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1.
Nat Commun ; 15(1): 5140, 2024 Jun 17.
Artigo em Inglês | MEDLINE | ID: mdl-38886375

RESUMO

Holliday junction resolution is a crucial process in homologous recombination and DNA double-strand break repair. Complete Holliday junction resolution requires two stepwise incisions across the center of the junction, but the precise mechanism of metal ion-catalyzed Holliday junction cleavage remains elusive. Here, we perform a metal ion-triggered catalysis in crystals to investigate the mechanism of Holliday junction cleavage by MOC1. We capture the structures of MOC1 in complex with a nicked Holliday junction at various catalytic states, including the ground state, the one-metal ion binding state, and the two-metal ion binding state. Moreover, we also identify a third metal ion that may aid in the nucleophilic attack on the scissile phosphate. Further structural and biochemical analyses reveal a metal ion-mediated allosteric regulation between the two active sites, contributing to the enhancement of the second strand cleavage following the first strand cleavage, as well as the precise symmetric cleavage across the Holliday junction. Our work provides insights into the mechanism of metal ion-catalyzed Holliday junction resolution by MOC1, with implications for understanding how cells preserve genome integrity during the Holliday junction resolution phase.


Assuntos
DNA Cruciforme , DNA Cruciforme/metabolismo , DNA Cruciforme/química , DNA Cruciforme/genética , Metais/metabolismo , Metais/química , Resolvases de Junção Holliday/metabolismo , Resolvases de Junção Holliday/química , Domínio Catalítico , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Cristalografia por Raios X , Íons/metabolismo , Quebras de DNA de Cadeia Dupla , Modelos Moleculares , Regulação Alostérica
2.
J Phys Chem B ; 128(23): 5642-5657, 2024 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-38812070

RESUMO

The integration host factor (IHF) in Escherichia coli is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160° upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Förster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.


Assuntos
DNA Cruciforme , Fatores Hospedeiros de Integração , DNA Cruciforme/química , DNA Cruciforme/metabolismo , Fatores Hospedeiros de Integração/metabolismo , Fatores Hospedeiros de Integração/química , Escherichia coli/metabolismo , Conformação de Ácido Nucleico , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Ligação Proteica
3.
Langmuir ; 40(19): 10195-10207, 2024 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-38690801

RESUMO

With recent advances in DNA-templated dye aggregation for leveraging and engineering molecular excitons, a need exists for minimizing structural heterogeneity. Holliday Junction complexes (HJ) are commonly used to covalently template dye aggregates on their core; however, the global conformation of HJ is detrimentally dynamic. Here, the global conformation of the HJ is selectively tuned by restricting its position and orientation by using a sheet-like DNA origami construct (DOC) physisorbed on glass. The HJ arms are fixed with four different designed interduplex angles (IDAs). Atomic force microscopy confirmed that the HJs are bound to the surface of DOC with tuned IDAs. Dye orientation distributions were determined by combining dipole imaging and super-resolution microscopy. All IDAs led to dye orientations having dispersed distributions along planes perpendicular to the HJ plane, suggesting that stacking occurred between the dye and the neighboring DNA bases. The dye-base stacking interpretation was supported by increasing the size of the core cavity. The narrowest IDA minimizes structural heterogeneity and suggests dye intercalation. A strong correlation is found between the IDA and the orientation of the dye along the HJ plane. These results show that the HJ imposes restrictions on the dye and that the dye-DNA interactions are always present regardless of global conformation. The implications of our results are discussed for the scalability of dye aggregates using DNA self-assembly. Our methodology provides an avenue for the solid-supported single-molecule characterization of molecular assemblies templated on biomolecules─such as DNA and protein templates involved in light-harvesting and catalysis─with tuned conformations and restricted in position and orientation.


Assuntos
DNA Cruciforme , Conformação de Ácido Nucleico , DNA Cruciforme/química , DNA/química , Corantes/química , Microscopia de Força Atômica
4.
J Mol Biol ; 436(10): 168550, 2024 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-38575054

RESUMO

The class 2 CRISPR-Cas9 and CRISPR-Cas12a systems, originally described as adaptive immune systems of bacteria and archaea, have emerged as versatile tools for genome-editing, with applications in biotechnology and medicine. However, significantly less is known about their substrate specificity, but such knowledge may provide instructive insights into their off-target cleavage and previously unrecognized mechanism of action. Here, we document that the Acidaminococcus sp. Cas12a (AsCas12a) binds preferentially, and independently of crRNA, to a suite of branched DNA structures, such as the Holliday junction (HJ), replication fork and D-loops, compared with single- or double-stranded DNA, and promotes their degradation. Further, our study revealed that AsCas12a binds to the HJ, specifically at the crossover region, protects it from DNase I cleavage and renders a pair of thymine residues in the HJ homologous core hypersensitive to KMnO4 oxidation, suggesting DNA melting and/or distortion. Notably, these structural changes enabled AsCas12a to resolve HJ into nonligatable intermediates, and subsequently their complete degradation. We further demonstrate that crRNA impedes HJ cleavage by AsCas12a, and that of Lachnospiraceae bacterium Cas12a, without affecting their DNA-binding ability. We identified a separation-of-function variant, which uncouples DNA-binding and DNA cleavage activities of AsCas12a. Importantly, we found robust evidence that AsCas12a endonuclease also has 3'-to-5' and 5'-to-3' exonuclease activity, and that these two activities synergistically promote degradation of DNA, yielding di- and mononucleotides. Collectively, this study significantly advances knowledge about the substrate specificity of AsCas12a and provides important insights into the degradation of different types of DNA substrates.


Assuntos
Acidaminococcus , Proteínas de Bactérias , Proteínas Associadas a CRISPR , Sistemas CRISPR-Cas , Endodesoxirribonucleases , Acidaminococcus/enzimologia , Acidaminococcus/genética , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas Associadas a CRISPR/química , Proteínas Associadas a CRISPR/genética , DNA Cruciforme/química , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/genética , Especificidade por Substrato
5.
J Mol Biol ; 434(21): 167822, 2022 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-36108776

RESUMO

RecG, belonging to the category of Superfamily-2 plays a vital role in rescuing different kinds of stalled fork. The elemental mechanism of the helicase activity of RecG with several non-homologous stalled fork structures resembling intermediates formed during the process of DNA repair has been investigated in the present study to capture the dynamic stages of genetic rearrangement. The functional characterization has been exemplified through quantifying the response of the substrate in terms of their molecular heterogeneity and dynamical response by employing single-molecule fluorescence methods. An elevated processivity of RecG is observed for the stalled fork where progression of lagging daughter strand is ahead as compared to that of the leading strand. Through precise alteration of its function in terms of unwinding, depending upon the substrate DNA, RecG catalyzes the formation of Holliday junction from a stalled fork DNA. RecG is found to adopt an asymmetric mode of locomotion to unwind the lagging daughter strand for facilitating formation of Holliday junction that acts as a suitable intermediate for recombinational repair pathway. Our results emphasize the mechanism adopted by RecG during its 'sliding back' mode along the lagging daughter strand to be 'active translocation and passive unwinding'. This also provide clues as to how this helicase decides and controls the mode of translocation along the DNA to unwind.


Assuntos
Proteínas de Bactérias , DNA Helicases , Replicação do DNA , DNA Cruciforme , DNA Helicases/química , DNA Cruciforme/química , Proteínas de Bactérias/química
6.
Nature ; 609(7927): 630-639, 2022 09.
Artigo em Inglês | MEDLINE | ID: mdl-36002576

RESUMO

The Holliday junction is a key intermediate formed during DNA recombination across all kingdoms of life1. In bacteria, the Holliday junction is processed by two homo-hexameric AAA+ ATPase RuvB motors, which assemble together with the RuvA-Holliday junction complex to energize the strand-exchange reaction2. Despite its importance for chromosome maintenance, the structure and mechanism by which this complex facilitates branch migration are unknown. Here, using time-resolved cryo-electron microscopy, we obtained structures of the ATP-hydrolysing RuvAB complex in seven distinct conformational states, captured during assembly and processing of a Holliday junction. Five structures together resolve the complete nucleotide cycle and reveal the spatiotemporal relationship between ATP hydrolysis, nucleotide exchange and context-specific conformational changes in RuvB. Coordinated motions in a converter formed by DNA-disengaged RuvB subunits stimulate hydrolysis and nucleotide exchange. Immobilization of the converter enables RuvB to convert the ATP-contained energy into a lever motion, which generates the pulling force driving the branch migration. We show that RuvB motors rotate together with the DNA substrate, which, together with a progressing nucleotide cycle, forms the mechanistic basis for DNA recombination by continuous branch migration. Together, our data decipher the molecular principles of homologous recombination by the RuvAB complex, elucidate discrete and sequential transition-state intermediates for chemo-mechanical coupling of hexameric AAA+ motors and provide a blueprint for the design of state-specific compounds targeting AAA+ motors.


Assuntos
ATPases Associadas a Diversas Atividades Celulares , Proteínas de Bactérias , DNA Helicases , DNA Cruciforme , ATPases Associadas a Diversas Atividades Celulares/química , ATPases Associadas a Diversas Atividades Celulares/metabolismo , ATPases Associadas a Diversas Atividades Celulares/ultraestrutura , Trifosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/ultraestrutura , Microscopia Crioeletrônica , DNA Helicases/química , DNA Helicases/metabolismo , DNA Helicases/ultraestrutura , DNA Cruciforme/química , DNA Cruciforme/metabolismo , DNA Cruciforme/ultraestrutura , DNA de Cadeia Simples/química , DNA de Cadeia Simples/metabolismo , DNA de Cadeia Simples/ultraestrutura , Recombinação Homóloga , Hidrólise , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Nucleotídeos , Conformação Proteica , Rotação
7.
Nat Commun ; 13(1): 654, 2022 02 03.
Artigo em Inglês | MEDLINE | ID: mdl-35115525

RESUMO

Homologous recombination (HR) is a ubiquitous and efficient process that serves the repair of severe forms of DNA damage and the generation of genetic diversity during meiosis. HR can proceed via multiple pathways with different outcomes that may aid or impair genome stability and faithful inheritance, underscoring the importance of HR quality control. Human Bloom's syndrome (BLM, RecQ family) helicase plays central roles in HR pathway selection and quality control via unexplored molecular mechanisms. Here we show that BLM's multi-domain structural architecture supports a balance between stabilization and disruption of displacement loops (D-loops), early HR intermediates that are key targets for HR regulation. We find that this balance is markedly shifted toward efficient D-loop disruption by the presence of BLM's interaction partners Topoisomerase IIIα-RMI1-RMI2, which have been shown to be involved in multiple steps of HR-based DNA repair. Our results point to a mechanism whereby BLM can differentially process D-loops and support HR control depending on cellular regulatory mechanisms.


Assuntos
DNA Topoisomerases Tipo I/metabolismo , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/metabolismo , RecQ Helicases/metabolismo , DNA Topoisomerases Tipo I/genética , DNA Cruciforme/química , DNA Cruciforme/genética , Proteínas de Ligação a DNA/genética , Humanos , Cinética , Modelos Genéticos , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Ligação Proteica , RecQ Helicases/genética , Reparo de DNA por Recombinação/genética
8.
ACS Appl Mater Interfaces ; 13(27): 32013-32021, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34212714

RESUMO

The reported donor donor-acceptor ("DD-A") fluorescence resonance energy transfer (FRET) was typically achieved through random collisions and interactions of DNA molecules in the bulk solution, which has inevitable defects, including weak biological stability, slow reaction kinetics, and low hybridization efficiency. In order to overcome these deficiencies, this work developed a quadrivalent cruciform DNA nanostructure (qCDN)-mediated cascaded catalyzed hairpin assembly (CHA) amplifier for the fluorescence detection of amyloid ß oligomer species (AßOs). First, four H1 and four H2 hairpins were assembled on one qCDN to obtain qCDNH1 and qCDNH2, respectively. In the presence of AßOs, strand C was released from the P1-C hybrid hairpin and then alternately opened qCDNH1 and qCDNH2 to trigger the qCDN-mediated CHA. As a result, double donors in H1 and one acceptor in H2 were mutually closed, and the porous DNA nanonet with a high loading of "DD-A" FRET binary probes was formed. The FRET efficiency was approximately 78%, and the initial reaction rate was 25-fold faster than the conventional CHA. The detection limit of AßOs was as low as 0.69 pM. The combination of the "DD-A" FRET binary probes and qCDN-mediated cascaded amplifier exhibited great promise for detecting biomarkers with trace levels.


Assuntos
Peptídeos beta-Amiloides/química , DNA Cruciforme/química , Transferência Ressonante de Energia de Fluorescência/métodos , Limite de Detecção , Nanoestruturas/química , Multimerização Proteica , Estrutura Quaternária de Proteína
9.
Molecules ; 26(4)2021 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-33670583

RESUMO

Blocking the PD-1/PD-L1 pathway can diminish immunosuppression and enhance anticancer immunity. PD-1/PD-L1 blockade can be realized by aptamers, which have good biocompatibility and can be synthesized in quantity economically. For in vivo applications, aptamers need to evade renal clearance and nuclease digestion. Here we investigated whether DNA nanostructures could be used to enhance the function of PD-L1 aptamers. Four PD-L1 aptamers (Apt) were built into a Holliday Junction (HJ) to form a tetravalent DNA nanostructure (Apt-HJ). The average size of Apt-HJ was 13.22 nm, which was above the threshold for renal clearance. Apt-HJ also underwent partial phosphorothioate modification and had improved nuclease resistance. Compared with the monovalent PD-L1 aptamer, the tetravalent Apt-HJ had stronger affinity to CT26 colon cancer cells. Moreover, Apt-HJ markedly boosted the antitumor efficacy in vivo vs. free PD-L1 aptamers without raising systemic toxicity. The results indicate that multiple aptamers attached to a DNA nanostructure may significantly improve the function of PD-L1 aptamers in vivo.


Assuntos
Antineoplásicos/farmacologia , Aptâmeros de Nucleotídeos/química , Antígeno B7-H1/metabolismo , DNA Cruciforme/química , Animais , Linhagem Celular Tumoral , Camundongos Endogâmicos BALB C , Nanoestruturas/química
10.
Nucleic Acids Res ; 49(5): 2803-2815, 2021 03 18.
Artigo em Inglês | MEDLINE | ID: mdl-33619520

RESUMO

Homologous recombination forms and resolves an entangled DNA Holliday Junction (HJ) crucial for achieving genetic reshuffling and genome repair. To maintain genomic integrity, specialized resolvase enzymes cleave the entangled DNA into two discrete DNA molecules. However, it is unclear how two similar stacking isomers are distinguished, and how a cognate sequence is found and recognized to achieve accurate recombination. We here use single-molecule fluorescence observation and cluster analysis to examine how prototypic bacterial resolvase RuvC singles out two of the four HJ strands and achieves sequence-specific cleavage. We find that RuvC first exploits, then constrains the dynamics of intrinsic HJ isomer exchange at a sampled branch position to direct cleavage toward the catalytically competent HJ conformation and sequence, thus controlling recombination output at minimal energetic cost. Our model of rapid DNA scanning followed by 'snap-locking' of a cognate sequence is strikingly consistent with the conformational proofreading of other DNA-modifying enzymes.


Assuntos
DNA Helicases/metabolismo , DNA Cruciforme/química , Proteínas de Escherichia coli/metabolismo , Resolvases de Junção Holliday/metabolismo , Recombinação Homóloga , Clivagem do DNA , Transferência Ressonante de Energia de Fluorescência , Magnésio
11.
Biochem Biophys Res Commun ; 534: 47-52, 2021 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-33310187

RESUMO

HU, a DNA-binding protein, has a helical N-terminal region (NTR) of ∼44 residues and a beta strand- and IDR-rich C-terminal region (CTR) of ∼46 residues. CTR binds to DNA through (i) a clasp (two arginine/lysine-rich, IDR-rich beta hairpins that bind to phosphate groups in the minor groove), (ii) a flat surface (comprising four antiparallel beta strands that abut the major groove), and (iii) a charge cluster (two lysine residues upon a short C-terminal helix). HU forms a dimer displaying extensive inter-subunit CTR-CTR contacts. A single-chain simulacrum of these contacts (HU-Simul) incorporating all DNA-binding elements was created by fusing together the CTRs of Escherichia coli HU-A and Thermus thermophilus HU. HU-Simul is monomeric, binds to dsDNA and cruciform DNA, but not to ssDNA.


Assuntos
Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , DNA/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Dicroísmo Circular , DNA/química , DNA Cruciforme/química , DNA Cruciforme/metabolismo , DNA de Cadeia Simples/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Engenharia de Proteínas/métodos , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Thermus thermophilus/genética
12.
Nucleic Acids Res ; 48(21): 12407-12414, 2020 12 02.
Artigo em Inglês | MEDLINE | ID: mdl-33152066

RESUMO

The axial stiffness of DNA origami is determined as a function of key nanostructural characteristics. Different constructs of two-helix nanobeams with specified densities of nicks and Holliday junctions are synthesized and stretched by fluid flow. Implementing single particle tracking to extract force-displacement curves enables the measurement of DNA origami stiffness values at the enthalpic elasticity regime, i.e. for forces larger than 15 pN. Comparisons between ligated and nicked helices show that the latter exhibit nearly a two-fold decrease in axial stiffness. Numerical models that treat the DNA helices as elastic rods are used to evaluate the local loss of stiffness at the locations of nicks and Holliday junctions. It is shown that the models reproduce the experimental data accurately, indicating that both of these design characteristics yield a local stiffness two orders of magnitude smaller than the corresponding value of the intact double-helix. This local degradation in turn leads to a macroscopic loss of stiffness that is evaluated numerically for multi-helix DNA bundles.


Assuntos
DNA Cruciforme/química , DNA de Cadeia Simples/química , DNA Viral/química , Nanoestruturas/química , Bacteriófago M13/química , Bacteriófago M13/genética , Fenômenos Biomecânicos , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Elasticidade , Polinucleotídeo 5'-Hidroxiquinase/química , Termodinâmica
13.
Molecules ; 25(21)2020 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-33153073

RESUMO

Immobile Holliday junctions represent not only the most fundamental building block of structural DNA nanotechnology but are also of tremendous importance for the in vitro investigation of genetic recombination and epigenetics. Here, we present a detailed study on the room-temperature assembly of immobile Holliday junctions with the help of the single-strand annealing protein Redß. Individual DNA single strands are initially coated with protein monomers and subsequently hybridized to form a rigid blunt-ended four-arm junction. We investigate the efficiency of this approach for different DNA/protein ratios, as well as for different DNA sequence lengths. Furthermore, we also evaluate the potential of Redß to anneal sticky-end modified Holliday junctions into hierarchical assemblies. We demonstrate the Redß-mediated annealing of Holliday junction dimers, multimers, and extended networks several microns in size. While these hybrid DNA-protein nanostructures may find applications in the crystallization of DNA-protein complexes, our work shows the great potential of Redß to aid in the synthesis of functional DNA nanostructures under mild reaction conditions.


Assuntos
DNA Cruciforme/química , DNA de Cadeia Simples/química , Proteínas de Ligação a DNA/química , DNA/química , Temperatura
14.
Biochem Biophys Res Commun ; 533(4): 919-924, 2020 12 17.
Artigo em Inglês | MEDLINE | ID: mdl-33010889

RESUMO

The SWI/SNF chromatin remodeling complex plays important roles in gene regulation and it is classified as the SWI/SNF complex in yeast and BAF complex in vertebrates. BAF57, one of the subunits that forms the chromatin remodeling complex core, is well conserved in the BAF complex of vertebrates, which is replaced by bap111 in the Drosophila BAP complex and does not have a counterpart in the yeast SWI/SNF complex. This suggests that BAF57 is a key component of the chromatin remodeling complex in higher eukaryotes. BAF57 contains a HMG domain, which is widely distributed among various proteins and functions as a DNA binding motif. Most proteins with HMG domain bind to four-way junction (4WJ) DNA. Here, we report the crystal structure of the HMG domain of BAF57 (BAF57HMG) at a resolution of 2.55 Å. The structure consists of three α-helices and adopts an L-shaped form. The overall structure is stabilized by a hydrophobic core, which is formed by hydrophobic residues. The binding affinity between BAF57HMG and 4WJ DNA is determined as a 295.83 ± 1.05 nM using a fluorescence quenching assay, and the structure revealed 4WJ DNA binding site of BAF57HMG. Our data will serve structural basis in understanding the roles of BAF57 during chromatin remodeling process.


Assuntos
Proteínas Cromossômicas não Histona/química , Proteínas de Ligação a DNA/química , DNA/química , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Cristalografia por Raios X , DNA/genética , DNA/metabolismo , DNA Cruciforme/química , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Domínios HMG-Box , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , Espectrometria de Fluorescência , Eletricidade Estática
15.
FEBS Lett ; 594(24): 4320-4337, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-32936932

RESUMO

The Saccharomyces cerevisiae Mus81-Mms4 complex is a highly conserved DNA structure-specific endonuclease that plays essential roles in the processing of recombination intermediates that arise during the repair of stalled replication forks and double-stranded breaks. To identify novel factors functioning conjointly with Mus81-Mms4, we performed a biochemical screen and found that Crp1, a cruciform DNA-recognizing protein that specifically binds to DNA four-way junction structures, could stimulate the Mus81-Mms4 endonuclease. The specific protein interaction between Mus81-Mms4 and Crp1 was responsible for the stimulation observed. Multicopy expression of Crp1 could partially rescue the sensitivity to DNA-damaging agents of the sgs1∆mus81∆21-24N mutant. Our results provide insight into the functional role and interaction of Crp1 with other proteins involved in DNA repair.


Assuntos
DNA Cruciforme/metabolismo , Proteínas de Ligação a DNA/metabolismo , Endonucleases/metabolismo , Endonucleases Flap/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Cruciforme/química , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Endonucleases/química , Endonucleases/genética , Ativação Enzimática , Regulação Fúngica da Expressão Gênica , Cinética , Mutação , Conformação de Ácido Nucleico , Ligação Proteica , Domínios Proteicos , RecQ Helicases/genética , RecQ Helicases/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
16.
Nature ; 586(7830): 623-627, 2020 10.
Artigo em Inglês | MEDLINE | ID: mdl-32814343

RESUMO

During meiosis, crossover recombination connects homologous chromosomes to direct their accurate segregation1. Defective crossing over causes infertility, miscarriage and congenital disease. Each pair of chromosomes attains at least one crossover via the formation and biased resolution of recombination intermediates known as double Holliday junctions2,3. A central principle of crossover resolution is that the two Holliday junctions are resolved in opposite planes by targeting nuclease incisions to specific DNA strands4. The endonuclease activity of the MutLγ complex has been implicated in the resolution of crossovers5-10, but the mechanisms that activate and direct strand-specific cleavage remain unknown. Here we show that the sliding clamp PCNA is important for crossover-biased resolution. In vitro assays with human enzymes show that PCNA and its loader RFC are sufficient to activate the MutLγ endonuclease. MutLγ is further stimulated by a co-dependent activity of the pro-crossover factors EXO1 and MutSγ, the latter of which binds Holliday junctions11. MutLγ also binds various branched DNAs, including Holliday junctions, but does not show canonical resolvase activity, implying that the endonuclease incises adjacent to junction branch points to achieve resolution. In vivo, RFC facilitates MutLγ-dependent crossing over in budding yeast. Furthermore, PCNA localizes to prospective crossover sites along synapsed chromosomes. These data highlight similarities between crossover resolution and the initiation steps of DNA mismatch repair12,13 and evoke a novel model for crossover-specific resolution of double Holliday junctions during meiosis.


Assuntos
Troca Genética , Endonucleases/metabolismo , Meiose , Proteínas MutL/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , DNA Cruciforme/química , DNA Cruciforme/genética , DNA Cruciforme/metabolismo , Ativação Enzimática , Humanos , Hidrólise , Masculino , Camundongos , Proteínas MutS/metabolismo , Ligação Proteica , Proteína de Replicação C/metabolismo , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
17.
Nucleic Acids Res ; 48(14): 8090-8098, 2020 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-32597986

RESUMO

The DNA four-way (Holliday) junction is the central intermediate of genetic recombination, yet key aspects of its conformational and thermodynamic properties remain unclear. While multiple experimental approaches have been used to characterize the canonical X-shape conformers under specific ionic conditions, the complete conformational ensemble of this motif, especially at low ionic conditions, remains largely undetermined. In line with previous studies, our single-molecule Förster resonance energy transfer (smFRET) measurements of junction dynamics revealed transitions between two states under high salt conditions, but smFRET could not determine whether there are fast and unresolvable transitions between distinct conformations or a broad ensemble of related states under low and intermediate salt conditions. We therefore used an emerging technique, X-ray scattering interferometry (XSI), to directly probe the conformational ensemble of the Holliday junction across a wide range of ionic conditions. Our results demonstrated that the four-way junction adopts an out-of-plane geometry under low ionic conditions and revealed a conformational state at intermediate ionic conditions previously undetected by other methods. Our results provide critical information to build toward a full description of the conformational landscape of the Holliday junction and underscore the utility of XSI for probing conformational ensembles under a wide range of solution conditions.


Assuntos
DNA Cruciforme/química , Transferência Ressonante de Energia de Fluorescência , Simulação de Dinâmica Molecular , Concentração Osmolar , Difração de Raios X
18.
ACS Chem Biol ; 15(7): 1942-1948, 2020 07 17.
Artigo em Inglês | MEDLINE | ID: mdl-32469201

RESUMO

In this study, we report experimental (Protein Data Bank (PDB) search) and theoretical (RI-MP2/def2-TZVP level of theory) evidence of the nature, stability, and directionality properties of intramolecular halogen bonding interactions (HaBs) between 5-bromo/5-iodoracil bases and backbone phosphate groups in nucleic acids (NAs). A PDB survey revealed relevant examples where intramolecular HaBs are undertaken and serve as a structural source of stability in RNA and DNA molecules. In order to develop suitable energy predictors, we started this investigation by calculating the interaction energy values and both the potential V(r) and kinetic G(r) energy densities (using Bader's "atoms in molecules" theory) of several halogen bond complexes involving 5-bromo/5-iodoracil molecules and biologically relevant electron donors. Once the energy predictors based on V(r)/G(r) energy densities were developed, we analyzed the HaBs observed in the biological examples retrieved from the PDB search in order to estimate the strength of the interaction. As far as our knowledge extends, intramolecular halogen bonds in NAs have not been previously quantified in the literature using this methodology and may be of great importance in understanding their structural properties as well as in the construction of molecular materials with DNA and other biological macromolecules.


Assuntos
Bromouracila/química , DNA Cruciforme/química , RNA/química , Eletricidade Estática , Uracila/análogos & derivados , Bromo/química , Bromouracila/metabolismo , DNA Cruciforme/metabolismo , Bases de Dados de Proteínas , Escherichia coli/química , Exodesoxirribonuclease V/metabolismo , Humanos , Iodo/química , Modelos Químicos , Ligação Proteica , RNA/metabolismo , Fator de Processamento U2AF/metabolismo , Termodinâmica , Uracila/química , Uracila/metabolismo
19.
Int J Nanomedicine ; 15: 2119-2129, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32280210

RESUMO

PURPOSE: Chemotherapy is the primary treatment for advanced colon cancer, but its efficacy is often limited by severe toxicities. Targeted therapy in the form of selectively drug delivery system (SDDS) is an important strategy to reduce adverse effects. Here, we aim to design a novel SDDS with potential for practical application using biocompatible components and scalable production process, for targeted delivery of doxorubicin (Dox) to colon cancer cells. METHODS: The SDDS was made of a self-assembled DNA nano-cross (Holliday junction, or HJ) functionalized by four AS1411 aptamers (Apt-HJ) and loaded with Dox. RESULTS: Apt-HJ had an average size of 12.45 nm and a zeta potential of -11.6 mV. Compared with the monovalent AS1411 aptamer, the quadrivalent Apt-HJ showed stronger binding to target cancer cells (CT26). A complex of Apt-HJ and doxorubicin (Apt-HJ-Dox) was formed by intercalating Dox into the DNA structure of Apt-HJ, with each complex carrying approximately 17 Dox molecules. Confocal microscopy revealed that Apt-HJ-Dox selectively delivered Dox into CT26 colon cancer cells but not the control cells. Moreover, Apt-HJ-Dox achieved targeted killing of CT26 cancer cells in vitro and reduced the damage to control cells. Importantly, compared with free Dox, Apt-HJ-Dox significantly enhanced the antitumor efficacy in vivo without boosting the adverse effects. CONCLUSION: These results suggest that Apt-HJ-Dox has application potential in targeted treatment of colon cancer.


Assuntos
Antibióticos Antineoplásicos/administração & dosagem , Aptâmeros de Nucleotídeos/química , Neoplasias do Colo/tratamento farmacológico , Doxorrubicina/administração & dosagem , Sistemas de Liberação de Medicamentos/métodos , Animais , Aptâmeros de Nucleotídeos/administração & dosagem , Células CHO , Linhagem Celular Tumoral , Cricetulus , DNA Cruciforme/química , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Nanoestruturas/química , Neoplasias Experimentais/tratamento farmacológico , Oligodesoxirribonucleotídeos/administração & dosagem , Oligodesoxirribonucleotídeos/química
20.
Nat Commun ; 11(1): 1417, 2020 03 17.
Artigo em Inglês | MEDLINE | ID: mdl-32184398

RESUMO

Holliday junctions (HJs) are key DNA intermediates in genetic recombination and are eliminated by nuclease, termed resolvase, to ensure genome stability. HJ resolvases have been identified across all kingdoms of life, members of which exhibit sequence-dependent HJ resolution. However, the molecular basis of sequence selectivity remains largely unknown. Here, we present the chloroplast resolvase MOC1, which cleaves HJ in a cytosine-dependent manner. We determine the crystal structure of MOC1 with and without HJs. MOC1 exhibits an RNase H fold, belonging to the retroviral integrase family. MOC1 functions as a dimer, and the HJ is embedded into the basic cleft of the dimeric enzyme. We characterize a base recognition loop (BR loop) that protrudes into and opens the junction. Residues from the BR loop intercalate into the bases, disrupt the C-G base pairing at the crossover and recognize the cytosine, providing the molecular basis for sequence-dependent HJ resolution by a resolvase.


Assuntos
Arabidopsis/enzimologia , Cloroplastos/enzimologia , DNA Cruciforme/metabolismo , Glycine max/enzimologia , Oryza/enzimologia , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Recombinases/química , Recombinases/metabolismo , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Sequência de Bases , Cloroplastos/química , Cloroplastos/genética , DNA Cruciforme/química , DNA Cruciforme/genética , DNA de Plantas/química , DNA de Plantas/genética , DNA de Plantas/metabolismo , Oryza/química , Oryza/genética , Oryza/metabolismo , Recombinases/genética , Glycine max/química , Glycine max/genética , Glycine max/metabolismo
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