Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 79
Filtrar
1.
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
2.
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
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 ; 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
5.
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
6.
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
7.
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
8.
QRB Discov ; 3: e15, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-37529279

RESUMO

Interactions between proteins and single-stranded DNA (ssDNA) are crucial for many fundamental biological processes, including DNA replication and genetic recombination. Thus, understanding detailed mechanisms of these interactions is necessary to uncover regulatory rules occurring in all living cells. The RNA-binding Hfq is a pleiotropic bacterial regulator that mediates many aspects of nucleic acid metabolism. The protein notably mediates mRNA stability and translation efficiency by using stress-related small regulatory RNA as cofactors. In addition, Hfq helps to compact double-stranded DNA. In this paper, we focused on the action of Hfq on ssDNA. A combination of experimental methodologies, including spectroscopy and molecular imaging, has been used to probe the interactions of Hfq and its amyloid C-terminal region with ssDNA. Our analysis revealed that Hfq binds to ssDNA. Moreover, we demonstrate for the first time that Hfq drastically changes the structure and helical parameters of ssDNA, mainly due to its C-terminal amyloid-like domain. The formation of the nucleoprotein complexes between Hfq and ssDNA unveils important implications for DNA replication and recombination.

9.
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
10.
ACS Nano ; 15(3): 4186-4196, 2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33586425

RESUMO

Technological breakthroughs in electron microscopy (EM) have made it possible to solve structures of biological macromolecular complexes and to raise novel challenges, specifically related to sample preparation and heterogeneous macromolecular assemblies such as DNA-protein, protein-protein, and membrane protein assemblies. Here, we built a V-shaped DNA origami as a scaffolding molecular system to template proteins at user-defined positions in space. This template positions macromolecular assemblies of various sizes, juxtaposes combinations of biomolecules into complex arrangements, isolates biomolecules in their active state, and stabilizes membrane proteins in solution. In addition, the design can be engineered to tune DNA mechanical properties by exerting a controlled piconewton (pN) force on the molecular system and thus adapted to characterize mechanosensitive proteins. The binding site can also be specifically customized to accommodate the protein of interest, either interacting spontaneously with DNA or through directed chemical conjugation, increasing the range of potential targets for single-particle EM investigation. We assessed the applicability for five different proteins. Finally, as a proof of principle, we used RNAP protein to validate the approach and to explore the compatibility of the template with cryo-EM sample preparation.


Assuntos
DNA , Imagem Individual de Molécula , Microscopia Crioeletrônica , Substâncias Macromoleculares , Microscopia Eletrônica
11.
Biol Methods Protoc ; 5(1): bpaa012, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32913896

RESUMO

DNA intermediate structures are formed in all major pathways of DNA metabolism. Transmission electron microscopy (TEM) is a tool of choice to study their choreography and has led to major advances in the understanding of these mechanisms, particularly those of homologous recombination (HR) and replication. In this article, we describe specific TEM procedures dedicated to the structural characterization of DNA intermediates formed during these processes. These particular DNA species contain single-stranded DNA regions and/or branched structures, which require controlling both the DNA molecules spreading and their staining for subsequent visualization using dark-field imaging mode. Combining BAC (benzyl dimethyl alkyl ammonium chloride) film hyperphase with positive staining and dark-field TEM allows characterizing synthetic DNA substrates, joint molecules formed during not only in vitro assays mimicking HR, but also in vivo DNA intermediates.

12.
DNA Repair (Amst) ; 82: 102698, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31518879

RESUMO

Aerobic respiration generates reactive oxygen species (ROS), which can damage nucleic acids, proteins and lipids. A number of transcription factors (TFs) contain redox-sensitive cysteine residues at their DNA-binding sites, hence ROS-induced thiol oxidation strongly inhibits their recognition of the cognate DNA sequences. Major human apurinic/apyrimidinic (AP) endonuclease 1 (APE1/APEX1/HAP-1), referred also as a redox factor 1 (Ref-1), stimulates the DNA binding activities of the oxidized TFs such as AP-1 and NF-κB. Also, APE1 participates in the base excision repair (BER) and nucleotide incision repair (NIR) pathways to remove oxidative DNA base damage. At present, the molecular mechanism underlying the TF-stimulating/redox function of APE1 and its biological role remains disputed. Here, we provide evidence that, instead of direct cysteine reduction in TFs by APE1, APE1-catalyzed NIR and TF-stimulating activities may be based on transient cooperative binding of APE1 to DNA and induction of conformational changes in the helix. The structure of DNA duplex strongly influences NIR and TF-stimulating activities. Homologous plant AP endonucleases lacking conserved cysteine residues stimulate DNA binding of the p50 subunit of NF-κB. APE1 acts synergistically with low-molecular-weight reducing agents on TFs. Finally, APE1 stimulates DNA binding of the redox-insensitive p50-C62S mutant protein. Electron microscopy imaging of APE1 complexes with DNA revealed preferential polymerization of APE1 on the gapped and intrinsically curved DNA duplexes. Molecular modeling offers a structural explanation how full-length APE1 can oligomerize on DNA. In conclusion, we propose that DNA-directed APE1 oligomerization can be regarded as a substitute for diffusion of APE1 along the DNA contour to probe for anisotropic flexibility. APE1 oligomers exacerbate pre-existing distortions in DNA and enable both NIR activity and DNA binding by TFs regardless of their oxidation state.


Assuntos
DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/metabolismo , Fatores de Transcrição/metabolismo , Sequência de Aminoácidos , Biocatálise , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Humanos , Modelos Moleculares , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína
13.
Nat Commun ; 10(1): 4058, 2019 09 06.
Artigo em Inglês | MEDLINE | ID: mdl-31492866

RESUMO

Homologous recombination (HR) uses a homologous template to accurately repair DNA double-strand breaks and stalled replication forks to maintain genome stability. During homology search, Rad51 nucleoprotein filaments probe and interact with dsDNA, forming the synaptic complex that is stabilized on a homologous sequence. Strand intertwining leads to the formation of a displacement-loop (D-loop). In yeast, Rad54 is essential for HR in vivo and required for D-loop formation in vitro, but its exact role remains to be fully elucidated. Using electron microscopy to visualize the DNA-protein complexes, here we find that Rad54 is crucial for Rad51-mediated synaptic complex formation and homology search. The Rad54-K341R ATPase-deficient mutant protein promotes formation of synaptic complexes but not D-loops and leads to the accumulation of stable heterologous associations, suggesting that the Rad54 ATPase is involved in preventing non-productive intermediates. We propose that Rad51/Rad54 form a functional unit operating in homology search, synaptic complex and D-loop formation.


Assuntos
DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , DNA de Cadeia Simples/metabolismo , DNA/metabolismo , Substâncias Macromoleculares/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , DNA/química , DNA/ultraestrutura , DNA Helicases/química , DNA Helicases/genética , Enzimas Reparadoras do DNA/química , Enzimas Reparadoras do DNA/genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Recombinação Homóloga , Substâncias Macromoleculares/química , Substâncias Macromoleculares/ultraestrutura , Microscopia Eletrônica , Mutação , Ligação Proteica , Rad51 Recombinase/química , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
14.
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
15.
Nat Struct Mol Biol ; 25(10): 971-980, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-30291363

RESUMO

The Ku70-Ku80 (Ku) heterodimer binds rapidly and tightly to the ends of DNA double-strand breaks and recruits factors of the non-homologous end-joining (NHEJ) repair pathway through molecular interactions that remain unclear. We have determined crystal structures of the Ku-binding motifs (KBM) of the NHEJ proteins APLF (A-KBM) and XLF (X-KBM) bound to a Ku-DNA complex. The two KBM motifs bind remote sites of the Ku80 α/ß domain. The X-KBM occupies an internal pocket formed by an unprecedented large outward rotation of the Ku80 α/ß domain. We observe independent recruitment of the APLF-interacting protein XRCC4 and of XLF to laser-irradiated sites via binding of A- and X-KBMs, respectively, to Ku80. Finally, we show that mutation of the X-KBM and A-KBM binding sites in Ku80 compromises both the efficiency and accuracy of end joining and cellular radiosensitivity. A- and X-KBMs may represent two initial anchor points to build the intricate interaction network required for NHEJ.


Assuntos
Reparo do DNA por Junção de Extremidades , Enzimas Reparadoras do DNA/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , Proteínas de Ligação a DNA/química , Autoantígeno Ku/química , Proteínas de Ligação a Poli-ADP-Ribose/química , Sequência Conservada , Cristalografia por Raios X , Enzimas Reparadoras do DNA/metabolismo , Enzimas Reparadoras do DNA/fisiologia , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/fisiologia , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/fisiologia , Humanos , Autoantígeno Ku/metabolismo , Autoantígeno Ku/fisiologia , Modelos Moleculares , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/fisiologia , Domínios Proteicos
16.
Methods Mol Biol ; 1805: 251-270, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29971722

RESUMO

Transmission electron microscopy (TEM) and atomic force microscopy (AFM) are powerful tools to study the behavior of various actors in homologous recombination including molecular motors such as recombinases and helicases/translocases. Here we present specific approaches developed in terms of sample preparation and imaging methods to contribute to the understanding of homologous recombination process and its regulation focusing on the interplay between recombinases and other related proteins such as mediators or antirecombinase actors.Homologous recombination (HR) is a high-fidelity DNA repair pathway since it uses a homologous DNA as template. Recombinases such as RecA in bacteria, RadA in archaea, and Rad51 in eukaryotes are key proteins in the HR pathway: HR is initiated with formation of an ssDNA overhang on which recombinases polymerize and form a dynamic active nucleoprotein filament able to search for homology and to exchange DNA strand in an ATP-dependent manner. We provide practical methods to analyze presynaptic filament formation on ssDNA, its composition and regulation in presence of mediator partners, antirecombinase activity of translocase, and chromatin remodeling events.


Assuntos
Recombinação Homóloga , Microscopia/métodos , Recombinases/metabolismo , Montagem e Desmontagem da Cromatina , DNA/ultraestrutura , Quebras de DNA de Cadeia Simples , Humanos , Microscopia de Força Atômica , Nucleossomos/metabolismo , Polimerização , Proteínas/metabolismo
17.
Elife ; 72018 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-29985128

RESUMO

Homology search and strand exchange mediated by Rad51 nucleoprotein filaments are key steps of the homologous recombination process. In budding yeast, Rad52 is the main mediator of Rad51 filament formation, thereby playing an essential role. The current model assumes that Rad51 filament formation requires the interaction between Rad52 and Rad51. However, we report here that Rad52 mutations that disrupt this interaction do not affect γ-ray- or HO endonuclease-induced gene conversion frequencies. In vivo and in vitro studies confirmed that Rad51 filaments formation is not affected by these mutations. Instead, we found that Rad52-Rad51 association makes Rad51 filaments toxic in Srs2-deficient cells after exposure to DNA damaging agents, independently of Rad52 role in Rad51 filament assembly. Importantly, we also demonstrated that Rad52 is essential for protecting Rad51 filaments against dissociation by the Srs2 DNA translocase. Our findings open new perspectives in the understanding of the role of Rad52 in eukaryotes.


Assuntos
DNA Helicases/metabolismo , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Alelos , Sequência de Aminoácidos , Quebras de DNA de Cadeia Dupla , DNA Fúngico/metabolismo , Conversão Gênica , Recombinação Homóloga , Modelos Biológicos , Proteínas Mutantes/metabolismo , Mutação/genética , Ligação Proteica , Domínios Proteicos , Proteína Rad52 de Recombinação e Reparo de DNA/química , Proteínas de Saccharomyces cerevisiae/química , Sumoilação
18.
DNA Repair (Amst) ; 64: 10-25, 2018 04.
Artigo em Inglês | MEDLINE | ID: mdl-29475157

RESUMO

The base excision repair (BER) pathway consists of sequential action of DNA glycosylase and apurinic/apyrimidinic (AP) endonuclease necessary to remove a damaged base and generate a single-strand break in duplex DNA. Human multifunctional AP endonuclease 1 (APE1, a.k.a. APEX1, HAP-1, or Ref-1) plays essential roles in BER by acting downstream of DNA glycosylases to incise a DNA duplex at AP sites and remove 3'-blocking sugar moieties at DNA strand breaks. Human 8-oxoguanine-DNA glycosylase (OGG1), methyl-CpG-binding domain 4 (MBD4, a.k.a. MED1), and alkyl-N-purine-DNA glycosylase (ANPG, a.k.a. Aag or MPG) excise a variety of damaged bases from DNA. Here we demonstrated that the redox-deficient truncated APE1 protein lacking the first N-terminal 61 amino acid residues (APE1-NΔ61) cannot stimulate DNA glycosylase activities of OGG1, MBD4, and ANPG on duplex DNA substrates. Electron microscopy imaging of APE1-DNA complexes revealed oligomerization of APE1 along the DNA duplex and APE1-mediated DNA bridging followed by DNA aggregation. APE1 polymerizes on both undamaged and damaged DNA in cooperative mode. Association of APE1 with undamaged DNA may enable scanning for damage; however, this event reduces effective concentration of the enzyme and subsequently decreases APE1-catalyzed cleavage rates on long DNA substrates. We propose that APE1 oligomers on DNA induce helix distortions thereby enhancing molecular recognition of DNA lesions by DNA glycosylases via a conformational proofreading/selection mechanism. Thus, APE1-mediated structural deformations of the DNA helix stabilize the enzyme-substrate complex and promote dissociation of human DNA glycosylases from the AP site with a subsequent increase in their turnover rate. SIGNIFICANCE STATEMENT: The major human apurinic/apyrimidinic (AP) endonuclease, APE1, stimulates DNA glycosylases by increasing their turnover rate on duplex DNA substrates. At present, the mechanism of the stimulation remains unclear. We report that the redox domain of APE1 is necessary for the active mode of stimulation of DNA glycosylases. Electron microscopy revealed that full-length APE1 oligomerizes on DNA possibly via cooperative binding to DNA. Consequently, APE1 shows DNA length dependence with preferential repair of short DNA duplexes. We propose that APE1-catalyzed oligomerization along DNA induces helix distortions, which in turn enable conformational selection and stimulation of DNA glycosylases. This new biochemical property of APE1 sheds light on the mechanism of redox function and its role in DNA repair.


Assuntos
DNA Glicosilases/metabolismo , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , DNA/metabolismo , Domínios e Motivos de Interação entre Proteínas , DNA/química , Dano ao DNA , Endodesoxirribonucleases/metabolismo , Humanos , Conformação de Ácido Nucleico
19.
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
20.
Sci Rep ; 7(1): 5586, 2017 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-28717157

RESUMO

Sepiolite is a nanofibrous natural silicate that can be used as a nanocarrier because it can be naturally internalized into mammalian cells, due to its nano-size dimension. Therefore, deciphering the mechanisms of sepiolite cell internalization constitutes a question interesting biotechnology, for the use of sepiolite as nanocarrier, as well as environmental and public health concerns. Though it is low, the perfectly stable and natural intrinsic fluorescence of sepiolite nanofibers allows to follow their fate into cells by specifically sensitive technics. By combining fluorescence microscopy (including confocal analysis), time-lapse video microscopy, fluorescence activated cell sorting and transmission electron microscopy, we show that sepiolite can be spontaneously internalized into mammalian cells through both non-endocytic and endocytic pathways, macropinocytosis being one of the main pathways. Interestingly, exposure of the cells to endocytosis inhibitors, such as chloroquine, two-fold increase the efficiency of sepiolite-mediated gene transfer, in addition to the 100-fold increased resulting from sepiolite sonomechanical treatment. As sepiolite is able to bind various biological molecules, this nanoparticulate silicate could be a good candidate as a nanocarrier for simultaneous vectorization of diverse biological molecules.


Assuntos
DNA/química , DNA/genética , Portadores de Fármacos/química , Silicatos de Magnésio/química , Transfecção/métodos , Animais , Células CHO , Linhagem Celular , Separação Celular , Cloroquina/farmacologia , Cricetulus , Endocitose , Citometria de Fluxo , Humanos , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Imagem com Lapso de Tempo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...