RESUMEN
Generation of O6-methylguanine (O6-meG) by DNA-alkylating agents such as N-methyl N-nitrosourea (MNU) activates the multiprotein mismatch repair (MMR) complex and the checkpoint response involving ATR/CHK1 and ATM/CHK2 kinases, which may in turn trigger cell cycle arrest and apoptosis. The Bloom syndrome DNA helicase BLM interacts with the MMR complex, suggesting functional relevance to repair and checkpoint responses. We observed a strong interaction of BLM with MMR proteins in HeLa cells upon treatment with MNU as evidenced by co-immunoprecipitation as well as colocalization in the nucleus as revealed by dual immunofluorescence staining. Knockout of BLM sensitized HeLa MR cells to MNU-induced cell cycle disruption and enhanced expression of the apoptosis markers cleaved caspase-9 and PARP1. MNU-treated BLM-deficient cells also exhibited a greater number of 53BP1 foci and greater phosphorylation levels of H2AX at S139 and RPA32 at S8, indicating the accumulation of DNA double-strand breaks. These findings suggest that BLM prevents double-strand DNA breaks during the MMR-dependent DNA damage response and mitigates O6-meG-induced apoptosis.
Asunto(s)
Apoptosis , Reparación de la Incompatibilidad de ADN , RecQ Helicasas , Humanos , RecQ Helicasas/metabolismo , RecQ Helicasas/genética , Células HeLa , Roturas del ADN de Doble Cadena , Metilnitrosourea/toxicidad , Síndrome de Bloom/genética , Síndrome de Bloom/metabolismo , Síndrome de Bloom/patología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/genéticaRESUMEN
BACKGROUND: Sn1-type alkylating agents methylate the oxygen atom on guanine bases thereby producing O6-methylguanine. This modified base could pair with thymine and cytosine, resulting in the formation of O6-methylguanine/thymine mismatch during DNA replication, recognized by the mismatch repair (MMR) complex, which then initiates the DNA damage response and subsequent apoptotic processes. In our investigation of the molecular mechanisms underlying MMR-dependent apoptosis, we observed FANCD2 modification upon the activity of alkylating agent N-methyl-N-nitrosourea (MNU). This observation led us to hypothesize a relevant role for FANCD2 in the apoptosis induction process. METHODS AND RESULTS: We generated FANCD2 knockout cells using the CRISPR/Cas9 method in the human cervical cancer cell line HeLa MR. FANCD2-deficient cells exhibited MNU hypersensitivity. Upon MNU exposure, FANCD2 colocalized with the MMR complex. MNU-treated FANCD2 knockout cells displayed severe S phase delay followed by increased G2/M arrest and MMR-dependent apoptotic cell death. Moreover, FANCD2 knockout cells exhibited impaired CtIP and RAD51 recruitment to the damaged chromatin and DNA double-strand break accumulation, indicated by simultaneously observed increased γH2AX signal and 53BP1 foci. CONCLUSIONS: Our data suggest that FANCD2 is crucial for recruiting homologous recombination factors to the sites of the MMR-dependent replication stress to resolve the arrested replication fork and counteract O6-methylguanine-triggered MMR-dependent apoptosis.
Asunto(s)
Apoptosis , Reparación de la Incompatibilidad de ADN , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi , Guanina , Humanos , Reparación de la Incompatibilidad de ADN/genética , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/metabolismo , Proteína del Grupo de Complementación D2 de la Anemia de Fanconi/genética , Apoptosis/genética , Apoptosis/efectos de los fármacos , Guanina/metabolismo , Guanina/análogos & derivados , Células HeLa , Daño del ADN , Metilnitrosourea/toxicidad , Sistemas CRISPR-Cas , Técnicas de Inactivación de Genes , Recombinasa Rad51/metabolismo , Recombinasa Rad51/genética , Replicación del ADN/efectos de los fármacos , Replicación del ADN/genéticaRESUMEN
The mismatch repair (MMR) complex is composed of MutSα (MSH2-MSH6) and MutLα (MLH1-PMS2) and specifically recognizes mismatched bases during DNA replication. O6-Methylguanine is produced by treatment with alkylating agents, such as N-methyl-N-nitrosourea (MNU), and during DNA replication forms a DNA mismatch (i.e. an O6-methylguanine/thymine pair) and induces a G/C to A/T transition mutation. To prevent this outcome, cells carrying this DNA mismatch are eliminated by MMR-dependent apoptosis, but the underlying molecular mechanism is unclear. In this study, we provide evidence that the chromatin-regulatory and ATP-dependent nucleosome-remodeling protein SMARCAD1 is involved in the induction of MMR-dependent apoptosis in human cells. Unlike control cells, SMARCAD1-knockout cells (ΔSMARCAD1) were MNU-resistant, and the appearance of a sub-G1 population and caspase-9 activation were significantly suppressed in the ΔSMARCAD1 cells. Furthermore, the MNU-induced mutation frequencies were increased in these cells. Immunoprecipitation analyses revealed that the recruitment of MutLα to chromatin-bound MutSα, observed in SMARCAD1-proficient cells, is suppressed in ΔSMARCAD1 cells. Of note, the effect of SMARCAD1 on the recruitment of MutLα exclusively depended on the ATPase activity of the protein. On the basis of these findings, we propose that SMARCAD1 induces apoptosis via its chromatin-remodeling activity, which helps recruit MutLα to MutSα on damaged chromatin.
Asunto(s)
Apoptosis , Cromatina/metabolismo , Daño del ADN , ADN Helicasas/metabolismo , Reparación de la Incompatibilidad de ADN , Proteínas MutL/metabolismo , Proteína 2 Homóloga a MutS/metabolismo , Línea Celular Tumoral , Técnicas de Inactivación de Genes , Humanos , Metilnitrosourea , Modelos Biológicos , Tasa de Mutación , Transducción de SeñalRESUMEN
O6 -Methylguanines (O6 -meG), which are produced in DNA by the action of alkylating agents, are mutagenic and cytotoxic, and induce apoptosis in a mismatch repair (MMR) protein-dependent manner. To understand the molecular mechanism of O6 -meG-induced apoptosis, we performed functional analyses of FANCD2 and FANCI-associated nuclease 1 (FAN1), which was identified as an interacting partner of MLH1. Immunoprecipitation analyses showed that FAN1 interacted with both MLH1 and MSH2 after treatment with N-methyl-N-nitrosourea (MNU), indicating the formation of a FAN1-MMR complex. In comparison with control cells, FAN1-knockdown cells were more resistant to MNU, and the appearances of a sub-G1 population and caspase-9 activation were suppressed. FAN1 formed nuclear foci in an MLH1-dependent manner after MNU treatment, and some were colocalized with both MLH1 foci and single-stranded DNA (ssDNA) created at damaged sites. Under the same condition, FANCD2 also formed nuclear foci, although it was dispensable for the formation of FAN1 foci and ssDNA. MNU-induced formation of ssDNA was dramatically suppressed in FAN1-knockdown cells. We therefore propose that FAN1 is loaded on chromatin through the interaction with MLH1 and produces ssDNA by its exonuclease activity, which contributes to the activation of the DNA damage response followed by the induction of apoptosis triggered by O6 -meG.
Asunto(s)
Apoptosis/efectos de los fármacos , Cromatina/metabolismo , Endodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/metabolismo , Guanina/análogos & derivados , Enzimas Multifuncionales/metabolismo , Homólogo 1 de la Proteína MutL/metabolismo , Daño del ADN , Endodesoxirribonucleasas/genética , Exodesoxirribonucleasas/genética , Guanina/farmacología , Células HeLa , Humanos , Enzimas Multifuncionales/genéticaRESUMEN
Repeat destabilisation is variously associated with human disease. In neoplastic diseases, microsatellite instability (MSI) has been regarded as simply reflecting DNA mismatch repair (MMR) deficiency. However, several discrepancies have been pointed out. Firstly, the MSI+ phenotype is not uniform in human neoplasms. Established classification utilises the frequency of microsatellite changes, i.e. MSI-H (high) and -L (low), the former regarded as an authentic MMR-defective phenotype. In addition, we have observed the qualitatively distinct modes of MSI, i.e. Type A and Type B. One discrepancy we previously pointed out is that tumours occurring in MMR gene knockout mice exhibited not drastic microsatellite changes typical in MSI-H tumours (i.e. Type B mode) but minor and more subtle alterations (i.e. Type A mode). In the present study, MSH2 mutations reported in Lynch syndrome (LS) kindred have been introduced into HeLa cells using the CRISPR/Cas9 system. The established mutant clones clearly exhibited MMR-defective phenotypes with alkylating agent-tolerance and elevated mutation frequencies. Nevertheless, microsatellites were not markedly destabilised as in MSI-H tumours occurring in LS patients, and all the observed alterations were uniformly Type A, which confirms the results in mice. Our findings suggest added complexities to the molecular mechanisms underlying repeat destabilisation in human genome.
Asunto(s)
Sistemas CRISPR-Cas , Neoplasias Colorrectales Hereditarias sin Poliposis/patología , Edición Génica , Genómica/métodos , Inestabilidad de Microsatélites , Proteína 2 Homóloga a MutS/genética , Mutación , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Células HeLa , Humanos , FenotipoRESUMEN
In all organisms, DNA and protein are synthesized by dedicated, but unrelated, machineries that move along distinct templates with no apparent coordination. Therefore, connections between DNA replication and translation are a priori unexpected. However, recent findings support the existence of such connections throughout the three domains of life. In particular, we recently identified in archaeal genomes a conserved association between genes encoding DNA replication and ribosome-related proteins which all have eukaryotic homologs. We believe that this gene organization is biologically relevant and, moreover, that it suggests the existence of a mechanism coupling DNA replication and translation in Archaea and Eukarya.
Asunto(s)
Archaea/genética , Bacterias/genética , Replicación del ADN/genética , Biosíntesis de Proteínas/genética , Animales , Archaea/metabolismo , Bacterias/metabolismo , Proteínas Bacterianas/metabolismo , GTP Fosfohidrolasas/metabolismo , Proteínas de Unión al GTP/metabolismo , Orden Génico , Genoma Arqueal , Genoma Bacteriano , Genoma Humano , Guanosina Pentafosfato/metabolismo , Guanosina Tetrafosfato/metabolismo , Humanos , Familia de Multigenes , Neoplasias/etiologíaRESUMEN
Respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in children. Despite decades of efforts, no effective therapies are available. We recently reported that extracts of Ephedra Herb and Cinnamon Bark interacted with the G attachment protein of RSV to inhibit infectivity. The present in vitro study aimed to investigate the antiviral effect of ephedrine alkaloids-free Ephedra Herb extract (EFE), which is characterized by free of harmful effects of ephedrine alkaloids in Ephedra Herb, on experimental RSV infection. Infection of RSV into A549 cells simultaneously with EFE resulted the significant reduction of RSV RNA, viral protein, and viral titers after the incubation of the cells. We found that RSV attachment to the cell surface was inhibited both in the presence of EFE and when RSV particles were pre-treated with EFE. We also found that EFE specifically interacted with the central conserved domain of RSV G protein by surface plasmon resonance, demonstrating that specific binding of G protein to the cellular receptor was inhibited by EFE. Another mechanism was found in which a higher concentration of EFE inhibited the viral load immediately after the viral entry into host cells, suggesting the inhibition of viral RNA replication. These results demonstrate that EFE worked against RSV infection through multiple antiviral mechanisms, a unique feature of this crude drug extract.
RESUMEN
The DNA sliding clamp is a multifunctional protein involved in cellular DNA transactions. In Archaea and Eukaryota, proliferating cell nuclear antigen (PCNA) is the sliding clamp. The ring-shaped PCNA encircles double-stranded DNA within its central hole and tethers other proteins on DNA. The majority of Crenarchaeota, a subdomain of Archaea, have multiple PCNA homologues, and they are capable of forming heterotrimeric rings for their functions. In contrast, most organisms in Euryarchaeota, the other major subdomain, have a single PCNA forming a homotrimeric ring structure. Among the Euryarchaeota whose genome is sequenced, Thermococcus kodakarensis is the only species with two genes encoding PCNA homologues on its genome. We cloned the two genes from the T. kodakarensis genome, and the gene products, PCNA1 and PCNA2, were characterized. PCNA1 stimulated the DNA synthesis reactions of the two DNA polymerases, PolB and PolD, from T. kodakarensis in vitro. PCNA2, however, only had an effect on PolB. We were able to disrupt the gene for PCNA2, whereas gene disruption for PCNA1 was not possible, suggesting that PCNA1 is essential for DNA replication. The sensitivities of the Δpcna2 mutant strain to ultraviolet irradiation (UV), methyl methanesulfonate (MMS) and mitomycin C (MMC) were indistinguishable from those of the wild-type strain.
Asunto(s)
Proteínas Arqueales/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Thermococcus/metabolismo , Adenosina Trifosfatasas/química , Adenosina Trifosfatasas/aislamiento & purificación , Adenosina Trifosfatasas/metabolismo , Proteínas Arqueales/química , Proteínas Arqueales/genética , Proteínas Arqueales/aislamiento & purificación , Daño del ADN , ADN Polimerasa III/química , ADN Polimerasa beta/química , Reparación del ADN , Replicación del ADN , ADN de Archaea/química , ADN de Archaea/metabolismo , Técnicas de Inactivación de Genes , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/aislamiento & purificación , Unión Proteica , Subunidades de Proteína/química , Subunidades de Proteína/aislamiento & purificación , Subunidades de Proteína/metabolismo , Proteína de Replicación C/química , Proteína de Replicación C/aislamiento & purificación , Proteína de Replicación C/metabolismo , Thermococcus/genética , Thermococcus/crecimiento & desarrolloRESUMEN
Although respiratory syncytial virus (RSV) is a major cause of respiratory tract infection in children, no effective therapies are available. Recently, RSV G, the attachment glycoprotein, has become a major focus in the development of therapeutic strategies against RSV infection. Treatment of RSV-infected cultured cells with maoto, a traditional herbal medicine for acute febrile diseases, significantly reduced the viral RNA and titers. RSV attachment to the cell surface was inhibited both in the presence of maoto and when RSV particles were pre-treated with maoto. We demonstrated that maoto components, Ephedrae Herba (EH) and Cinnamomi Cortex (CC), specifically interacted with the central conserved domain (CCD) of G protein, and also found that this interaction blocked viral attachment to the cellular receptor CX3CR1. Genetic mutation of CX3C motif on the CCD, the epitope for CX3CR1, decreased the binding capacity to EH and CC, suggesting that CX3C motif was the target for EH and CC. Finally, oral administration of maoto for five days to RSV-infected mice significantly reduced the lung viral titers. These experiments clearly showed the anti-RSV activity of EH and CC mixed in maoto. Taken together, this study provides insights for the rational design of therapies against RSV infection.
Asunto(s)
Antivirales/uso terapéutico , Medicamentos Herbarios Chinos/farmacología , Infecciones por Virus Sincitial Respiratorio/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Antivirales/química , Antivirales/farmacología , Cinnamomum zeylanicum , Medicamentos Herbarios Chinos/química , Ratones , Modelos Moleculares , Conformación Proteica , Infecciones por Virus Sincitial Respiratorio/virología , Virus Sincitiales Respiratorios , Proteínas Virales de Fusión , Carga Viral , Acoplamiento ViralRESUMEN
Mismatch repair (MMR) is a highly conserved DNA repair pathway that corrects mismatched bases during DNA replication. The biological significance of MMR in human cells is underscored by the fact that dysfunction of the MMR pathway results in Lynch syndrome, which is associated with a genetic predisposition to different cancer types. We have previously established a reporter mismatch plasmid to evaluate MMR using fluorescent proteins in living cells. However, the preparation of these plasmids requires significant amounts of time and money, which reduces their broad applicability. To overcome the abovementioned limitations, we produced in this study a novel reporter plasmid, pBSII NLS-MC-EGFP-tdTomato (pBET2), that can be used in the oligo swapping method. In this method, a nicking endonuclease produces a single-stranded DNA gap on a double-stranded DNA plasmid that can be replaced by ligation with synthetic oligonucleotides. It is significantly easier and more user-friendly than previous assays, which require in vitro DNA synthesis with single-stranded plasmid DNA and purification using ultracentrifugation in cesium chloride-ethidium bromide gradients. The plasmid also contains a nicking site that allows the MMR repair machinery to efficiently distinguish the newly synthesized strand as a target for repair. In addition, a nuclear localization signal facilitates green fluorescent protein expression in the nucleus, which helps to verify the effectiveness of MMR using fluorescence microscopy. Similar to the previous reporter plasmid, this construct facilitates the assessment of MMR proficiency in human living cells via the expression of fluorescent proteins while overcoming many of the negative aspects of the previous protocol.
Asunto(s)
Reparación de la Incompatibilidad de ADN , Reparación del ADN , ADN/metabolismo , Fluorescencia , Humanos , Plásmidos/genéticaRESUMEN
In prokaryotes and yeasts, DNA polymerase proofreading (PPR) and DNA mismatch repair (MMR) cooperatively counteracts replication errors leading to repeat sequence destabilization (i.e. insertions/deletions of repeat units). However, PPR has not thus far been regarded as a mechanism stabilizing repeat sequences in higher eukaryotic cells. In a human cancer cell line, DLD-1, which carries mutations in both MSH6 and the Exo domain of POLD1, we previously observed that mononucleotide microsatellites were markedly destabilized whereas being stable in the simple MMR-defective backgrounds. In this study, we introduced the Exo domain mutation found in DLD-1 cells into MSH2-null HeLa cell clones, using CRISPR/Cas9 system. In the established Exo-/MMR-mutated HeLa clones, mononucleotide repeat sequences were remarkably destabilized as in DLD-1 cells. In contrast, dinucleotide microsatellites were readily destabilized in the parental MMR-deficient backgrounds, and the instability was not notably increased in the genome-edited HeLa clones. Here, we show an involvement of the Exo domain functions of DNA polymerase delta in mononucleotide repeat stabilization in human cells, which also suggests a possible role division between DNA polymerase and MMR in repeat maintenance in the human genome.
Asunto(s)
Reparación de la Incompatibilidad de ADN , ADN Polimerasa III , Repeticiones de Microsatélite , Línea Celular Tumoral , ADN Polimerasa III/genética , Células HeLa , Humanos , Mutación , Dominios ProteicosRESUMEN
BACKGROUND: Pyrococcus furiosus Hjm (PfuHjm) is a structure-specific DNA helicase that was originally identified by in vitro screening for Holliday junction migration activity. It belongs to helicase superfamily 2, and shares homology with the human DNA polymerase Theta (PolTheta), HEL308, and Drosophila Mus308 proteins, which are involved in DNA repair. Previous biochemical and genetic analyses revealed that PfuHjm preferentially binds to fork-related Y-structured DNAs and unwinds their double-stranded regions, suggesting that this helicase is a functional counterpart of the bacterial RecQ helicase, which is essential for genome maintenance. Elucidation of the DNA unwinding and translocation mechanisms by PfuHjm will require its three-dimensional structure at atomic resolution. RESULTS: We determined the crystal structures of PfuHjm, in two apo-states and two nucleotide bound forms, at resolutions of 2.0-2.7 A. The overall structures and the local conformations around the nucleotide binding sites are almost the same, including the side-chain conformations, irrespective of the nucleotide-binding states. The architecture of Hjm was similar to that of Archaeoglobus fulgidus Hel308 complexed with DNA. An Hjm-DNA complex model, constructed by fitting the five domains of Hjm onto the corresponding Hel308 domains, indicated that the interaction of Hjm with DNA is similar to that of Hel308. Notably, sulphate ions bound to Hjm lie on the putative DNA binding surfaces. Electron microscopic analysis of an Hjm-DNA complex revealed substantial flexibility of the double stranded region of DNA, presumably due to particularly weak protein-DNA interactions. Our present structures allowed reasonable homology model building of the helicase region of human PolTheta, indicating the strong conformational conservation between archaea and eukarya. CONCLUSION: The detailed comparison between our DNA-free PfuHjm structure and the structure of Hel308 complexed with DNA suggests similar DNA unwinding and translocation mechanisms, which could be generalized to all of the members in the same family. Structural comparison also implied a minor rearrangement of the five domains during DNA unwinding reaction. The unexpected small contact between the DNA duplex region and the enzyme appears to be advantageous for processive helicase activity.
Asunto(s)
Proteínas Arqueales/química , Pyrococcus furiosus/enzimología , RecQ Helicasas/química , Proteínas Arqueales/ultraestructura , Archaeoglobus fulgidus/enzimología , ADN de Archaea/metabolismo , ADN de Archaea/ultraestructura , Humanos , Microscopía Electrónica , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , RecQ Helicasas/ultraestructura , Alineación de Secuencia , Homología Estructural de ProteínaRESUMEN
Archaeal RNA:pseudouridine-synthase (PUS) Cbf5 in complex with proteins L7Ae, Nop10 and Gar1, and guide box H/ACA sRNAs forms ribonucleoprotein (RNP) catalysts that insure the conversion of uridines into pseudouridines (Ψs) in ribosomal RNAs (rRNAs). Nonetheless, in the absence of guide RNA, Cbf5 catalyzes the in vitro formation of Ψ2603 in Pyrococcus abyssi 23S rRNA and of Ψ55 in tRNAs. Using gene-disrupted strains of the hyperthermophilic archaeon Thermococcus kodakarensis, we studied the in vivo contribution of proteins Nop10 and Gar1 to the dual RNA guide-dependent and RNA-independent activities of Cbf5 on 23S rRNA. The single-null mutants of the cbf5, nop10, and gar1 genes are viable, but display a thermosensitive slow growth phenotype. We also generated a single-null mutant of the gene encoding Pus10, which has redundant activity with Cbf5 for in vitro formation of Ψ55 in tRNA. Analysis of the presence of Ψs within the rRNA peptidyl transferase center (PTC) of the mutants demonstrated that Cbf5 but not Pus10 is required for rRNA modification. Our data reveal that, in contrast to Nop10, Gar1 is crucial for in vivo and in vitro RNA guide-independent formation of Ψ2607 (Ψ2603 in P. abyssi) by Cbf5. Furthermore, our data indicate that pseudouridylation at orphan position 2589 (2585 in P. abyssi), for which no PUS or guide sRNA has been identified so far, relies on RNA- and Gar1-dependent activity of Cbf5.
Asunto(s)
Seudouridina/metabolismo , ARN de Archaea/biosíntesis , ARN de Archaea/genética , Proteínas Arqueales/metabolismo , Genes Arqueales/genética , Transferasas Intramoleculares/metabolismo , Conformación de Ácido Nucleico , ARN/metabolismo , ARN Guía de Kinetoplastida/metabolismo , ARN Ribosómico , ARN de Transferencia , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas Nucleolares Pequeñas/genética , Ribonucleoproteínas Nucleolares Pequeñas/metabolismo , Uridina/metabolismoRESUMEN
O(6)-Methylguanine produced in DNA can pair with thymine during DNA replication, thus leading to a G-to-A transition mutation. To prevent such outcomes, cells harboring O(6)-methylguanine-containing mispair undergo apoptosis that requires the function of mismatch repair (MMR) protein complex. To identify the genes involved in the induction of apoptosis, we performed gene-trap mutagenesis and isolated a clone of mouse cells exhibiting an increased resistance to the killing effect of an alkylating agent, N-methyl-N-nitrosourea (MNU). The mutant carries an insertion in the Hmga2 gene, which belongs to a gene family encoding the high-mobility group A non-histone chromatin proteins. To elucidate the function of HMGA proteins in the apoptosis pathway, we introduced siRNAs for HMGA1 and/or HMGA2 into human HeLa MR cells defective in O(6)-methylguanine-DNA methyltransferase. HMGA1- and HMGA2-single knockdown cells showed an increased resistance to MNU, and HMGA1/HMGA2-double knockdown cells exhibited further increased tolerance compared to the control. The phosphorylation of ATR and CHK1, the appearance of a sub-G1 population, and caspase-9 activation were suppressed in the knockdown cells, although the formation of mismatch recognition complex was unaffected. These results suggest that HMGA family proteins function at the step following the damage recognition in the process of apoptosis triggered by O(6)-methylguanine.
Asunto(s)
Apoptosis , Daño del ADN , Proteínas HMGA/metabolismo , Transducción de Señal , Animales , Caspasa 9/genética , Caspasa 9/metabolismo , Fase G1/genética , Proteínas HMGA/genética , Células HeLa , Humanos , Ratones NoqueadosRESUMEN
We identified a novel structure-specific endonuclease in Pyrococcus furiosus. This nuclease contains two distinct domains, which are similar to the DEAH helicase family at the N-terminal two-third and the XPF endonuclease superfamily at the C-terminal one-third of the protein, respectively. The C-terminal domain has an endonuclease activity cleaving the DNA strand at the 5'-side of nicked or flapped positions in the duplex DNA. The nuclease also incises in the proximity of the 5'-side of a branch point in the template strand for leading synthesis in the fork-structured DNA. The N-terminal helicase may work cooperatively to change the fork structure suitable for cleavage by the C-terminal endonuclease. This protein, designated as Hef (helicase-associated endonuclease for fork-structured DNA), may be a prototypical enzyme for resolving stalled forks during DNA replication, as well as working at nucleotide excision repair.
Asunto(s)
Reparación del ADN/genética , ADN/metabolismo , Endonucleasas/genética , Pyrococcus furiosus/genética , Secuencia de Aminoácidos , Reparación del ADN/fisiología , Replicación del ADN/fisiología , Proteínas de Unión al ADN/genética , Endodesoxirribonucleasas/genética , Endonucleasas/metabolismo , Resolvasas de Unión Holliday , Datos de Secuencia Molecular , Pyrococcus furiosus/enzimologíaRESUMEN
O6-Methylguanine, one of alkylated DNA bases, is especially mutagenic. Cells containing this lesion are eliminated by induction of apoptosis, associated with the function of mismatch repair (MMR) proteins. A retrovirus-mediated gene-trap mutagenesis was used to isolate new genes related to the induction of apoptosis, triggered by the treatment with an alkylating agent, N-methyl-N-nitrosourea (MNU). This report describes the identification of a novel gene, MAPO2 (O6-methylguanine-induced apoptosis 2), which is originally annotated as C1orf201. The MAPO2 gene is conserved among a wide variety of multicellular organisms and encodes a protein containing characteristic PxPxxY repeats. To elucidate the function of the gene product in the apoptosis pathway, a human cell line derived from HeLa MR cells, in which the MAPO2 gene was stably knocked down by expressing specific miRNA, was constructed. The knockdown cells grew at the same rate as HeLa MR, thus indicating that MAPO2 played no role in the cellular growth. After exposure to MNU, HeLa MR cells and the knockdown cells underwent cell cycle arrest at G2/M phase, however, the production of the sub-G1 population in the knockdown cells was significantly suppressed in comparison to that in HeLa MR cells. Moreover, the activation of BAK and caspase-3, and depolarization of mitochondrial membrane, hallmarks for the induction of apoptosis, were also suppressed in the knockdown cells. These results suggest that the MAPO2 gene product might positively contribute to the induction of apoptosis triggered by O6-methylguanine.
Asunto(s)
Proteínas Reguladoras de la Apoptosis/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Guanina/análogos & derivados , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Animales , Proteínas Reguladoras de la Apoptosis/química , Proteínas Reguladoras de la Apoptosis/deficiencia , Proteínas Reguladoras de la Apoptosis/metabolismo , Puntos de Control de la Fase G2 del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase G2 del Ciclo Celular/genética , Técnicas de Silenciamiento del Gen , Guanina/farmacología , Células HeLa , Humanos , Puntos de Control de la Fase M del Ciclo Celular/efectos de los fármacos , Puntos de Control de la Fase M del Ciclo Celular/genética , Metilnitrosourea/farmacología , Ratones , Datos de Secuencia Molecular , RatasRESUMEN
O6-methylguanine produced in DNA by the action of simple alkylating agents, such as N-methyl-N-nitrosourea (MNU), causes base-mispairing during DNA replication, thus leading to mutations and cancer. To prevent such outcomes, the cells carrying O6-methylguanine undergo apoptosis in a mismatch repair protein-dependent manner. We previously identified MAPO1 as one of the components required for the induction of apoptosis triggered by O6-methylguanine. MAPO1, also known as FNIP2 and FNIPL, forms a complex with AMP-activated protein kinase (AMPK) and folliculin (FLCN), which is encoded by the BHD tumor suppressor gene. We describe here the involvement of the AMPK-MAPO1-FLCN complex in the signaling pathway of apoptosis induced by O6-methylguanine. By the introduction of siRNAs specific for these genes, the transition of cells to a population with sub-G1 DNA content following MNU treatment was significantly suppressed. After MNU exposure, phosphorylation of AMPKα occurred in an MLH1-dependent manner, and this activation of AMPK was not observed in cells in which the expression of either the Mapo1 or the Flcn gene was downregulated. When cells were treated with AICA-ribose (AICAR), a specific activator of AMPK, activation of AMPK was also observed in a MAPO1- and FLCN-dependent manner, thus leading to cell death which was accompanied by the depolarization of the mitochondrial membrane, a hallmark of the apoptosis induction. It is therefore likely that MAPO1, in its association with FLCN, may regulate the activation of AMPK to control the induction of apoptosis triggered by O6-methylguanine.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Reguladoras de la Apoptosis/metabolismo , Apoptosis , Disparidad de Par Base , Proteínas Portadoras/metabolismo , ADN/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Alquilación/efectos de los fármacos , Animales , Apoptosis/efectos de los fármacos , Disparidad de Par Base/efectos de los fármacos , Línea Celular , Activación Enzimática/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Metilnitrosourea , Ratones , Unión Proteica/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , ARN Interferente Pequeño/metabolismoRESUMEN
Extensive biochemical and structural analyses have been performed on the putative DNA repair proteins of hyperthermophilic archaea, in contrast to the few genetic analyses of the genes encoding these proteins. Accordingly, little is known about the repair pathways used by archaeal cells at high temperature. Here, we attempted to disrupt the genes encoding the potential repair proteins in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. We succeeded in isolating null mutants of the hjc, hef, hjm, xpb, and xpd genes, but not the radA, rad50, mre11, herA, nurA, and xpg/fen1 genes. Phenotypic analyses of the gene-disrupted strains showed that the xpb and xpd null mutants are only slightly sensitive to ultraviolet (UV) irradiation, methyl methanesulfonate (MMS) and mitomycin C (MMC), as compared with the wild-type strain. The hjm null mutant showed sensitivity specifically to mitomycin C. On the other hand, the null mutants of the hjc gene lacked increasing sensitivity to any type of DNA damage. The Hef protein is particularly important for maintaining genome homeostasis, by functioning in the repair of a wide variety of DNA damage in T. kodakaraensis cells. Deletion of the entire hef gene or of the segments encoding either its nuclease or helicase domain produced similar phenotypes. The high sensitivity of the Δhef mutants to MMC suggests that Hef performs a critical function in the repair process of DNA interstrand cross-links. These damage-sensitivity profiles suggest that the archaeal DNA repair system has processes depending on repair-related proteins different from those of eukaryotic and bacterial DNA repair systems using homologous repair proteins analyzed here.
Asunto(s)
Proteínas Arqueales/genética , Reparación del ADN , ADN de Archaea/genética , Thermococcaceae/genética , Proteínas Arqueales/aislamiento & purificación , ADN Helicasas/genética , ADN Helicasas/aislamiento & purificación , Análisis Mutacional de ADN , ADN de Archaea/efectos de los fármacos , ADN de Archaea/efectos de la radiación , Endonucleasas/genética , Endonucleasas/aislamiento & purificación , Rayos gamma , Eliminación de Gen , Técnicas de Inactivación de Genes , Metilmetanosulfonato/toxicidad , Mitomicina/toxicidad , Mutación , Thermococcaceae/efectos de los fármacos , Thermococcaceae/efectos de la radiación , Rayos UltravioletaRESUMEN
Pyrococcus furiosus, a hyperthermophilic Archaea, has homologs of the eukaryotic MCM (mini-chromosome maintenance) helicase and GINS complex. The MCM and GINS proteins are both essential factors to initiate DNA replication in eukaryotic cells. Many biochemical characterizations of the replication-related proteins have been reported, but it has not been proved that the homologs of each protein are also essential for replication in archaeal cells. Here, we demonstrated that the P. furiosus GINS complex interacts with P. furiosus MCM. A chromatin immunoprecipitation assay revealed that the GINS complex is detected preferentially at the oriC region on Pyrococcus chromosomal DNA during the exponential growth phase but not in the stationary phase. Furthermore, the GINS complex stimulates both the ATPase and DNA helicase activities of MCM in vitro. These results strongly suggest that the archaeal GINS is involved in both the initiation and elongation processes of DNA replication in P. furiosus, as observed in eukaryotic cells.