Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.838
Filtrar
1.
Nat Commun ; 11(1): 5241, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-33067443

RESUMO

To understand how the RuvC catalytic domain of Class 2 Cas proteins cleaves DNA, it will be necessary to elucidate the structures of RuvC-containing Cas complexes in their catalytically competent states. Cas12i2 is a Class 2 type V-I CRISPR-Cas endonuclease that cleaves target dsDNA by an unknown mechanism. Here, we report structures of Cas12i2-crRNA-DNA complexes and a Cas12i2-crRNA complex. We reveal the mechanism of DNA recognition and cleavage by Cas12i2, and activation of the RuvC catalytic pocket induced by a conformational change of the Helical-II domain. The seed region (nucleotides 1-8) is dispensable for RuvC activation, but the duplex of the central spacer (nucleotides 9-15) is required. We captured the catalytic state of Cas12i2, with both metal ions and the ssDNA substrate bound in the RuvC catalytic pocket. Together, our studies provide significant insights into the DNA cleavage mechanism by RuvC-containing Cas proteins.


Assuntos
DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/química , Desoxirribonuclease I/metabolismo , Metais/metabolismo , Catálise , Domínio Catalítico , Clivagem do DNA , DNA de Cadeia Simples/química , DNA de Cadeia Simples/genética , Desoxirribonuclease I/genética , Íons/química , Íons/metabolismo , Metais/química
2.
Nucleic Acids Res ; 48(14): 7818-7833, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32609828

RESUMO

The tumor suppressor BRCA2 plays a key role in initiating homologous recombination by facilitating RAD51 filament formation on single-stranded DNA. The small acidic protein DSS1 is a crucial partner to BRCA2 in this process. In vitro and in cells (1,2), BRCA2 associates into oligomeric complexes besides also existing as monomers. A dimeric structure was further characterized by electron microscopic analysis (3), but the functional significance of the different BRCA2 assemblies remains to be determined. Here, we used biochemistry and electron microscopic imaging to demonstrate that the multimerization of BRCA2 is counteracted by DSS1 and ssDNA. When validating the findings, we identified three self-interacting regions and two types of self-association, the N-to-C terminal and the N-to-N terminal interactions. The N-to-C terminal self-interaction of BRCA2 is sensitive to DSS1 and ssDNA. The N-to-N terminal self-interaction is modulated by ssDNA. Our results define a novel role of DSS1 to regulate BRCA2 in an RPA-independent fashion. Since DSS1 is required for BRCA2 function in recombination, we speculate that the monomeric and oligomeric forms of BRCA2 might be active for different cellular events in recombinational DNA repair and replication fork stabilization.


Assuntos
Proteína BRCA2/metabolismo , DNA de Cadeia Simples/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Proteína BRCA2/química , Proteína BRCA2/genética , Proteína BRCA2/ultraestrutura , Linhagem Celular , Cricetulus , Humanos , Multimerização Proteica
3.
Nat Commun ; 11(1): 3713, 2020 07 24.
Artigo em Inglês | MEDLINE | ID: mdl-32709841

RESUMO

A ring-shaped helicase unwinds DNA during chromosome replication in all organisms. Replicative helicases generally unwind duplex DNA an order of magnitude slower compared to their in vivo replication fork rates. However, the origin of slow DNA unwinding rates by replicative helicases and the mechanism by which other replication components increase helicase speed are unclear. Here, we demonstrate that engagement of the eukaryotic CMG helicase with template DNA at the replication fork impairs its helicase activity, which is alleviated by binding of the single-stranded DNA binding protein, RPA, to the excluded DNA strand. Intriguingly, we found that, when stalled due to interaction with the parental duplex, DNA rezipping-induced helicase backtracking reestablishes productive helicase-fork engagement, underscoring the significance of plasticity in helicase action. Our work provides a mechanistic basis for relatively slow duplex unwinding by replicative helicases and explains how replisome components that interact with the excluded DNA strand stimulate fork rates.


Assuntos
DNA Helicases/metabolismo , Replicação do DNA/fisiologia , DNA/química , DNA/metabolismo , Animais , Bacteriófago T4 , Microscopia Crioeletrônica , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Drosophila melanogaster/genética , Escherichia coli/genética
4.
Mol Cell ; 79(5): 741-757.e7, 2020 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-32730741

RESUMO

Cmr-ß is a type III-B CRISPR-Cas complex that, upon target RNA recognition, unleashes a multifaceted immune response against invading genetic elements, including single-stranded DNA (ssDNA) cleavage, cyclic oligoadenylate synthesis, and also a unique UA-specific single-stranded RNA (ssRNA) hydrolysis by the Cmr2 subunit. Here, we present the structure-function relationship of Cmr-ß, unveiling how binding of the target RNA regulates the Cmr2 activities. Cryoelectron microscopy (cryo-EM) analysis revealed the unique subunit architecture of Cmr-ß and captured the complex in different conformational stages of the immune response, including the non-cognate and cognate target-RNA-bound complexes. The binding of the target RNA induces a conformational change of Cmr2, which together with the complementation between the 5' tag in the CRISPR RNAs (crRNA) and the 3' antitag of the target RNA activate different configurations in a unique loop of the Cmr3 subunit, which acts as an allosteric sensor signaling the self- versus non-self-recognition. These findings highlight the diverse defense strategies of type III complexes.


Assuntos
Imunidade Adaptativa/fisiologia , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/fisiologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Proteínas Arqueais/química , Proteínas Arqueais/fisiologia , Proteínas Arqueais/ultraestrutura , Proteínas Associadas a CRISPR/ultraestrutura , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/fisiologia , Microscopia Crioeletrônica , DNA de Cadeia Simples/metabolismo , Modelos Moleculares , Ligação Proteica , Conformação Proteica , RNA Mensageiro/metabolismo , Relação Estrutura-Atividade , Sulfolobus/genética , Sulfolobus/fisiologia
5.
Nucleic Acids Res ; 48(14): 7991-8005, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32621607

RESUMO

DNA2 is an essential enzyme involved in DNA replication and repair in eukaryotes. In a search for homologues of this protein, we identified and characterised Geobacillus stearothermophilus Bad, a bacterial DNA helicase-nuclease with similarity to human DNA2. We show that Bad contains an Fe-S cluster and identify four cysteine residues that are likely to co-ordinate the cluster by analogy to DNA2. The purified enzyme specifically recognises ss-dsDNA junctions and possesses ssDNA-dependent ATPase, ssDNA binding, ssDNA endonuclease, 5' to 3' ssDNA translocase and 5' to 3' helicase activity. Single molecule analysis reveals that Bad is a processive DNA motor capable of moving along DNA for distances of >4 kb at a rate of ∼200 bp per second at room temperature. Interestingly, as reported for the homologous human and yeast DNA2 proteins, the DNA unwinding activity of Bad is cryptic and can be unmasked by inactivating the intrinsic nuclease activity. Strikingly, our experiments show that the enzyme loops DNA while translocating, which is an emerging feature of processive DNA unwinding enzymes. The bacterial Bad enzymes will provide an excellent model system for understanding the biochemical properties of DNA2-like helicase-nucleases and DNA looping motor proteins in general.


Assuntos
Proteínas de Bactérias/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Desoxirribonuclease I/metabolismo , Geobacillus stearothermophilus/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/isolamento & purificação , DNA , DNA Helicases/química , DNA Helicases/isolamento & purificação , Desoxirribonuclease I/química , Desoxirribonuclease I/isolamento & purificação
6.
Nucleic Acids Res ; 48(14): 7834-7843, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32621611

RESUMO

RADX is a mammalian single-stranded DNA-binding protein that stabilizes telomeres and stalled replication forks. Cellular biology studies have shown that the balance between RADX and Replication Protein A (RPA) is critical for DNA replication integrity. RADX is also a negative regulator of RAD51-mediated homologous recombination at stalled forks. However, the mechanism of RADX acting on DNA and its interactions with RPA and RAD51 are enigmatic. Using single-molecule imaging of the key proteins in vitro, we reveal that RADX condenses ssDNA filaments, even when the ssDNA is coated with RPA at physiological protein ratios. RADX compacts RPA-coated ssDNA filaments via higher-order assemblies that can capture ssDNA in trans. Furthermore, RADX blocks RPA displacement by RAD51 and prevents RAD51 loading on ssDNA. Our results indicate that RADX is an ssDNA condensation protein that inhibits RAD51 filament formation and may antagonize other ssDNA-binding proteins on RPA-coated ssDNA.


Assuntos
DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Rad51 Recombinase/metabolismo , Humanos , Proteína de Replicação A/metabolismo , Imagem Individual de Molécula
7.
Mol Cell ; 79(4): 689-701.e10, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32610038

RESUMO

Meiotic recombination proceeds via binding of RPA, RAD51, and DMC1 to single-stranded DNA (ssDNA) substrates created after formation of programmed DNA double-strand breaks. Here we report high-resolution in vivo maps of RPA and RAD51 in meiosis, mapping their binding locations and lifespans to individual homologous chromosomes using a genetically engineered hybrid mouse. Together with high-resolution microscopy and DMC1 binding maps, we show that DMC1 and RAD51 have distinct spatial localization on ssDNA: DMC1 binds near the break site, and RAD51 binds away from it. We characterize inter-homolog recombination intermediates bound by RPA in vivo, with properties expected for the critical displacement loop (D-loop) intermediates. These data support the hypothesis that DMC1, not RAD51, performs strand exchange in mammalian meiosis. RPA-bound D-loops can be resolved as crossovers or non-crossovers, but crossover-destined D-loops may have longer lifespans. D-loops resemble crossover gene conversions in size, but their extent is similar in both repair pathways.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Recombinação Homóloga , Meiose , Proteínas de Ligação a Fosfato/metabolismo , Rad51 Recombinase/metabolismo , Proteína de Replicação A/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Cromossomos/genética , Cromossomos/metabolismo , Troca Genética , DNA de Cadeia Simples/metabolismo , Genoma , Masculino , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Proteínas de Ligação a Fosfato/genética , Rad51 Recombinase/genética , Proteína de Replicação A/genética , Testículo
8.
Nat Commun ; 11(1): 2948, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528060

RESUMO

Homologous recombination (HR) mediates the error-free repair of DNA double-strand breaks to maintain genomic stability. Here we characterize C17orf53/MCM8IP, an OB-fold containing protein that binds ssDNA, as a DNA repair factor involved in HR. MCM8IP-deficient cells exhibit HR defects, especially in long-tract gene conversion, occurring downstream of RAD51 loading, consistent with a role for MCM8IP in HR-dependent DNA synthesis. Moreover, loss of MCM8IP confers cellular sensitivity to crosslinking agents and PARP inhibition. Importantly, we report that MCM8IP directly associates with MCM8-9, a helicase complex mutated in primary ovarian insufficiency, and RPA1. We additionally show that the interactions of MCM8IP with MCM8-9 and RPA facilitate HR and promote replication fork progression and cellular viability in response to treatment with crosslinking agents. Mechanistically, MCM8IP stimulates the helicase activity of MCM8-9. Collectively, our work identifies MCM8IP as a key regulator of MCM8-9-dependent DNA synthesis during DNA recombination and replication.


Assuntos
Dano ao DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Manutenção de Minicromossomo/metabolismo , Reparo de DNA por Recombinação , Linhagem Celular Tumoral , Sobrevivência Celular/genética , Cromatina/genética , Cromatina/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Células HCT116 , Células HEK293 , Humanos , Proteínas de Manutenção de Minicromossomo/genética , Mutação , Ligação Proteica , Rad51 Recombinase/metabolismo , Proteína de Replicação A/genética , Proteína de Replicação A/metabolismo
9.
Nat Commun ; 11(1): 2828, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32504003

RESUMO

The TATA-binding protein (TBP) and a transcription factor (TF) IIB-like factor are important constituents of all eukaryotic initiation complexes. The reason for the emergence and strict requirement of the additional initiation factor Bdp1 in the RNA polymerase (RNAP) III system, however, remained elusive. A poorly studied aspect in this context is the effect of DNA strain arising from DNA compaction and transcriptional activity on initiation complex formation. We made use of a DNA origami-based force clamp to follow the assembly of human initiation complexes in the RNAP II and RNAP III systems at the single-molecule level under piconewton forces. We demonstrate that TBP-DNA complexes are force-sensitive and TFIIB is sufficient to stabilise TBP on a strained promoter. In contrast, Bdp1 is the pivotal component that ensures stable anchoring of initiation factors, and thus the polymerase itself, in the RNAP III system. Thereby, we offer an explanation for the crucial role of Bdp1 for the high transcriptional output of RNAP III.


Assuntos
DNA de Cadeia Simples/metabolismo , RNA Polimerase III/metabolismo , Imagem Individual de Molécula/métodos , Fator de Transcrição TFIIIB/metabolismo , Transcrição Genética , DNA de Cadeia Simples/química , DNA de Cadeia Simples/ultraestrutura , Transferência Ressonante de Energia de Fluorescência , Cinética , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Sondas Moleculares/química , Sondas Moleculares/metabolismo , Sondas Moleculares/ultraestrutura , Conformação de Ácido Nucleico , Regiões Promotoras Genéticas , Estabilidade Proteica , RNA Polimerase III/química , Proteínas Recombinantes/metabolismo , Proteína de Ligação a TATA-Box/metabolismo
10.
Mol Cell ; 79(1): 115-126.e6, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32497497

RESUMO

Extension of telomeres is a critical step in the immortalization of cancer cells. This complex reaction requires proper spatiotemporal coordination of telomerase and telomeres and remains poorly understood at the cellular level. To understand how cancer cells execute this process, we combine CRISPR genome editing and MS2 RNA tagging to image single molecules of telomerase RNA (hTR). Real-time dynamics and photoactivation experiments of hTR in Cajal bodies (CBs) reveal that hTERT controls the exit of hTR from CBs. Single-molecule tracking of hTR at telomeres shows that TPP1-mediated recruitment results in short telomere-telomerase scanning interactions, and then base pairing between hTR and telomere ssDNA promotes long interactions required for stable telomerase retention. Interestingly, POT1 OB-fold mutations that result in abnormally long telomeres in cancers act by enhancing this retention step. In summary, single-molecule imaging unveils the life cycle of telomerase RNA and provides a framework to reveal how cancer-associated mutations mechanistically drive defects in telomere homeostasis.


Assuntos
Corpos Enovelados/metabolismo , DNA de Cadeia Simples/metabolismo , RNA/metabolismo , Imagem Individual de Molécula/métodos , Telomerase/metabolismo , Homeostase do Telômero , Telômero/metabolismo , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , DNA de Cadeia Simples/genética , Edição de Genes , Células HeLa , Humanos , Mutação , RNA/genética , Telomerase/genética , Telômero/genética , Proteínas de Ligação a Telômeros/genética , Proteínas de Ligação a Telômeros/metabolismo
11.
Nat Commun ; 11(1): 3114, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32561744

RESUMO

Revealing antibody-antigen interactions at the single-molecule level will deepen our understanding of immunology. However, structural determination under crystal or cryogenic conditions does not provide temporal resolution for resolving transient, physiologically or pathologically relevant functional antibody-antigen complexes. Here, we develop a triangular DNA origami framework with site-specifically anchored and spatially organized artificial epitopes to capture transient conformations of immunoglobulin Gs (IgGs) at room temperature. The DNA origami epitopes (DOEs) allows programmed spatial distribution of epitope spikes, which enables direct imaging of functional complexes with atomic force microscopy (AFM). We establish the critical dependence of the IgG avidity on the lateral distance of epitopes within 3-20 nm at the single-molecule level. High-speed AFM imaging of transient conformations further provides structural and dynamic evidence for the IgG avidity from monovalent to bivalent in a single event, which sheds light on various applications including virus neutralization, diagnostic detection and cancer immunotherapy.


Assuntos
Afinidade de Anticorpos , Epitopos/ultraestrutura , Imunoglobulina G/ultraestrutura , Sondas Moleculares/ultraestrutura , Imagem Individual de Molécula/métodos , Complexo Antígeno-Anticorpo/ultraestrutura , DNA de Cadeia Simples/imunologia , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Epitopos/imunologia , Epitopos/metabolismo , Transferência Ressonante de Energia de Fluorescência/métodos , Humanos , Imunoglobulina G/imunologia , Imunoglobulina G/metabolismo , Microscopia de Força Atômica/métodos , Simulação de Dinâmica Molecular , Sondas Moleculares/imunologia , Sondas Moleculares/metabolismo , Nanotecnologia , Relação Estrutura-Atividade
12.
Proc Natl Acad Sci U S A ; 117(21): 11257-11264, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32404423

RESUMO

Dmc1 recombinases are essential to homologous recombination in meiosis. Here, we studied the kinetics of the nucleoprotein filament assembly of Saccharomyces cerevisiae Dmc1 using single-molecule tethered particle motion experiments and in vitro biochemical assay. ScDmc1 nucleoprotein filaments are less stable than the ScRad51 ones because of the kinetically much reduced nucleation step. The lower nucleation rate of ScDmc1 results from its lower single-stranded DNA (ssDNA) affinity, compared to that of ScRad51. Surprisingly, ScDmc1 nucleates mostly on the DNA structure containing the single-stranded and duplex DNA junction with the allowed extension in the 5'-to-3' polarity, while ScRad51 nucleation depends strongly on ssDNA lengths. This nucleation preference is also conserved for mammalian RAD51 and DMC1. In addition, ScDmc1 nucleation can be stimulated by short ScRad51 patches, but not by EcRecA ones. Pull-down experiments also confirm the physical interactions of ScDmc1 with ScRad51 in solution, but not with EcRecA. Our results are consistent with a model that Dmc1 nucleation can be facilitated by a structural component (such as DNA junction and protein-protein interaction) and DNA polarity. They provide direct evidence of how Rad51 is required for meiotic recombination and highlight a regulation strategy in Dmc1 nucleoprotein filament assembly.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas de Ligação a DNA/metabolismo , Meiose , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Ciclo Celular/genética , Citoesqueleto/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/genética , Nucleoproteínas/metabolismo , Rad51 Recombinase/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética , Imagem Individual de Molécula/métodos
14.
Nucleic Acids Res ; 48(8): 4448-4462, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32232337

RESUMO

Type IA topoisomerases interact with G-strand and T-strand ssDNA to regulate DNA topology. However, simultaneous binding of two ssDNA segments to a type IA topoisomerase has not been observed previously. We report here the crystal structure of a type IA topoisomerase with ssDNA segments bound in opposite polarity to the N- and C-terminal domains. Titration of small ssDNA oligonucleotides to Mycobacterium smegmatis topoisomerase I with progressive C-terminal deletions showed that the C-terminal region has higher affinity for ssDNA than the N-terminal active site. This allows the C-terminal domains to capture one strand of underwound negatively supercoiled DNA substrate first and position the N-terminal domains to bind and cleave the opposite strand in the relaxation reaction. Efficiency of negative supercoiling relaxation increases with the number of domains that bind ssDNA primarily with conserved aromatic residues and possibly with assistance from polar/basic residues. A comparison of bacterial topoisomerase I structures showed that a conserved transesterification unit (N-terminal toroid structure) for cutting and rejoining of a ssDNA strand can be combined with two different types of C-terminal ssDNA binding domains to form diverse bacterial topoisomerase I enzymes that are highly efficient in their physiological role of preventing excess negative supercoiling in the genome.


Assuntos
DNA Topoisomerases Tipo I/química , DNA de Cadeia Simples/metabolismo , Mycobacterium smegmatis/enzimologia , Cristalografia por Raios X , DNA Topoisomerases Tipo I/genética , DNA Topoisomerases Tipo I/metabolismo , Modelos Moleculares , Domínios Proteicos , Deleção de Sequência
15.
Nucleic Acids Res ; 48(9): 4976-4991, 2020 05 21.
Artigo em Inglês | MEDLINE | ID: mdl-32232414

RESUMO

The reaction mechanism by which the shelterin protein POT1 (Protection of Telomeres 1) unfolds human telomeric G-quadruplex structures is not fully understood. We report here kinetic, thermodynamic, hydrodynamic and computational studies that show that a conformational selection mechanism, in which POT1 binding is coupled to an obligatory unfolding reaction, is the most plausible mechanism. Stopped-flow kinetic and spectroscopic titration studies, along with isothermal calorimetry, were used to show that binding of the single-strand oligonucleotide d[TTAGGGTTAG] to POT1 is both fast (80 ms) and strong (-10.1 ± 0.3 kcal mol-1). In sharp contrast, kinetic studies showed the binding of POT1 to an initially folded 24 nt G-quadruplex structure is four orders of magnitude slower. Fluorescence, circular dichroism and analytical ultracentrifugation studies showed that POT1 binding is coupled to quadruplex unfolding, with a final complex with a stoichiometry of 2 POT1 per 24 nt DNA. The binding isotherm for the POT1-quadruplex interaction was sigmoidal, indicative of a complex reaction. A conformational selection model that includes equilibrium constants for both G-quadruplex unfolding and POT1 binding to the resultant single-strand provided an excellent quantitative fit to the experimental binding data. POT1 unfolded and bound to any conformational form of human telomeric G-quadruplex (antiparallel, hybrid, parallel monomers or a 48 nt sequence with two contiguous quadruplexes), but did not avidly interact with duplex DNA or with other G-quadruplex structures. Finally, molecular dynamics simulations provided a detailed structural model of a 2:1 POT1:DNA complex that is fully consistent with experimental biophysical results.


Assuntos
Quadruplex G , Proteínas de Ligação a Telômeros/metabolismo , Telômero/química , DNA/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Cinética , Simulação de Dinâmica Molecular , Ligação Proteica , Proteínas de Ligação a Telômeros/química , Termodinâmica
16.
Proc Natl Acad Sci U S A ; 117(16): 8859-8869, 2020 04 21.
Artigo em Inglês | MEDLINE | ID: mdl-32241893

RESUMO

To repair a DNA double-strand break by homologous recombination, 5'-terminated DNA strands must first be resected to reveal 3'-overhangs. This process is initiated by a short-range resection catalyzed by MRE11-RAD50-NBS1 (MRN) stimulated by CtIP, which is followed by a long-range step involving EXO1 or DNA2 nuclease. DNA2 is a bifunctional enzyme that contains both single-stranded DNA (ssDNA)-specific nuclease and motor activities. Upon DNA unwinding by Bloom (BLM) or Werner (WRN) helicase, RPA directs the DNA2 nuclease to degrade the 5'-strand. RPA bound to ssDNA also represents a barrier, explaining the need for the motor activity of DNA2 to displace RPA prior to resection. Using ensemble and single-molecule biochemistry, we show that CtIP also dramatically stimulates the adenosine 5'-triphosphate (ATP) hydrolysis-driven motor activity of DNA2 involved in the long-range resection step. This activation in turn strongly promotes the degradation of RPA-coated ssDNA by DNA2. Accordingly, the stimulatory effect of CtIP is only observed with wild-type DNA2, but not the helicase-deficient variant. Similarly to the function of CtIP to promote MRN, also the DNA2 stimulatory effect is facilitated by CtIP phosphorylation. The domain of CtIP required to promote DNA2 is located in the central region lacking in lower eukaryotes and is fully separable from domains involved in the stimulation of MRN. These results establish how CtIP couples both MRE11-dependent short-range and DNA2-dependent long-range resection and define the involvement of the motor activity of DNA2 in this process. Our data might help explain the less severe resection defects of MRE11 nuclease-deficient cells compared to those lacking CtIP.


Assuntos
DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Endodesoxirribonucleases/metabolismo , Reparo de DNA por Recombinação , Hidrolases Anidrido Ácido/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Ensaios Enzimáticos , Hidrólise , Proteína Homóloga a MRE11/metabolismo , Proteínas Nucleares/metabolismo , Domínios Proteicos , Proteínas Recombinantes/metabolismo , Células Sf9
17.
Biochem Biophys Res Commun ; 525(3): 755-758, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32145916

RESUMO

We purified and characterized a prokaryotic argonaute (pAgo) (KjMP) and its associated protein (KjAA) from a bacterium Kordia jejudonensis. The two proteins present as a complex were revealed by the copurification of KjAA with His-tagged KjMP by Ni-NTA affinity column. The KjAA/KjMP complex was a heterodimer evaluated from the molecular weight estimated using size exclusion chromatography. The pAgo complex presented a guide-dependent target DNA cleavage. RNA was the preferred guide; however, DNA also functioned, albeit weakly. Additionally, 5'-phosphorylate or non-phosphorylated guide was equally effective. The purified complex exhibited nonspecific nuclease activity on dsDNA and ssDNA. This is the first study to report that short pAgo and its associated protein form a complex, which has a nucleic acid-guided target recognition and cleavage.


Assuntos
Proteínas Argonauta/metabolismo , Endonucleases/metabolismo , Flavobacteriaceae/metabolismo , Ácidos Nucleicos/metabolismo , Multimerização Proteica , Proteínas Argonauta/genética , Proteínas Argonauta/isolamento & purificação , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/metabolismo
18.
Sci Adv ; 6(10): eaaz2309, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32181363

RESUMO

Adenine base editors, which were developed by engineering a transfer RNA adenosine deaminase enzyme (TadA) into a DNA editing enzyme (TadA*), enable precise modification of A:T to G⋮C base pairs. Here, we use molecular dynamics simulations to uncover the structural and functional roles played by the initial mutations in the onset of the DNA editing activity by TadA*. Atomistic insights reveal that early mutations lead to intricate conformational changes in the structure of TadA*. In particular, the first mutation, Asp108Asn, induces an enhancement in the binding affinity of TadA to DNA. In silico and in vivo reversion analyses verify the importance of this single mutation in imparting functional promiscuity to TadA* and demonstrate that TadA* performs DNA base editing as a monomer rather than a dimer.


Assuntos
Desaminases APOBEC/química , Adenina/metabolismo , Adenosina Desaminase/química , DNA de Cadeia Simples/química , DNA/química , Proteínas de Escherichia coli/química , Edição de Genes/métodos , Desaminases APOBEC/genética , Desaminases APOBEC/metabolismo , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Sítios de Ligação , Clonagem Molecular , DNA/genética , DNA/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Células HEK293 , Humanos , Cinética , Simulação de Dinâmica Molecular , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidade por Substrato , Termodinâmica
19.
Nat Commun ; 11(1): 1318, 2020 03 12.
Artigo em Inglês | MEDLINE | ID: mdl-32165630

RESUMO

Persistent protein obstacles on genomic DNA, such as DNA-protein crosslinks (DPCs) and tight nucleoprotein complexes, can block replication forks. DPCs can be removed by the proteolytic activities of the metalloprotease SPRTN or the proteasome in a replication-coupled manner; however, additional proteolytic mechanisms may exist to cope with the diversity of protein obstacles. Here, we show that FAM111A, a PCNA-interacting protein, plays an important role in mitigating the effect of protein obstacles on replication forks. This function of FAM111A requires an intact trypsin-like protease domain, the PCNA interaction, and the DNA-binding domain that is necessary for protease activity in vivo. FAM111A, but not SPRTN, protects replication forks from stalling at poly(ADP-ribose) polymerase 1 (PARP1)-DNA complexes trapped by PARP inhibitors, thereby promoting cell survival after drug treatment. Altogether, our findings reveal a role of FAM111A in overcoming protein obstacles to replication forks, shedding light on cellular responses to anti-cancer therapies.


Assuntos
Replicação do DNA , Receptores Virais/metabolismo , Tripsina/química , Camptotecina/farmacologia , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dano ao DNA , DNA Topoisomerases Tipo I/metabolismo , DNA de Cadeia Simples/metabolismo , Humanos , Mutação/genética , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Receptores Virais/química , Receptores Virais/genética
20.
Nucleic Acids Res ; 48(7): 3657-3677, 2020 04 17.
Artigo em Inglês | MEDLINE | ID: mdl-32128579

RESUMO

DNA replication is a central process in all living organisms. Polyomavirus DNA replication serves as a model system for eukaryotic DNA replication and has considerably contributed to our understanding of basic replication mechanisms. However, the details of the involved processes are still unclear, in particular regarding lagging strand synthesis. To delineate the complex mechanism of coordination of various cellular proteins binding simultaneously or consecutively to DNA to initiate replication, we investigated single-stranded DNA (ssDNA) interactions by the SV40 large T antigen (Tag). Using single molecule imaging by atomic force microscopy (AFM) combined with biochemical and spectroscopic analyses we reveal independent activity of monomeric and oligomeric Tag in high affinity binding to ssDNA. Depending on ssDNA length, we obtain dissociation constants for Tag-ssDNA interactions (KD values of 10-30 nM) that are in the same order of magnitude as ssDNA binding by human replication protein A (RPA). Furthermore, we observe the formation of RPA-Tag-ssDNA complexes containing hexameric as well as monomeric Tag forms. Importantly, our data clearly show stimulation of primase function in lagging strand Okazaki fragment synthesis by monomeric Tag whereas hexameric Tag inhibits the reaction, redefining DNA replication initiation on the lagging strand.


Assuntos
Antígenos Transformantes de Poliomavirus/metabolismo , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteína de Replicação A/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/metabolismo , DNA Polimerase I/metabolismo , DNA Primase/metabolismo , DNA de Cadeia Simples/química , Ligação Proteica , Vírus 40 dos Símios/imunologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA