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1.
Cancer Discov ; 14(6): 953-964, 2024 Jun 03.
Artículo en Inglés | MEDLINE | ID: mdl-38501975

RESUMEN

Pediatric cancers are rare diseases, and children without known germline predisposing conditions who develop a second malignancy during developmental ages are extremely rare. We present four such clinical cases and, through whole-genome and error-correcting ultra-deep duplex sequencing of tumor and normal samples, we explored the origin of the second malignancy in four children, uncovering different routes of development. The exposure to cytotoxic therapies was linked to the emergence of a secondary acute myeloid leukemia. A common somatic mutation acquired early during embryonic development was the driver of two solid malignancies in another child. In two cases, the two tumors developed from completely independent clones diverging during embryogenesis. Importantly, we demonstrate that platinum-based therapies contributed at least one order of magnitude more mutations per day of exposure than aging to normal tissues in these children. SIGNIFICANCE: Using whole-genome and error-correcting ultra-deep duplex sequencing, we uncover different origins for second neoplasms in four children. We also uncover the presence of platinum-related mutations across 10 normal tissues of exposed individuals, highlighting the impact that the use of cytotoxic therapies may have on cancer survivors. See related commentary by Pacyna and Nangalia, p. 900. This article is featured in Selected Articles from This Issue, p. 897.


Asunto(s)
Mutación , Neoplasias Primarias Secundarias , Adolescente , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Antineoplásicos/uso terapéutico , Secuenciación de Nucleótidos de Alto Rendimiento , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Neoplasias Primarias Secundarias/genética , Secuenciación Completa del Genoma
3.
Cancers (Basel) ; 14(3)2022 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-35159100

RESUMEN

There is a growing body of evidence that cancer causes systemic changes. These influences are most evident in the bone marrow and the blood, particularly in the myeloid compartment. Here, we show that there is an increase in the number of bone marrow, circulating and splenic monocytes by using mouse models of breast cancer caused by the mammary epithelial expression of the polyoma middle T antigen. Cancer does not affect ratios of classical to non-classical populations of monocytes in the circulation nor does it affect their half-lives. Single cell RNA sequencing also indicates that cancer does not induce any new monocyte populations. Cancer does not change the monocytic progenitor number in the bone marrow, but the proliferation rate of monocytes is higher, thus providing an explanation for the expansion of the circulating numbers. Deep RNA sequencing of these monocytic populations reveals that cancer causes changes in the classical monocyte compartment, with changes evident in bone marrow monocytes and even more so in the blood, suggesting influences in both compartments, with the down-regulation of interferon type 1 signaling and antigen presentation being the most prominent of these. Consistent with this analysis, down-regulated genes are enriched with STAT1/STAT2 binding sites in their promoter, which are transcription factors required for type 1 interferon signaling. However, these transcriptome changes in mice did not replicate those found in patients with breast cancer. Consequently, this mouse model of breast cancer may be insufficient to study the systemic influences of human cancer.

4.
PLoS One ; 16(10): e0258269, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34614038

RESUMEN

Ionizing radiation (IR) is widely used in cancer therapy and accidental or environmental exposure is a major concern. However, little is known about the genome-wide effects IR exerts on germ cells and the relative contribution of DNA repair pathways for mending IR-induced lesions. Here, using C. elegans as a model system and using primary sequencing data from our recent high-level overview of the mutagenic consequences of 11 genotoxic agents, we investigate in detail the genome-wide mutagenic consequences of exposing wild-type and 43 DNA repair and damage response defective C. elegans strains to a Caesium (Cs-137) source, emitting γ-rays. Cs-137 radiation induced single nucleotide variants (SNVs) at a rate of ~1 base substitution per 3 Gy, affecting all nucleotides equally. In nucleotide excision repair mutants, this frequency increased 2-fold concurrently with increased dinucleotide substitutions. As observed for DNA damage induced by bulky DNA adducts, small deletions were increased in translesion polymerase mutants, while base changes decreased. Structural variants (SVs) were augmented with dose, but did not arise with significantly higher frequency in any DNA repair mutants tested. Moreover, 6% of all mutations occurred in clusters, but clustering was not significantly altered in any DNA repair mutant background. Our data is relevant for better understanding how DNA repair pathways modulate IR-induced lesions.


Asunto(s)
Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de la radiación , Reparación del ADN/genética , Reparación del ADN/efectos de la radiación , Genoma de los Helmintos , Radiación Ionizante , Animales , Caenorhabditis elegans/efectos de los fármacos , Cisplatino/farmacología , Reparación del ADN/efectos de los fármacos , Humanos , Mutación/genética , Neoplasias/genética , Polimorfismo de Nucleótido Simple/genética , Rayos Ultravioleta
5.
PLoS One ; 16(4): e0250291, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33905417

RESUMEN

Maintaining genome integrity is particularly important in germ cells to ensure faithful transmission of genetic information across generations. Here we systematically describe germ cell mutagenesis in wild-type and 61 DNA repair mutants cultivated over multiple generations. ~44% of the DNA repair mutants analysed showed a >2-fold increased mutagenesis with a broad spectrum of mutational outcomes. Nucleotide excision repair deficiency led to higher base substitution rates, whereas polh-1(Polη) and rev-3(Polζ) translesion synthesis polymerase mutants resulted in 50-400 bp deletions. Signatures associated with defective homologous recombination fall into two classes: 1) brc-1/BRCA1 and rad-51/RAD51 paralog mutants showed increased mutations across all mutation classes, 2) mus-81/MUS81 and slx-1/SLX1 nuclease, and him-6/BLM, helq-1/HELQ or rtel-1/RTEL1 helicase mutants primarily accumulated structural variants. Repetitive and G-quadruplex sequence-containing loci were more frequently mutated in specific DNA repair backgrounds. Tandem duplications embedded in inverted repeats were observed in helq-1 helicase mutants, and a unique pattern of 'translocations' involving homeologous sequences occurred in rip-1 recombination mutants. atm-1/ATM checkpoint mutants harboured structural variants specifically enriched in subtelomeric regions. Interestingly, locally clustered mutagenesis was only observed for combined brc-1 and cep-1/p53 deficiency. Our study provides a global view of how different DNA repair pathways contribute to prevent germ cell mutagenesis.


Asunto(s)
Caenorhabditis elegans/genética , Reparación del ADN , ADN de Helmintos/genética , Regulación de la Expresión Génica , Genoma de los Helmintos , Células Germinativas/metabolismo , Mutación , Animales , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Proliferación Celular , Mapeo Cromosómico , Daño del ADN , ADN Helicasas/genética , ADN Helicasas/metabolismo , Replicación del ADN , ADN de Helmintos/metabolismo , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Desoxirribonucleasas/genética , Desoxirribonucleasas/metabolismo , Endonucleasas/genética , Endonucleasas/metabolismo , Células Germinativas/citología , Isoenzimas/genética , Isoenzimas/metabolismo , Recombinasa Rad51/genética , Recombinasa Rad51/metabolismo
6.
Life Sci Alliance ; 4(1)2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33268347

RESUMEN

The association of increased levels of tumour-infiltrating gamma-delta (γδ) T cells with favorable prognosis across many cancer types and their ability to recognize stress antigens in an MHC unrestricted manner has led to an increased interest in exploiting them for cancer immunotherapy. We performed single-cell RNA sequencing (scRNA-seq) of peripheral blood γδ T cells from healthy adult donors and from fresh tumour biopsies of breast cancer patients. We identified five γδ T cells subtypes in blood and three subtypes of γδ T cells in breast tumour. These subtypes differed in the expression of genes contributing to effector functions such as antigen presentation, cytotoxicity, and IL17A and IFNγ production. Compared with the blood γδ T cells, the breast tumour-infiltrating γδ T cells were more activated, expressed higher levels of cytotoxic genes, yet were immunosuppressed. One subtype in the breast tumour that was IFNγ-positive had no obvious similarity to any of the subtypes observed in the blood γδ T cell and was the only subtype associated with improved overall survival of breast cancer patients. Taken together, our study has identified markers of subtypes of human blood γδ T cells and uncovered a tumour-infiltrating γδ T cells subtype associated improved overall cancer survival.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/inmunología , Linfocitos Intraepiteliales/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , RNA-Seq/métodos , Receptores de Antígenos de Linfocitos T gamma-delta/genética , Análisis de la Célula Individual/métodos , Adulto , Secuencia de Bases , Donantes de Sangre , Neoplasias de la Mama/mortalidad , Neoplasias de la Mama/patología , Estudios de Casos y Controles , Células Cultivadas , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Estimación de Kaplan-Meier , Pronóstico , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología
7.
Nat Commun ; 11(1): 2169, 2020 05 01.
Artículo en Inglés | MEDLINE | ID: mdl-32358516

RESUMEN

Cells possess an armamentarium of DNA repair pathways to counter DNA damage and prevent mutation. Here we use C. elegans whole genome sequencing to systematically quantify the contributions of these factors to mutational signatures. We analyse 2,717 genomes from wild-type and 53 DNA repair defective backgrounds, exposed to 11 genotoxins, including UV-B and ionizing radiation, alkylating compounds, aristolochic acid, aflatoxin B1, and cisplatin. Combined genotoxic exposure and DNA repair deficiency alters mutation rates or signatures in 41% of experiments, revealing how different DNA alterations induced by the same genotoxin are mended by separate repair pathways. Error-prone translesion synthesis causes the majority of genotoxin-induced base substitutions, but averts larger deletions. Nucleotide excision repair prevents up to 99% of point mutations, almost uniformly across the mutation spectrum. Our data show that mutational signatures are joint products of DNA damage and repair and suggest that multiple factors underlie signatures observed in cancer genomes.


Asunto(s)
Daño del ADN/fisiología , Reparación del ADN/fisiología , Animales , Caenorhabditis elegans/genética , Daño del ADN/genética , Reparación del ADN/genética , Genómica/métodos , Humanos , Mutación/genética , Mutación Puntual/genética
8.
Genetics ; 206(4): 1853-1864, 2017 08.
Artículo en Inglés | MEDLINE | ID: mdl-28634159

RESUMEN

Ionizing radiation (IR) is commonly used in cancer therapy and is a main source of DNA double-strand breaks (DSBs), one of the most toxic forms of DNA damage. We have used Caenorhabditis elegans as an invertebrate model to identify novel factors required for repair of DNA damage inflicted by IR. We have performed an unbiased genetic screen, finding that smg-1 mutations confer strong hyper-sensitivity to IR. SMG-1 is a phosphoinositide-3 kinase (PI3K) involved in mediating nonsense-mediated mRNA decay (NMD) of transcripts containing premature stop codons and related to the ATM and ATR kinases which are at the apex of DNA damage signaling pathways. Hyper-sensitivity to IR also occurs when other genes mediating NMD are mutated. The hyper-sensitivity to bleomycin, a drug known to induce DSBs, further supports that NMD pathway mutants are defective in DSB repair. Hyper-sensitivity was not observed upon treatment with alkylating agents or UV irradiation. We show that SMG-1 mainly acts in mitotically dividing germ cells, and during late embryonic and larval development. Based on epistasis experiments, SMG-1 does not appear to act in any of the three major pathways known to mend DNA DSBs, namely homologous recombination (HR), nonhomologous end-joining (NHEJ), and microhomology-mediated end-joining (MMEJ). We speculate that SMG-1 kinase activity could be activated following DNA damage to phosphorylate specific DNA repair proteins and/or that NMD inactivation may lead to aberrant mRNAs leading to synthesis of malfunctioning DNA repair proteins.


Asunto(s)
Inestabilidad Genómica , Degradación de ARNm Mediada por Codón sin Sentido , Animales , Caenorhabditis elegans/genética , Caenorhabditis elegans/efectos de la radiación , Proteínas de Caenorhabditis elegans/genética , Roturas del ADN de Doble Cadena , Daño del ADN , Reparación del ADN , Epistasis Genética , Recombinación Homóloga , Mitosis , Proteínas Serina-Treonina Quinasas/genética , Radiación Ionizante
9.
EMBO J ; 33(4): 327-40, 2014 Feb 18.
Artículo en Inglés | MEDLINE | ID: mdl-24473148

RESUMEN

DNA replication is sensitive to damage in the template. To bypass lesions and complete replication, cells activate recombination-mediated (error-free) and translesion synthesis-mediated (error-prone) DNA damage tolerance pathways. Crucial for error-free DNA damage tolerance is template switching, which depends on the formation and resolution of damage-bypass intermediates consisting of sister chromatid junctions. Here we show that a chromatin architectural pathway involving the high mobility group box protein Hmo1 channels replication-associated lesions into the error-free DNA damage tolerance pathway mediated by Rad5 and PCNA polyubiquitylation, while preventing mutagenic bypass and toxic recombination. In the process of template switching, Hmo1 also promotes sister chromatid junction formation predominantly during replication. Its C-terminal tail, implicated in chromatin bending, facilitates the formation of catenations/hemicatenations and mediates the roles of Hmo1 in DNA damage tolerance pathway choice and sister chromatid junction formation. Together, the results suggest that replication-associated topological changes involving the molecular DNA bender, Hmo1, set the stage for dedicated repair reactions that limit errors during replication and impact on genome stability.


Asunto(s)
Cromosomas Fúngicos/ultraestructura , Daño del ADN , ADN de Hongos/genética , Proteínas del Grupo de Alta Movilidad/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Saccharomyces cerevisiae/metabolismo , Cromátides/genética , Cromátides/ultraestructura , Cromatina/ultraestructura , Cromosomas Fúngicos/genética , ADN Helicasas/metabolismo , Replicación del ADN , ADN Cruciforme , ADN de Hongos/efectos de los fármacos , Inestabilidad Genómica , Proteínas del Grupo de Alta Movilidad/química , Proteínas del Grupo de Alta Movilidad/genética , Metilmetanosulfonato/farmacología , Mutágenos/farmacología , Antígeno Nuclear de Célula en Proliferación/metabolismo , Proteína de Replicación A/metabolismo , Fase S , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Enzimas Ubiquitina-Conjugadoras/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación
10.
Cell ; 138(5): 870-84, 2009 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-19737516

RESUMEN

Specialized topoisomerases solve the topological constraints arising when replication forks encounter transcription. We have investigated the contribution of Top2 in S phase transcription. Specifically in S phase, Top2 binds intergenic regions close to transcribed genes. The Top2-bound loci exhibit low nucleosome density and accumulate gammaH2A when Top2 is defective. These intergenic loci associate with the HMG protein Hmo1 throughout the cell cycle and are refractory to the histone variant Htz1. In top2 mutants, Hmo1 is deleterious and accumulates at pericentromeric regions in G2/M. Our data indicate that Top2 is dispensable for transcription and that Hmo1 and Top2 bind in the proximity of genes transcribed in S phase suppressing chromosome fragility at the M-G1 transition. We propose that an Hmo1-dependent epigenetic signature together with Top2 mediate an S phase architectural pathway to preserve genome integrity.


Asunto(s)
Replicación del ADN , ADN-Topoisomerasas de Tipo II/metabolismo , Proteínas del Grupo de Alta Movilidad/metabolismo , Fase S , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transcripción Genética , Fragilidad Cromosómica , Epigénesis Genética , Genoma Fúngico , ARN Polimerasa II/metabolismo , Saccharomyces cerevisiae/citología , Saccharomyces cerevisiae/enzimología
11.
J Bacteriol ; 189(15): 5542-9, 2007 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-17526715

RESUMEN

It is becoming clear that in vivo phage DNA ejection is not a mere passive process. In most cases, both phage and host proteins seem to be involved in pulling at least part of the viral DNA inside the cell. The DNA ejection mechanism of Bacillus subtilis bacteriophage phi29 is a two-step process where the linear DNA penetrates the cell with a right-left polarity. In the first step approximately 65% of the DNA is pushed into the cell. In the second step, the remaining DNA is actively pulled into the cytoplasm. This step requires protein p17, which is encoded by the right-side early operon that is ejected during the first push step. The membrane protein p16.7, also encoded by the right-side early operon, is known to play an important role in membrane-associated phage DNA replication. In this work we show that, in addition, p16.7 is required for efficient execution of the second pull step of DNA ejection.


Asunto(s)
Fagos de Bacillus/fisiología , ADN Viral/metabolismo , Proteínas de la Membrana/fisiología , Proteínas Virales/fisiología , Fagos de Bacillus/genética , Bacillus subtilis/virología , Replicación del ADN/fisiología , Regiones Promotoras Genéticas
12.
Gene ; 374: 19-25, 2006 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-16517096

RESUMEN

Phage phi29 infects Bacillus subtilis and ejects its linear DNA with a right to left polarity in a two-step, "push-pull" mechanism. In the first step 65% of the DNA is pushed inside the cell, presumably by the pressure built inside the capsid. In the second step, the remaining DNA is pulled by a hypothetical motor that comprises at least viral protein p17, encoded by the right early operon, in an energy-dependent process. We have further studied phi29 DNA ejection by using energy poisons and DNA replication and transcription inhibitors. The first step is passive, as it does not require an external energy source. The second step is transcription-independent and is completely abolished by novobiocin, suggesting a requirement for negatively supercoiled DNA. Viral DNA pulling also requires an electrochemical proton gradient, as the process is highly impaired by specific energy poisons such as gramicidin and CCCP (carbonyl cyanide m-chlorophenylhydrazone). The fact that azide has no effect in the absence of p17 suggests that this protein is essential for energy transduction.


Asunto(s)
Fagos de Bacillus/metabolismo , Bacillus subtilis/virología , ADN Viral/metabolismo , Proteínas Virales/metabolismo , Fagos de Bacillus/genética , Bacillus subtilis/genética , Carbonil Cianuro m-Clorofenil Hidrazona/farmacología , Inmunoprecipitación de Cromatina , Replicación del ADN , Metabolismo Energético , Inhibidores Enzimáticos/farmacología , Gramicidina/farmacología , Modelos Biológicos , Novobiocina/farmacología , Protones , Desacopladores/farmacología , Proteínas Virales/genética
13.
J Bacteriol ; 186(24): 8401-6, 2004 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-15576790

RESUMEN

Bacteriophage phi29 protein p6 is a viral architectural protein, which binds along the whole linear phi29 DNA in vivo and is involved in initiation of DNA replication and transcription control. Protein p1 is a membrane-associated viral protein, proposed to attach the viral genome to the cell membrane. Protein p17 is involved in pulling phi29 DNA into the cell during the injection process. We have used chromatin immunoprecipitation and real-time PCR to analyze in vivo p6 binding to DNA in cells infected with phi29 sus1 or sus17 mutants; in both cases p6 binding is significantly decreased all along phi29 DNA. phi29 DNA is topologically constrained in vivo, and p6 binding is highly increased in the presence of novobiocin, a gyrase inhibitor that produces a loss of DNA negative superhelicity. Here we show that, in cells infected with phi29 sus1 or sus17 mutants, the increase of p6 binding by novobiocin is even higher than in cells containing p1 and p17, alleviating the p6 binding deficiency. Therefore, proteins p1 and p17 could be required to restrain the proper topology of phi29 DNA, which would explain the impaired DNA replication observed in cells infected with sus1 or sus17 mutants.


Asunto(s)
Fagos de Bacillus/metabolismo , Bacillus subtilis/virología , ADN Viral/metabolismo , Proteínas Virales/metabolismo , Fagos de Bacillus/genética , Inmunoprecipitación de Cromatina , Replicación del ADN , Regulación Viral de la Expresión Génica , Mutación , Reacción en Cadena de la Polimerasa , Proteínas Virales/genética
14.
J Mol Recognit ; 17(5): 390-6, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15362097

RESUMEN

Protein p6 of B. subtilis bacteriophage Ø29 binds to DNA forming a nucleoprotein complex in which the DNA wraps a protein core forming a right-handed superhelix, therefore restraining positive supercoiling and compacting the DNA. The protein does not specifically recognize a nucleotide sequence but rather a structural feature and it binds as a dimer through the minor groove. Protein p6 is in a monomer-dimer equilibrium that shifts to higher-order structures at a concentration of about 1 mM. These structures are probably present in vivo as the intracellular concentration of p6 is estimated to be in this range, and in fact the effective concentration should be still higher due to the macromolecular crowding. The p6 oligomers show an elongated shape compatible with a helical structure reminiscent of the superhelical DNA of the nucleoprotein complex, therefore it was proposed that protein p6 forms a scaffold on which the DNA folds. Since protein p6 is very abundant in infected cells, enough to bind the entire viral progeny, it was proposed to have an architectural role organizing and compacting the viral genome. It has been demonstrated that protein p6 binds in vivo to most, if not all, the Ø29 genome, although with different affinity, the highest one corresponding to the genome ends. Binding to plasmidic DNA was much lower, although it increased dramatically when the negative superhelicity was decreased. Hence, protein p6 binding specificity for Ø29 DNA is based on supercoiling, providing that the Ø29 genome, although topologically constrained, has a negative superhelicity lower than that of plasmid DNA. The formation of the nucleoprotein complex has functional implications in DNA replication and the control of transcription. It activates the initiation of replication that occurs at the genome ends for which the binding affinity is highest. It represses early transcription from promoter C2, and, together with protein p4, it represses transcription from promoters A2b and A2c and activates late transcription from promoter A3; therefore, protein p6 is involved in the early to late transcription switch.


Asunto(s)
Fagos de Bacillus/genética , Replicación del ADN , Transcripción Genética , Proteínas Virales/fisiología , Replicación Viral , Fagos de Bacillus/metabolismo , Fagos de Bacillus/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/fisiología , Genoma Viral , Proteínas Reguladoras y Accesorias Virales/metabolismo , Proteínas Reguladoras y Accesorias Virales/fisiología
15.
Nucleic Acids Res ; 32(11): 3493-502, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15247336

RESUMEN

Bacillus subtilis phage Phi29 protein p6 is required for DNA replication and promotes the switch from early to late transcription. In vivo it binds all along the viral linear DNA, which suggests a global role as an architectural protein; in contrast, binding to bacterial DNA is negligible. This specificity could be due to the p6 binding preference for less negatively supercoiled DNA, as is presumably the case with viral (with respect to bacterial) DNA. Here we demonstrate that p6 binding to Phi29 DNA is greatly increased when negative supercoiling is decreased by novobiocin; in addition, gyrase is required for DNA replication. This indicates that, although non-covalently closed, the viral genome is topologically constrained in vivo. We also show that the p6 binding to different Phi29 DNA regions is modulated by the structural properties of their nucleotide sequences. The higher affinity for DNA ends is possibly related to the presence of sequences in which their bendability properties favor the formation of the p6-DNA complex, whereas the lower affinity for the transcription control region is most probably due to the presence of a rigid intrinsic DNA curvature.


Asunto(s)
Fagos de Bacillus/genética , ADN Viral/química , Proteínas Virales/metabolismo , Fagos de Bacillus/metabolismo , Fagos de Bacillus/fisiología , Secuencia de Bases , Clonación Molecular , ADN Viral/metabolismo , Escherichia coli/genética , Genoma Viral , Conformación de Ácido Nucleico , Unión Proteica , Replicación Viral
16.
Nucleic Acids Res ; 32(8): 2306-14, 2004.
Artículo en Inglés | MEDLINE | ID: mdl-15118076

RESUMEN

Protein p6 of Bacillus subtilis bacteriophage Phi29 is essential for phage development. In vitro it activates the initiation of DNA replication and is involved in the early to late transcriptional switch. These activities require the formation of a nucleoprotein complex in which the DNA forms a right-handed superhelix wrapping around a multimeric protein core. However, there was no evidence of p6 binding to Phi29 DNA in vivo. By crosslinking, chromatin immunoprecipitation and real-time PCR we show that protein p6 binds to most, if not all, the viral genome in vivo, although with higher affinity for both DNA ends, which contain the replication origins. In contrast, the affinity for plasmid DNA is negligible, but greatly increases when the negative supercoiling decreases, as shown in vivo by treatment of cells with novobiocin and in vitro by fluorescence quenching with plasmids with different topology. In conclusion, binding of protein p6 all along the Phi29 genome strongly suggests that its functions in replication and transcription control could be local outcomes of a more global role as a histone-like protein. The p6 binding dependence on DNA topology could explain its preferential binding to viral with respect to bacterial DNA, whose level of negative supercoiling is presumably higher than that of Phi29 DNA.


Asunto(s)
Fagos de Bacillus/genética , ADN Superhelicoidal/metabolismo , ADN Viral/química , ADN Viral/metabolismo , Proteínas Virales/metabolismo , Fagos de Bacillus/metabolismo , ADN Viral/biosíntesis , Regulación Viral de la Expresión Génica , Genoma Viral , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Transcripción Genética , Proteínas Virales/fisiología , Replicación Viral
17.
Mol Microbiol ; 52(2): 529-40, 2004 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-15066038

RESUMEN

The mechanism of bacteriophage DNA injection is poorly understood, often considered a simple process, driven merely by the packing pressure inside the capsid. In contrast to the well-established DNA packaging mechanism of Bacillus subtilis phage Ø29, that involves a molecular motor formed by the connector and a viral ATPase, nothing is known about its DNA injection into the cell. We have studied this process measuring DNA binding of p6, a viral genome organization protein. The linear DNA penetrates with a right-left polarity, in a two-step process. In the first step approximately 65% of the genome is pushed into the cell most probably by the pressure built inside the viral capsid. Thus, synthesis of viral proteins from the right early operon is allowed. This step is controlled, probably by bacterial protein(s) that slow down DNA entry. In the second step at least one of the viral early proteins, p17, participates in the molecular machinery that pulls the remaining DNA inside the cell. Both steps are energy-dependent, as treatment of cells with azide overrides the whole mechanism, leading to a deregulated, passive entry of DNA.


Asunto(s)
Fagos de Bacillus/metabolismo , ADN Viral/metabolismo , Podoviridae/metabolismo , Proteínas Virales/metabolismo , Fagos de Bacillus/genética , Sitios de Unión , Cápside/fisiología , Metabolismo Energético , Modelos Biológicos , Podoviridae/genética , Proteínas Virales/biosíntesis
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