA1-depleted and mutant cells showed accumulation in the S phase, leading to hyperproliferation, which is a characteristic of hamartomatous lesions. Specifically, the AMBRA1 Q30R mutation disturbed the G1/S transition of cells, leading to continuous mitotic entry of mutant cells, irrespective of the extracellular condition. From our analysis of primary ciliogenesis in these cells, we speculated that the mitotic entry of AMBRA1 Q30R mutants could be due to non-functional primary cilia that lead to impaired processing of extracellular sensory signals. Additionally, we observed a situs inversus phenotype in ambra1-depleted zebrafish, a developmental abnormality resulting from dysregulated primary ciliogenesis. Taken together, we established that the AMBRA1 Q30R mutation that we observed in CS patients might play an important role in inducing the hyperproliferative potential of cells through regulating primary ciliogenesis.
Assuntos
Síndrome do Hamartoma Múltiplo , Animais , Proteína Beclina-1/genética , Mutação em Linhagem Germinativa , Síndrome do Hamartoma Múltiplo/complicações , Síndrome do Hamartoma Múltiplo/genética , Síndrome do Hamartoma Múltiplo/patologia , Mutação , PTEN Fosfo-Hidrolase/genética , DNA Polimerase Dirigida por RNA/genética , Tensinas/genética , Peixe-Zebra/genéticaRESUMO
Cancer stem-like cells (CSCs) induce drug resistance and recurrence of tumors when they experience DNA replication stress. However, the mechanisms underlying DNA replication stress in CSCs and its compensation remain unclear. Here, we demonstrate that upregulated c-Myc expression induces stronger DNA replication stress in patient-derived breast CSCs than in differentiated cancer cells. Our results suggest critical roles for mini-chromosome maintenance protein 10 (MCM10), a firing (activating) factor of DNA replication origins, to compensate for DNA replication stress in CSCs. MCM10 expression is upregulated in CSCs and is maintained by c-Myc. c-Myc-dependent collisions between RNA transcription and DNA replication machinery may occur in nuclei, thereby causing DNA replication stress. MCM10 may activate dormant replication origins close to these collisions to ensure the progression of replication. Moreover, patient-derived breast CSCs were found to be dependent on MCM10 for their maintenance, even after enrichment for CSCs that were resistant to paclitaxel, the standard chemotherapeutic agent. Further, MCM10 depletion decreased the growth of cancer cells, but not of normal cells. Therefore, MCM10 may robustly compensate for DNA replication stress and facilitate genome duplication in cancer cells in the S-phase, which is more pronounced in CSCs. Overall, we provide a preclinical rationale to target the c-Myc-MCM10 axis for preventing drug resistance and recurrence of tumors.
Assuntos
Neoplasias da Mama/genética , Proteínas de Manutenção de Minicromossomo/metabolismo , Recidiva Local de Neoplasia/genética , Células-Tronco Neoplásicas/patologia , Proteínas Proto-Oncogênicas c-myc/metabolismo , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Mama/patologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Dano ao DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Humanos , Proteínas de Manutenção de Minicromossomo/genética , Recidiva Local de Neoplasia/patologia , Recidiva Local de Neoplasia/prevenção & controle , Células-Tronco Neoplásicas/efeitos dos fármacos , Cultura Primária de Células , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Esferoides Celulares , Células Tumorais Cultivadas , Regulação para CimaRESUMO
Chk1 (encoded by CHEK1 in mammals) is an evolutionarily conserved protein kinase that transduces checkpoint signals from ATR to Cdc25A during the DNA damage response (DDR). In mammals, Chk1 also controls cellular proliferation even in the absence of exogenous DNA damage. However, little is known about how Chk1 regulates unperturbed cell cycle progression, and how this effect under physiological conditions differs from its regulatory role in DDR. Here, we have established near-diploid HCT116 cell lines containing endogenous Chk1 protein tagged with a minimum auxin-inducible degron (mAID) through CRISPR/Cas9-based gene editing. Establishment of these cells enabled us to induce specific and rapid depletion of the endogenous Chk1 protein, which resulted in aberrant accumulation of DNA damage factors that induced cell cycle arrest at S or G2 phase. Cdc25A was stabilized upon Chk1 depletion before the accumulation of DNA damage factors. Simultaneous depletion of Chk1 and Cdc25A partially suppressed the defects caused by Chk1 single depletion. These results indicate that, similar to its function in DDR, Chk1 controls normal cell cycle progression mainly by inducing Cdc25A degradation.
Assuntos
Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Pontos de Checagem da Fase G2 do Ciclo Celular , Proteólise , Pontos de Checagem da Fase S do Ciclo Celular , Fosfatases cdc25/metabolismo , Sistemas CRISPR-Cas , Quinase 1 do Ponto de Checagem/genética , Edição de Genes , Células HCT116 , Humanos , Fosfatases cdc25/genéticaRESUMO
At the onset of procentriole formation, a structure called the cartwheel is formed adjacent to the pre-existing centriole. SAS-6 proteins are thought to constitute the hub of the cartwheel structure. However, the exact function of the cartwheel in the process of centriole formation has not been well characterized. In this study, we focused on the functions of human SAS-6 (HsSAS-6, also known as SASS6). By using an in vitro reconstitution system with recombinant HsSAS-6, we first observed its conserved molecular property of forming the central part of the cartwheel structure. Furthermore, we uncovered critical functions of HsSAS-6 by using a combination of an auxin-inducible HsSAS-6-degron (AID) system and super-resolution microscopy in human cells. Our results demonstrate that the HsSAS-6 is required not only for the initiation of centriole formation, but also for the stabilization of centriole intermediates. Moreover, after procentriole formation, HsSAS-6 is necessary for limiting Plk4 accumulation at the centrioles and thereby suppressing the formation of initiation sites that would otherwise promote the development of extra procentrioles. Overall, these findings illustrate the conserved and fundamental functions of the cartwheel in centriole duplication.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Centríolos/metabolismo , Técnicas de Cultura de Células , Ciclo Celular/fisiologia , Humanos , Proteínas Serina-Treonina Quinases/metabolismoRESUMO
Centromeres play several important roles in ensuring proper chromosome segregation. Not only do they promote kinetochore assembly for microtubule attachment, but they also support robust sister chromatid cohesion at pericentromeres and facilitate replication of centromeric DNA early in S phase. However, it is still elusive how centromeres orchestrate all these functions at the same site. Here, we show that the budding yeast Dbf4-dependent kinase (DDK) accumulates at kinetochores in telophase, facilitated by the Ctf19 kinetochore complex. This promptly recruits Sld3-Sld7 replication initiator proteins to pericentromeric replication origins so that they initiate replication early in S phase. Furthermore, DDK at kinetochores independently recruits the Scc2-Scc4 cohesin loader to centromeres in G1 phase. This enhances cohesin loading and facilitates robust pericentromeric cohesion in S phase. Thus, we have found the central mechanism by which kinetochores orchestrate early S phase DNA replication and robust sister chromatid cohesion at microtubule attachment sites.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Replicação do DNA , Cinetocoros/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/genética , Centrômero/genética , Centrômero/metabolismo , Cromátides/genética , Cromátides/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Serina-Treonina Quinases/genética , Fase S/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Although condensins play essential roles in mitotic chromosome assembly, Ki-67 (also known as MKI67), a protein localizing to the periphery of mitotic chromosomes, had also been shown to make a contribution to the process. To examine their respective roles, we generated a set of HCT116-based cell lines expressing Ki-67 and/or condensin subunits that were fused with an auxin-inducible degron for their conditional degradation. Both the localization and the dynamic behavior of Ki-67 on mitotic chromosomes were not largely affected upon depletion of condensin subunits, and vice versa. When both Ki-67 and SMC2 (a core subunit of condensins) were depleted, ball-like chromosome clusters with no sign of discernible thread-like structures were observed. This severe defective phenotype was distinct from that observed in cells depleted of either Ki-67 or SMC2 alone. Our results show that Ki-67 and condensins, which localize to the external surface and the central axis of mitotic chromosomes, respectively, have independent yet cooperative functions in supporting the structural integrity of mitotic chromosomes.
Assuntos
Adenosina Trifosfatases/metabolismo , Cromossomos Humanos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Antígeno Ki-67/metabolismo , Mitose , Complexos Multiproteicos/metabolismo , Adenosina Trifosfatases/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular , Linhagem Celular Tumoral , Cromossomos Humanos/genética , Proteínas de Ligação a DNA/genética , Humanos , Ácidos Indolacéticos/metabolismo , Antígeno Ki-67/genética , Complexos Multiproteicos/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Transporte ProteicoRESUMO
Controlling protein expression using a degron is advantageous because the protein of interest can be rapidly depleted in a reversible manner. We pioneered the development of the auxin-inducible degron (AID) technology by transplanting a plant-specific degradation pathway to non-plant cells. In human cells expressing an E3 ligase component, OsTIR1, it is possible to degrade a degron-fused protein with a half-life of 15-45â¯min in the presence of the phytohormone auxin. We reported previously the generation of human HCT116 mutants in which the C terminus of endogenous proteins was fused with the degron by CRISPR-Cas9-based knock-in. Here, we show new plasmids for N-terminal tagging and describe a detailed protocol for the generation of AID mutants of human HCT116 and DLD1 cells. Moreover, we report the use of an OsTIR1 inhibitor, auxinole, to suppress leaky degradation of degron-fused proteins. The addition of auxinole is also useful for rapid re-expression after depletion of degron-fused proteins. These improvements enhance the utility of AID technology for studying protein function in living human cells.
Assuntos
Ácidos Indolacéticos/farmacologia , Proteólise/efeitos dos fármacos , Proteínas Recombinantes de Fusão/metabolismo , Sistemas CRISPR-Cas/genética , Técnicas de Cultura de Células/métodos , Vetores Genéticos/genética , Células HCT116 , Meia-Vida , Humanos , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas de Plantas/antagonistas & inibidores , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plasmídeos/genética , Proteínas Recombinantes de Fusão/genética , Transfecção/métodos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
MCM8 and MCM9 are paralogues of the MCM2-7 eukaryotic DNA replication helicase proteins and play a crucial role in a homologous recombination-mediated repair process to resolve replication stress by fork stalling. Thus, deficiency of MCM8-9 sensitizes cells to replication stress caused, for example, by platinum compounds that induce interstrand cross-links. It is suggested that cancer cells undergo more replication stress than normal cells due to hyperstimulation of growth. Therefore, it is possible that inhibiting MCM8-9 selectively hypersensitizes cancer cells to platinum compounds and poly(ADP-ribose) polymerase inhibitors, both of which hamper replication fork progression. Here, we inhibited MCM8-9 in transformed and nontransformed cells and examined their sensitivity to cisplatin and olaparib. We found that knockout of MCM9 or knockdown of MCM8 selectively hypersensitized transformed cells to cisplatin and olaparib. In agreement with reported findings, RAS- and human papilloma virus type 16 E7-mediated transformation of human fibroblasts increased replication stress, as indicated by induction of multiple DNA damage responses (including formation of Rad51 foci). Such replication stress induced by oncogenes was further increased by knockdown of MCM8, providing a rationale for cancer-specific hypersensitization to cisplatin and olaparib. Finally, we showed that knocking out MCM9 increased the sensitivity of HCT116 xenograft tumors to cisplatin. Taken together, the data suggest that conceptual MCM8-9 inhibitors will be powerful cancer-specific chemosensitizers for platinum compounds and poly(ADP-ribose) polymerase inhibitors, thereby opening new avenues to the design of novel cancer chemotherapeutic strategies.
Assuntos
Cisplatino/farmacologia , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Ftalazinas/farmacologia , Piperazinas/farmacologia , Animais , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , Feminino , Células HCT116 , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neoplasias/metabolismo , Compostos Organoplatínicos/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo de DNA por Recombinação/efeitos dos fármacosRESUMO
Although the condensin complexes and topoisomerase IIα (TopoIIα) are the central players in mitotic chromosome formation, they are insufficient for its completion, and additional factors involved in the process have been extensively sought. In this study, we examined the possibility that Ki67, a perichromosomal protein widely used as a cell proliferation marker, is one such factor. Using a combination of auxin-inducible degron and CRISPR-Cas9-based gene editing technologies, we generated a human HCT116 cell line in which Ki67 is rapidly depleted in a few hours. The removal of Ki67 before mitotic entry did not impact the early mitotic chromosome assembly observed in prophase but subsequently resulted in the formation of misshapen mitotic chromosomes. When Ki67 was removed after mitotic entry, preassembled rod-shaped mitotic chromosomes became disorganized. In addition, we show that Ki67 and TopoIIα are reciprocally coimmunoprecipitated from mitotic cell extracts. These observations indicate that Ki67 aids the finalization of mitotic chromosome formation and helps maintain rod-shaped chromosome architecture, likely in collaboration with TopoIIα. Together, these findings represent a new model in which mitotic chromosome architecture is supported both internally and externally.
Assuntos
Cromossomos Humanos/fisiologia , Antígeno Ki-67/fisiologia , Mitose/fisiologia , Antígenos de Neoplasias/fisiologia , DNA Topoisomerases Tipo II/fisiologia , Proteínas de Ligação a DNA/fisiologia , Células HCT116 , Células HeLa , Humanos , Antígeno Ki-67/genética , Modelos BiológicosRESUMO
Homologous recombination (HR) is initiated by double-strand break (DSB) resection, during which DSBs are processed by nucleases to generate 3' single-strand DNA. DSB resection is initiated by CtIP and Mre11 followed by long-range resection by Dna2 and Exo1 in Saccharomyces cerevisiae. To analyze the relative contribution of four nucleases, CtIP, Mre11, Dna2 and Exo1, to DSB resection, we disrupted genes encoding these nucleases in chicken DT40 cells. CtIP and Dna2 are required for DSB resection, whereas Exo1 is dispensable even in the absence of Dna2, which observation agrees with no developmental defect in Exo1-deficient mice. Despite the critical role of Mre11 in DSB resection in S. cerevisiae, loss of Mre11 only modestly impairs DSB resection in DT40 cells. To further test the role of CtIP and Mre11 in other species, we conditionally disrupted CtIP and MRE11 genes in the human TK6 B cell line. As with DT40 cells, CtIP contributes to DSB resection considerably more significantly than Mre11 in TK6 cells. Considering the critical role of Mre11 in HR, this study suggests that Mre11 is involved in a mechanism other than DSB resection. In summary, CtIP and Dna2 are sufficient for DSB resection to ensure efficient DSB repair by HR.
Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Desoxirribonucleases/genética , Desoxirribonucleases/metabolismo , Recombinação Homóloga , Animais , Proteínas de Transporte/metabolismo , Linhagem Celular , Galinhas , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exodesoxirribonucleases/metabolismo , HumanosRESUMO
The main function of the centromere is to promote kinetochore assembly for spindle microtubule attachment. Two additional functions of the centromere, however, are becoming increasingly clear: facilitation of robust sister-chromatid cohesion at pericentromeres and advancement of replication of centromeric regions. The combination of these three centromere functions ensures correct chromosome segregation during mitosis. Here, we review the mechanisms of the kinetochore-microtubule interaction, focusing on sister-kinetochore bi-orientation (or chromosome bi-orientation). We also discuss the biological importance of robust pericentromeric cohesion and early centromere replication, as well as the mechanisms orchestrating these two functions at the microtubule attachment site.
Assuntos
Segregação de Cromossomos , Quebras de DNA , Cinetocoros/metabolismo , Animais , Aurora Quinase B/metabolismo , Replicação do DNA , Humanos , Microtúbulos/metabolismo , Leveduras/genética , Leveduras/metabolismoRESUMO
For accurate mitotic cell division, replicated chromatin must be assembled into chromosomes and faithfully segregated into daughter cells. While protein factors like condensin play key roles in this process, it is unclear how chromosome assembly proceeds as molecular events of nucleosomes in living cells and how condensins act on nucleosomes to organize chromosomes. To approach these questions, we investigate nucleosome behavior during mitosis of living human cells using single-nucleosome tracking, combined with rapid-protein depletion technology and computational modeling. Our results show that local nucleosome motion becomes increasingly constrained during mitotic chromosome assembly, which is functionally distinct from condensed apoptotic chromatin. Condensins act as molecular crosslinkers, locally constraining nucleosomes to organize chromosomes. Additionally, nucleosome-nucleosome interactions via histone tails constrain and compact whole chromosomes. Our findings elucidate the physical nature of the chromosome assembly process during mitosis.
Assuntos
Adenosina Trifosfatases , Cromatina , Proteínas de Ligação a DNA , Mitose , Complexos Multiproteicos , Nucleossomos , Humanos , Nucleossomos/metabolismo , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/genética , Complexos Multiproteicos/metabolismo , Cromatina/metabolismo , Histonas/metabolismo , Células HeLa , Cromossomos Humanos/metabolismo , Cromossomos Humanos/genética , Cromossomos/metabolismoRESUMO
To ensure timely duplication of the entire eukaryotic genome, thousands of replication machineries (replisomes) act on genomic DNA at any time during S phase. In the final stages of this process, replisomes are unloaded from chromatin. Unloading is driven by polyubiquitylation of MCM7, a subunit of the terminated replicative helicase, and processed by p97/VCP segregase. Most of our knowledge of replication termination comes from model organisms, and little is known about how this process is executed and regulated in human somatic cells. Here we show that replisome disassembly in this system requires CUL2LRR1-driven MCM7 ubiquitylation, p97, and UBXN7 for unloading and provide evidence for "backup" mitotic replisome disassembly, demonstrating conservation of such mechanisms. Finally, we find that small-molecule inhibitors against Cullin ubiquitin ligases (CULi) and p97 (p97i) affect replisome unloading but also lead to induction of replication stress in cells, which limits their usefulness to specifically target replisome disassembly processes.
RESUMO
Cell division is the basis for the propagation of life and requires accurate duplication of all genetic information. DNA damage created during replication (replication stress) is a major cause of cancer, premature aging and a spectrum of other human disorders. Over the years, TRAIP E3 ubiquitin ligase has been shown to play a role in various cellular processes that govern genome integrity and faultless segregation. TRAIP is essential for cell viability, and mutations in TRAIP ubiquitin ligase activity lead to primordial dwarfism in patients. Here, we have determined the mechanism of inhibition of cell proliferation in TRAIP-depleted cells. We have taken advantage of the auxin induced degron system to rapidly degrade TRAIP within cells and to dissect the importance of various functions of TRAIP in different stages of the cell cycle. We conclude that upon rapid TRAIP degradation, specifically in S-phase, cells cease to proliferate, arrest in G2 stage of the cell cycle and undergo senescence. Our findings reveal that TRAIP works in S-phase to prevent DNA damage at transcription start sites, caused by replication-transcription conflicts.
Assuntos
Ubiquitina-Proteína Ligases , Humanos , Fase S/genética , Divisão Celular/genética , Proliferação de Células/genética , Ciclo Celular , Sobrevivência Celular , Ubiquitina-Proteína Ligases/genéticaRESUMO
The division of labour among DNA polymerase underlies the accuracy and efficiency of replication. However, the roles of replicative polymerases have not been directly established in human cells. We developed polymerase usage sequencing (Pu-seq) in HCT116 cells and mapped Polε and Polα usage genome wide. The polymerase usage profiles show Polε synthesises the leading strand and Polα contributes mainly to lagging strand synthesis. Combining the Polε and Polα profiles, we accurately predict the genome-wide pattern of fork directionality plus zones of replication initiation and termination. We confirm that transcriptional activity contributes to the pattern of initiation and termination and, by separately analysing the effect of transcription on co-directional and converging forks, demonstrate that coupled DNA synthesis of leading and lagging strands is compromised by transcription in both co-directional and convergent forks. Polymerase uncoupling is particularly evident in the vicinity of large genes, including the two most unstable common fragile sites, FRA3B and FRA3D, thus linking transcription-induced polymerase uncoupling to chromosomal instability. Together, our result demonstrated that Pu-seq in human cells provides a powerful and straightforward methodology to explore DNA polymerase usage and replication fork dynamics.