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1.
EMBO J ; 42(20): e110844, 2023 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-37661798

RESUMO

Homologous recombination (HR) is a prominent DNA repair pathway maintaining genome integrity. Mutations in many HR genes lead to cancer predisposition. Paradoxically, the implication of the pivotal HR factor RAD51 on cancer development remains puzzling. Particularly, no RAD51 mouse models are available to address the role of RAD51 in aging and carcinogenesis in vivo. We engineered a mouse model with an inducible dominant-negative form of RAD51 (SMRad51) that suppresses RAD51-mediated HR without stimulating alternative mutagenic repair pathways. We found that in vivo expression of SMRad51 led to replicative stress, systemic inflammation, progenitor exhaustion, premature aging and reduced lifespan, but did not trigger tumorigenesis. Expressing SMRAD51 in a breast cancer predisposition mouse model (PyMT) decreased the number and the size of tumors, revealing an anti-tumor activity of SMRAD51. We propose that these in vivo phenotypes result from chronic endogenous replication stress caused by HR decrease, which preferentially targets progenitors and tumor cells. Our work underlines the importance of RAD51 activity for progenitor cell homeostasis, preventing aging and more generally for the balance between cancer and aging.


Assuntos
Neoplasias , Rad51 Recombinase , Animais , Camundongos , Envelhecimento/genética , Carcinogênese/genética , Transformação Celular Neoplásica , Dano ao DNA , Reparo do DNA , Recombinação Homóloga , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
2.
Nucleic Acids Res ; 51(6): 2800-2817, 2023 04 11.
Artigo em Inglês | MEDLINE | ID: mdl-36806960

RESUMO

RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes.


Assuntos
Recombinases Rec A , Streptococcus pneumoniae , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Recombinases Rec A/metabolismo , Recombinases Rec A/ultraestrutura , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/metabolismo , Microscopia Crioeletrônica
3.
Nucleic Acids Res ; 51(11): 5864-5882, 2023 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-37207342

RESUMO

The compaction of mitochondrial DNA (mtDNA) is regulated by architectural HMG-box proteins whose limited cross-species similarity suggests diverse underlying mechanisms. Viability of Candida albicans, a human antibiotic-resistant mucosal pathogen, is compromised by altering mtDNA regulators. Among them, there is the mtDNA maintenance factor Gcf1p, which differs in sequence and structure from its human and Saccharomyces cerevisiae counterparts, TFAM and Abf2p. Our crystallographic, biophysical, biochemical and computational analysis showed that Gcf1p forms dynamic protein/DNA multimers by a combined action of an N-terminal unstructured tail and a long helix. Furthermore, an HMG-box domain canonically binds the minor groove and dramatically bends the DNA while, unprecedentedly, a second HMG-box binds the major groove without imposing distortions. This architectural protein thus uses its multiple domains to bridge co-aligned DNA segments without altering the DNA topology, revealing a new mechanism of mtDNA condensation.


Assuntos
Candida albicans , DNA Mitocondrial , Proteínas de Ligação a DNA , Proteínas Fúngicas , Humanos , Candida albicans/genética , Candida albicans/metabolismo , DNA Mitocondrial/metabolismo , Proteínas de Ligação a DNA/metabolismo , Mitocôndrias/metabolismo , Proteínas Mitocondriais/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Fatores de Transcrição/metabolismo , Proteínas Fúngicas/metabolismo
4.
Nucleic Acids Res ; 50(17): 9909-9929, 2022 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-36107774

RESUMO

DNA lesions in S phase threaten genome stability. The DNA damage tolerance (DDT) pathways overcome these obstacles and allow completion of DNA synthesis by the use of specialised translesion (TLS) DNA polymerases or through recombination-related processes. However, how these mechanisms coordinate with each other and with bulk replication remains elusive. To address these issues, we monitored the variation of replication intermediate architecture in response to ultraviolet irradiation using transmission electron microscopy. We show that the TLS polymerase η, able to accurately bypass the major UV lesion and mutated in the skin cancer-prone xeroderma pigmentosum variant (XPV) syndrome, acts at the replication fork to resolve uncoupling and prevent post-replicative gap accumulation. Repriming occurs as a compensatory mechanism when this on-the-fly mechanism cannot operate, and is therefore predominant in XPV cells. Interestingly, our data support a recombination-independent function of RAD51 at the replication fork to sustain repriming. Finally, we provide evidence for the post-replicative commitment of recombination in gap repair and for pioneering observations of in vivo recombination intermediates. Altogether, we propose a chronology of UV damage tolerance in human cells that highlights the key role of polη in shaping this response and ensuring the continuity of DNA synthesis.


Assuntos
Reparo do DNA , Xeroderma Pigmentoso , Dano ao DNA , Replicação do DNA , DNA Polimerase Dirigida por DNA/genética , DNA Polimerase Dirigida por DNA/metabolismo , Humanos , Raios Ultravioleta , Xeroderma Pigmentoso/genética
5.
Nucleic Acids Res ; 50(5): 2651-2666, 2022 03 21.
Artigo em Inglês | MEDLINE | ID: mdl-35137208

RESUMO

Selection of the appropriate DNA double-strand break (DSB) repair pathway is decisive for genetic stability. It is proposed to act according to two steps: 1-canonical nonhomologous end-joining (C-NHEJ) versus resection that generates single-stranded DNA (ssDNA) stretches; 2-on ssDNA, gene conversion (GC) versus nonconservative single-strand annealing (SSA) or alternative end-joining (A-EJ). Here, we addressed the mechanisms by which RAD51 regulates this second step, preventing nonconservative repair in human cells. Silencing RAD51 or BRCA2 stimulated both SSA and A-EJ, but not C-NHEJ, validating the two-step model. Three different RAD51 dominant-negative forms (DN-RAD51s) repressed GC and stimulated SSA/A-EJ. However, a fourth DN-RAD51 repressed SSA/A-EJ, although it efficiently represses GC. In living cells, the three DN-RAD51s that stimulate SSA/A-EJ failed to load efficiently onto damaged chromatin and inhibited the binding of endogenous RAD51, while the fourth DN-RAD51, which inhibits SSA/A-EJ, efficiently loads on damaged chromatin. Therefore, the binding of RAD51 to DNA, rather than its ability to promote GC, is required for SSA/A-EJ inhibition by RAD51. We showed that RAD51 did not limit resection of endonuclease-induced DSBs, but prevented spontaneous and RAD52-induced annealing of complementary ssDNA in vitro. Therefore, RAD51 controls the selection of the DSB repair pathway, protecting genome integrity from nonconservative DSB repair through ssDNA occupancy, independently of the promotion of CG.


Assuntos
Quebras de DNA de Cadeia Dupla , Rad51 Recombinase , Cromatina , Reparo do DNA por Junção de Extremidades , Reparo do DNA , DNA de Cadeia Simples/genética , Humanos , Rad51 Recombinase/metabolismo
6.
Nucleic Acids Res ; 49(11): 6569-6586, 2021 06 21.
Artigo em Inglês | MEDLINE | ID: mdl-34107018

RESUMO

Replicative helicases are essential proteins that unwind DNA in front of replication forks. Their loading depends on accessory proteins and in bacteria, DnaC and DnaI are well characterized loaders. However, most bacteria do not express either of these two proteins. Instead, they are proposed to rely on DciA, an ancestral protein unrelated to DnaC/I. While the DciA structure from Vibrio cholerae shares no homology with DnaC, it reveals similarities with DnaA and DnaX, two proteins involved during replication initiation. As other bacterial replicative helicases, VcDnaB adopts a toroid-shaped homo-hexameric structure, but with a slightly open dynamic conformation in the free state. We show that VcDnaB can load itself on DNA in vitro and that VcDciA stimulates this function, resulting in an increased DNA unwinding. VcDciA interacts with VcDnaB with a 3/6 stoichiometry and we show that a determinant residue, which discriminates DciA- and DnaC/I-helicases, is critical in vivo. Our work is the first step toward the understanding of the ancestral mode of loading of bacterial replicative helicases on DNA. It sheds light on the strategy employed by phage helicase loaders to hijack bacterial replicative helicases and may explain the recurrent domestication of dnaC/I through evolution in bacteria.


Assuntos
Proteínas de Bactérias/química , Proteínas de Ligação a DNA/química , DnaB Helicases/química , Vibrio cholerae/enzimologia , Proteínas de Bactérias/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/metabolismo , DnaB Helicases/metabolismo , Modelos Moleculares , Conformação Proteica , Serina/química
7.
Int J Mol Sci ; 24(2)2023 Jan 11.
Artigo em Inglês | MEDLINE | ID: mdl-36674944

RESUMO

DciA is the ancestral bacterial replicative helicase loader, punctually replaced during evolution by the DnaC/I loaders of phage origin. DnaC helps the helicase to load onto DNA by cracking open the hexameric ring, but the mechanism of loading by DciA remains unknown. We demonstrate by electron microscopy, nuclear magnetic resonance (NMR) spectroscopy, and biochemistry experiments that DciA, which folds into a KH-like domain, interacts with not only single-stranded but also double-stranded DNA, in an atypical mode. Some point mutations of the long α-helix 1 demonstrate its importance in the interaction of DciA for various DNA substrates mimicking single-stranded, double-stranded, and forked DNA. Some of these mutations also affect the loading of the helicase by DciA. We come to the hypothesis that DciA could be a DNA chaperone by intercalating itself between the two DNA strands to stabilize it. This work allows us to propose that the direct interaction of DciA with DNA could play a role in the loading mechanism of the helicase.


Assuntos
Proteínas de Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , DNA Helicases/metabolismo , DNA , Replicação do DNA , Bactérias/metabolismo , DNA de Cadeia Simples , Proteínas de Bactérias/genética , Proteínas de Bactérias/química
8.
Nucleic Acids Res ; 47(12): 6195-6207, 2019 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-31114898

RESUMO

DNA folding and dynamics along with major nuclear functions are determined by chromosome structural properties, which remain, thus far, elusive in vivo. Here, we combine polymer modeling and single particle tracking experiments to determine the physico-chemical parameters of chromatin in vitro and in living yeast. We find that the motion of reconstituted chromatin fibers can be recapitulated by the Rouse model using mechanical parameters of nucleosome arrays deduced from structural simulations. Conversely, we report that the Rouse model shows some inconsistencies to analyze the motion and structural properties inferred from yeast chromosomes determined with chromosome conformation capture techniques (specifically, Hi-C). We hence introduce the Rouse model with Transient Internal Contacts (RouseTIC), in which random association and dissociation occurs along the chromosome contour. The parametrization of this model by fitting motion and Hi-C data allows us to measure the kinetic parameters of the contact formation reaction. Chromosome contacts appear to be transient; associated to a lifetime of seconds and characterized by an attractive energy of -0.3 to -0.5 kBT. We suggest attributing this energy to the occurrence of histone tail-DNA contacts and notice that its amplitude sets chromosomes in 'theta' conditions, in which they are poised for compartmentalization and phase separation.


Assuntos
Cromossomos Fúngicos/química , Modelos Genéticos , Cromatina/química , DNA Fúngico/química , Cinética , Movimento (Física) , Nucleossomos/química
9.
Chem Rec ; 18(7-8): 849-857, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29286197

RESUMO

Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier for DNA transfer thanks to its strong interaction with DNA molecules and its ability to be naturally internalized into mammalian cells through both non-endocytic and endocytic pathways. Sepiolite, due to its ability to bind various biomolecules, could be a good candidate for use as a nanocarrier for the simultaneous vectorization of diverse biological molecules. In this paper, we review our recent work, issued from a starting collaboration with Prof. Ruiz-Hitzky, that includes diverse aspects on the characterization and main features of sepiolite/DNA nanohybrids, and we present an outlook for the further development of sepiolite for DNA transfer.


Assuntos
DNA/química , Técnicas de Transferência de Genes , Silicatos de Magnésio/química , Nanoestruturas/química , Adsorção , Animais , DNA/metabolismo , Humanos , Silicatos de Magnésio/metabolismo , Silicatos de Magnésio/toxicidade , Nanoestruturas/toxicidade , Tamanho da Partícula , Estudo de Prova de Conceito , Proteínas/química
10.
Nucleic Acids Res ; 44(10): 4785-4806, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-26961308

RESUMO

Non-homologous end joining is a ligation process repairing DNA double strand breaks in eukaryotes and many prokaryotes. The ring structured eukaryotic Ku binds DNA ends and recruits other factors which can access DNA ends through the threading of Ku inward the DNA, making this protein a key ingredient for the scaffolding of the NHEJ machinery. However, this threading ability seems unevenly conserved among bacterial Ku. As bacterial Ku differ mainly by their C-terminus, we evaluate the role of this region in the loading and the threading abilities of Bacillus subtilis Ku and the stimulation of the DNA ligase LigD. We identify two distinct sub-regions: a ubiquitous minimal C-terminal region and a frequent basic C-terminal extension. We show that truncation of one or both of these sub-regions in Bacillus subtilis Ku impairs the stimulation of the LigD end joining activity in vitro. We further demonstrate that the minimal C-terminus is required for the Ku-LigD interaction, whereas the basic extension controls the threading and DNA bridging abilities of Ku. We propose that the Ku basic C-terminal extension increases the concentration of Ku near DNA ends, favoring the recruitment of LigD at the break, thanks to the minimal C-terminal sub-region.

11.
Haematologica ; 101(12): 1469-1478, 2016 12.
Artigo em Inglês | MEDLINE | ID: mdl-27515249

RESUMO

Megakaryocytes are naturally polyploid cells that increase their ploidy by endomitosis. However, very little is known regarding the mechanism by which they escape the tetraploid checkpoint to become polyploid. Recently, it has been shown that the tetraploid checkpoint was regulated by the Hippo-p53 pathway in response to a downregulation of Rho activity. We therefore analyzed the role of Hippo-p53 pathway in the regulation of human megakaryocyte polyploidy. Our results revealed that Hippo-p53 signaling pathway proteins are present and are functional in megakaryocytes. Although this pathway responds to the genotoxic stress agent etoposide, it is not activated in tetraploid or polyploid megakaryocytes. Furthermore, Hippo pathway was observed to be uncoupled from Rho activity. Additionally, polyploid megakaryocytes showed increased expression of YAP target genes when compared to diploid and tetraploid megakaryocytes. Although p53 knockdown increased both modal ploidy and proplatelet formation in megakaryocytes, YAP knockdown caused no significant change in ploidy while moderately affecting proplatelet formation. Interestingly, YAP knockdown reduced the mitochondrial mass in polyploid megakaryocytes and decreased expression of PGC1α, an important mitochondrial biogenesis regulator. Thus, the Hippo pathway is functional in megakaryocytes, but is not induced by tetraploidy. Additionally, YAP regulates the mitochondrial mass in polyploid megakaryocytes.


Assuntos
Diferenciação Celular , Megacariócitos/citologia , Megacariócitos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Tetraploidia , Proteínas rho de Ligação ao GTP/metabolismo , Biomarcadores , Plaquetas/citologia , Plaquetas/metabolismo , Proteínas de Ciclo Celular , Diferenciação Celular/genética , Expressão Gênica , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Via de Sinalização Hippo , Humanos , Modelos Biológicos , Proteínas Nucleares/metabolismo , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/genética , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Poliploidia , Proteínas Serina-Treonina Quinases/genética , Trombopoese/genética , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Proteínas rho de Ligação ao GTP/genética
12.
PLoS Genet ; 9(10): e1003833, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24130504

RESUMO

The budding yeast Srs2 is the archetype of helicases that regulate several aspects of homologous recombination (HR) to maintain genomic stability. Srs2 inhibits HR at replication forks and prevents high frequencies of crossing-over. Additionally, sensitivity to DNA damage and synthetic lethality with replication and recombination mutants are phenotypes that can only be attributed to another role of Srs2: the elimination of lethal intermediates formed by recombination proteins. To shed light on these intermediates, we searched for mutations that bypass the requirement of Srs2 in DNA repair without affecting HR. Remarkably, we isolated rad52-L264P, a novel allele of RAD52, a gene that encodes one of the most central recombination proteins in yeast. This mutation suppresses a broad spectrum of srs2Δ phenotypes in haploid cells, such as UV and γ-ray sensitivities as well as synthetic lethality with replication and recombination mutants, while it does not significantly affect Rad52 functions in HR and DNA repair. Extensive analysis of the genetic interactions between rad52-L264P and srs2Δ shows that rad52-L264P bypasses the requirement for Srs2 specifically for the prevention of toxic Rad51 filaments. Conversely, this Rad52 mutant cannot restore viability of srs2Δ cells that accumulate intertwined recombination intermediates which are normally processed by Srs2 post-synaptic functions. The avoidance of toxic Rad51 filaments by Rad52-L264P can be explained by a modification of its Rad51 filament mediator activity, as indicated by Chromatin immunoprecipitation and biochemical analysis. Remarkably, sensitivity to DNA damage of srs2Δ cells can also be overcome by stimulating Rad52 sumoylation through overexpression of the sumo-ligase SIZ2, or by replacing Rad52 by a Rad52-SUMO fusion protein. We propose that, like the rad52-L264P mutation, sumoylation modifies Rad52 activity thereby changing the properties of Rad51 filaments. This conclusion is strengthened by the finding that Rad52 is often associated with complete Rad51 filaments in vitro.


Assuntos
DNA Helicases/genética , Recombinação Homóloga/genética , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Citoesqueleto/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Saccharomyces cerevisiae/genética , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/genética , Sumoilação
13.
Mol Microbiol ; 92(6): 1313-25, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24779456

RESUMO

The exceptional genomic content and genome organization of the Acidianus filamentous virus 1 (AFV1) that infects the hyperthermophilic archaeon Acidianus hospitalis suggest that this virus might exploit an unusual mechanism of genome replication. An analysis of replicative intermediates of the viral genome by two-dimensional (2D) agarose gel electrophoresis revealed that viral genome replication starts by the formation of a D-loop and proceeds via strand displacement replication. Characterization of replicative intermediates using dark-field electron microscopy, in combination with the 2D agarose gel electrophoresis data, suggests that recombination plays a key role in the termination of AFV1 genome replication through the formation of terminal loops. A terminal protein was found to be attached to the ends of the viral genome. The results allow us to postulate a model of genome replication that relies on recombination events for initiation and termination.


Assuntos
Vírus de Archaea/fisiologia , Genoma Viral , Lipothrixviridae/fisiologia , Replicação Viral , Vírus de Archaea/genética , Eletroforese em Gel de Ágar , Eletroforese em Gel Bidimensional , Microscopia Eletrônica
14.
PLoS Pathog ; 9(6): e1003473, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23825953

RESUMO

Natural genetic transformation is widely distributed in bacteria and generally occurs during a genetically programmed differentiated state called competence. This process promotes genome plasticity and adaptability in Gram-negative and Gram-positive bacteria. Transformation requires the binding and internalization of exogenous DNA, the mechanisms of which are unclear. Here, we report the discovery of a transformation pilus at the surface of competent Streptococcus pneumoniae cells. This Type IV-like pilus, which is primarily composed of the ComGC pilin, is required for transformation. We provide evidence that it directly binds DNA and propose that the transformation pilus is the primary DNA receptor on the bacterial cell during transformation in S. pneumoniae. Being a central component of the transformation apparatus, the transformation pilus enables S. pneumoniae, a major Gram-positive human pathogen, to acquire resistance to antibiotics and to escape vaccines through the binding and incorporation of new genetic material.


Assuntos
DNA Bacteriano/metabolismo , Proteínas de Fímbrias/metabolismo , Fímbrias Bacterianas/metabolismo , Streptococcus pneumoniae/metabolismo , Transformação Bacteriana/fisiologia , DNA Bacteriano/genética , DNA Bacteriano/imunologia , Resistência a Medicamentos/fisiologia , Proteínas de Fímbrias/genética , Proteínas de Fímbrias/imunologia , Fímbrias Bacterianas/genética , Fímbrias Bacterianas/imunologia , Humanos , Evasão da Resposta Imune/fisiologia , Streptococcus pneumoniae/genética , Streptococcus pneumoniae/imunologia , Streptococcus pneumoniae/patogenicidade
15.
Environ Microbiol ; 16(4): 1167-75, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24034793

RESUMO

Cells from the three domains of life produce extracellular membrane vesicles (MVs), suggesting that MV production is a fundamental aspect of cellular physiology. We have recently shown that MVs produced by the hyperthermophilic archaeon Thermococcus kodakaraensis can be used as vehicles to transfer exogenous recombinant plasmid DNA from cell to cell. Here, we show that Thermococcus nautilus, which harbours three plasmids, pTN1, pTN2 and pTN3, produces MVs, and that some of them selectively incorporate pTN1 and pTN3. Interestingly, pTN3 represents the genome of a defective virus, which encodes signature proteins common to a large group of viruses infecting hosts from all three cellular domains. However, preparations of MVs produced by T. nautilus have a protein composition similar to that of classical MVs from Thermococcales and do not contain the viral major capsid protein encoded by pTN3. Our results suggest that MVs can serve as vehicles for the intercellular transport of viral genomes and facilitate recombination between viral, plasmid and/or cellular chromosomes in the absence of viral infection.


Assuntos
Membrana Celular/virologia , Genoma Viral/genética , Plasmídeos , Thermococcus/genética , Transporte Biológico , Membrana Celular/metabolismo , DNA Viral/genética , Dados de Sequência Molecular , Análise de Sequência de DNA , Thermococcus/metabolismo , Thermococcus/virologia , Proteínas Virais/genética
16.
RNA ; 18(9): 1702-15, 2012 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-22836354

RESUMO

Translational repression is achieved by protein complexes that typically bind 3' UTR mRNA motifs and interfere with the formation of the cap-dependent initiation complex, resulting in mRNPs with a closed-loop conformation. We demonstrate here that the human DEAD-box protein Rck/p54, which is a component of such complexes and central to P-body assembly, is in considerable molecular excess with respect to cellular mRNAs and enriched to a concentration of 0.5 mM in P-bodies, where it is organized in clusters. Accordingly, multiple binding of p54 proteins along mRNA molecules was detected in vivo. Consistently, the purified protein bound RNA with no sequence specificity and high nanomolar affinity. Moreover, bound RNA molecules had a relaxed conformation. While RNA binding was ATP independent, relaxing of bound RNA was dependent on ATP, though not on its hydrolysis. We propose that Rck/p54 recruitment by sequence-specific translational repressors leads to further binding of Rck/p54 along mRNA molecules, resulting in their masking, unwinding, and ultimately recruitment to P-bodies. Rck/p54 proteins located at the 5' extremity of mRNA can then recruit the decapping complex, thus coupling translational repression and mRNA degradation.


Assuntos
RNA Helicases DEAD-box/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA Mensageiro/metabolismo , Trifosfato de Adenosina/metabolismo , Células HeLa , Humanos , Modelos Biológicos , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica
17.
Org Biomol Chem ; 12(17): 2778-83, 2014 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-24668242

RESUMO

The interest in the functionalization of oligonucleotides with organic molecules has grown considerably over the last decade. In this work, we report on the synthesis and characterization of porphyrin-oligonucleotide hybrids containing one to four DNA strands (P1-P4). The hybrid P4, which inserts one porphyrin and four DNA fragments, was combined with gold nanoparticles and imaged by transmission electron microscopy.


Assuntos
DNA/química , Ouro/química , Nanopartículas Metálicas/química , Oligonucleotídeos/química , Porfirinas/química , Espectroscopia de Ressonância Magnética , Nanopartículas Metálicas/ultraestrutura , Microscopia Eletrônica de Transmissão , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz
18.
Proc Natl Acad Sci U S A ; 108(31): 12663-8, 2011 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-21768349

RESUMO

Cernunnos/XLF is a core protein of the nonhomologous DNA end-joining (NHEJ) pathway that processes the majority of DNA double-strand breaks in mammals. Cernunnos stimulates the final ligation step catalyzed by the complex between DNA ligase IV and Xrcc4 (X4). Here we present the crystal structure of the X4(1-157)-Cernunnos(1-224) complex at 5.5-Å resolution and identify the relative positions of the two factors and their binding sites. The X-ray structure reveals a filament arrangement for X4(1-157) and Cernunnos(1-224) homodimers mediated by repeated interactions through their N-terminal head domains. A filament arrangement of the X4-Cernunnos complex was confirmed by transmission electron microscopy analyses both with truncated and full-length proteins. We further modeled the interface and used structure-based site-directed mutagenesis and calorimetry to characterize the roles of various residues at the X4-Cernunnos interface. We identified four X4 residues (Glu(55), Asp(58), Met(61), and Phe(106)) essential for the interaction with Cernunnos. These findings provide new insights into the molecular bases for stimulatory and bridging roles of Cernunnos in the final DNA ligation step.


Assuntos
Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Sequência de Aminoácidos , Ácido Aspártico/química , Ácido Aspártico/genética , Ácido Aspártico/metabolismo , Sítios de Ligação/genética , Western Blotting , Calorimetria , Cristalografia por Raios X , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Ácido Glutâmico/química , Ácido Glutâmico/genética , Ácido Glutâmico/metabolismo , Humanos , Metionina/química , Metionina/genética , Metionina/metabolismo , Microscopia Eletrônica de Transmissão , Modelos Moleculares , Dados de Sequência Molecular , Mutação , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Fragmentos de Peptídeos/ultraestrutura , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Ligação Proteica , Multimerização Proteica , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos
19.
Life Sci Alliance ; 7(3)2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38081641

RESUMO

Homologous recombination (HR) is a DNA repair mechanism of double-strand breaks and blocked replication forks, involving a process of homology search leading to the formation of synaptic intermediates that are regulated to ensure genome integrity. RAD51 recombinase plays a central role in this mechanism, supported by its RAD52 and BRCA2 partners. If the mediator function of BRCA2 to load RAD51 on RPA-ssDNA is well established, the role of RAD52 in HR is still far from understood. We used transmission electron microscopy combined with biochemistry to characterize the sequential participation of RPA, RAD52, and BRCA2 in the assembly of the RAD51 filament and its activity. Although our results confirm that RAD52 lacks a mediator activity, RAD52 can tightly bind to RPA-coated ssDNA, inhibit the mediator activity of BRCA2, and form shorter RAD51-RAD52 mixed filaments that are more efficient in the formation of synaptic complexes and D-loops, resulting in more frequent multi-invasions as well. We confirm the in situ interaction between RAD51 and RAD52 after double-strand break induction in vivo. This study provides new molecular insights into the formation and regulation of presynaptic and synaptic intermediates by BRCA2 and RAD52 during human HR.


Assuntos
Rad51 Recombinase , Proteína de Replicação A , Humanos , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo , Rad51 Recombinase/genética , DNA de Cadeia Simples/genética , Reparo do DNA/genética , Recombinação Homóloga/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo
20.
Acta Crystallogr D Biol Crystallogr ; 69(Pt 3): 409-19, 2013 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-23519416

RESUMO

Repressor activator protein 1 (Rap1) is an essential factor involved in transcription and telomere stability in the budding yeast Saccharomyces cerevisiae. Its interaction with DNA causes hypersensitivity to potassium permanganate, suggesting local DNA melting and/or distortion. In this study, various Rap1-DNA crystal forms were obtained using specifically designed crystal screens. Analysis of the DNA conformation showed that its distortion was not sufficient to explain the permanganate reactivity. However, anomalous data collected at the Mn edge using a Rap1-DNA crystal soaked in potassium permanganate solution indicated that the DNA conformation in the crystal was compatible with interaction with permanganate ions. Sequence-conservation analysis revealed that double-Myb-containing Rap1 proteins all carry a fully conserved Arg580 at a position that may favour interaction with permanganate ions, although it is not involved in the hypersensitive cytosine distortion. Permanganate reactivity assays with wild-type Rap1 and the Rap1[R580A] mutant demonstrated that Arg580 is essential for hypersensitivity. AFM experiments showed that wild-type Rap1 and the Rap1[R580A] mutant interact with DNA over 16 successive binding sites, leading to local DNA stiffening but not to accumulation of the observed local distortion. Therefore, Rap1 may cause permanganate hypersensitivity of DNA by forming a pocket between the reactive cytosine and Arg580, driving the permanganate ion towards the C5-C6 bond of the cytosine.


Assuntos
DNA Fúngico/química , DNA Fúngico/metabolismo , Permanganato de Potássio/química , Permanganato de Potássio/farmacologia , Proteínas de Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/química , Proteínas de Ligação a Telômeros/química , Fatores de Transcrição/química , Arginina/química , Cristalografia por Raios X , Citosina/química , DNA Fúngico/efeitos dos fármacos , Ligação de Hidrogênio/efeitos dos fármacos , Conformação de Ácido Nucleico/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Complexo Shelterina , Soluções , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
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