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1.
Nat Commun ; 11(1): 4287, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855419

RESUMO

Warsaw Breakage Syndrome (WABS) is a rare disorder related to cohesinopathies and Fanconi anemia, caused by bi-allelic mutations in DDX11. Here, we report multiple compound heterozygous WABS cases, each displaying destabilized DDX11 protein and residual DDX11 function at the cellular level. Patient-derived cell lines exhibit sensitivity to topoisomerase and PARP inhibitors, defective sister chromatid cohesion and reduced DNA replication fork speed. Deleting DDX11 in RPE1-TERT cells inhibits proliferation and survival in a TP53-dependent manner and causes chromosome breaks and cohesion defects, independent of the expressed pseudogene DDX12p. Importantly, G-quadruplex (G4) stabilizing compounds induce chromosome breaks and cohesion defects which are strongly aggravated by inactivation of DDX11 but not FANCJ. The DNA helicase domain of DDX11 is essential for sister chromatid cohesion and resistance to G4 stabilizers. We propose that DDX11 is a DNA helicase protecting against G4 induced double-stranded breaks and concomitant loss of cohesion, possibly at DNA replication forks.


Assuntos
Anormalidades Múltiplas/etiologia , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Troca de Cromátide Irmã , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Proliferação de Células , RNA Helicases DEAD-box/química , DNA Helicases/química , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Estabilidade Proteica , Pseudogenes , RNA Helicases/genética , RNA Helicases/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Síndrome , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo
2.
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
3.
Proc Natl Acad Sci U S A ; 117(30): 17747-17756, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32669428

RESUMO

DNA replication origins serve as sites of replicative helicase loading. In all eukaryotes, the six-subunit origin recognition complex (Orc1-6; ORC) recognizes the replication origin. During late M-phase of the cell-cycle, Cdc6 binds to ORC and the ORC-Cdc6 complex loads in a multistep reaction and, with the help of Cdt1, the core Mcm2-7 helicase onto DNA. A key intermediate is the ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) complex in which DNA has been already inserted into the central channel of Mcm2-7. Until now, it has been unclear how the origin DNA is guided by ORC-Cdc6 and inserted into the Mcm2-7 hexamer. Here, we truncated the C-terminal winged-helix-domain (WHD) of Mcm6 to slow down the loading reaction, thereby capturing two loading intermediates prior to DNA insertion in budding yeast. In "semi-attached OCCM," the Mcm3 and Mcm7 WHDs latch onto ORC-Cdc6 while the main body of the Mcm2-7 hexamer is not connected. In "pre-insertion OCCM," the main body of Mcm2-7 docks onto ORC-Cdc6, and the origin DNA is bent and positioned adjacent to the open DNA entry gate, poised for insertion, at the Mcm2-Mcm5 interface. We used molecular simulations to reveal the dynamic transition from preloading conformers to the loaded conformers in which the loading of Mcm2-7 on DNA is complete and the DNA entry gate is fully closed. Our work provides multiple molecular insights into a key event of eukaryotic DNA replication.


Assuntos
Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Replicação do DNA , Origem de Replicação , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Sítios de Ligação , Microscopia Crioeletrônica , Componente 6 do Complexo de Manutenção de Minicromossomo/química , Componente 6 do Complexo de Manutenção de Minicromossomo/metabolismo , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Complexo de Reconhecimento de Origem , Ligação Proteica , Conformação Proteica , Relação Estrutura-Atividade
4.
Nucleic Acids Res ; 48(13): 7239-7251, 2020 07 27.
Artigo em Inglês | MEDLINE | ID: mdl-32542379

RESUMO

Telomeres cap the ends of eukaryotic chromosomes and distinguish them from broken DNA ends to suppress DNA damage response, cell cycle arrest and genomic instability. Telomeres are elongated by telomerase to compensate for incomplete replication and nuclease degradation and to extend the proliferation potential of germ and stem cells and most cancers. However, telomeres in somatic cells gradually shorten with age, ultimately leading to cellular senescence. Hoyeraal-Hreidarsson syndrome (HHS) is characterized by accelerated telomere shortening and diverse symptoms including bone marrow failure, immunodeficiency, and neurodevelopmental defects. HHS is caused by germline mutations in telomerase subunits, factors essential for its biogenesis and recruitment to telomeres, and in the helicase RTEL1. While diverse phenotypes were associated with RTEL1 deficiency, the telomeric role of RTEL1 affected in HHS is yet unknown. Inducible ectopic expression of wild-type RTEL1 in patient fibroblasts rescued the cells, enabled telomerase-dependent telomere elongation and suppressed the abnormal cellular phenotypes, while silencing its expression resulted in gradual telomere shortening. Our observations reveal an essential role of the RTEL1 C-terminus in facilitating telomerase action at the telomeric 3' overhang. Thus, the common etiology for HHS is the compromised telomerase action, resulting in telomere shortening and reduced lifespan of telomerase positive cells.


Assuntos
DNA Helicases/metabolismo , Disceratose Congênita/genética , Retardo do Crescimento Fetal/genética , Deficiência Intelectual/genética , Microcefalia/genética , Homeostase do Telômero , Células Cultivadas , DNA Helicases/química , DNA Helicases/genética , Fibroblastos/metabolismo , Humanos , Domínios Proteicos , Telomerase/genética , Telomerase/metabolismo , Encurtamento do Telômero
5.
Nat Commun ; 11(1): 2639, 2020 05 26.
Artigo em Inglês | MEDLINE | ID: mdl-32457312

RESUMO

Homologous recombination (HR) is important for error-free DNA double strand break repair and maintenance of genomic stability. However, upregulated HR is also used by cancer cells to promote therapeutic resistance. Therefore, inducing HR deficiency (HRD) is a viable strategy to sensitize HR proficient cancers to DNA targeted therapies in order to overcome therapeutic resistance. A bromodomain containing protein, BRD9, was previously reported to regulate chromatin remodeling and transcription. Here, we discover that following DNA damage, the bromodomain of BRD9 binds acetylated K515 on RAD54 and facilitates RAD54's interaction with RAD51, which is essential for HR. BRD9 is overexpressed in ovarian cancer and depleting BRD9 sensitizes cancer cells to olaparib and cisplatin. In addition, inhibitor of BRD9, I-BRD9, acts synergistically with olaparib in HR-proficient cancer cells. Overall, our results elucidate a role for BRD9 in HR and identify BRD9 as a potential therapeutic target to promote synthetic lethality and overcome chemoresistance.


Assuntos
DNA Helicases/metabolismo , Proteínas de Ligação a DNA/metabolismo , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , DNA Helicases/química , DNA Helicases/genética , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Técnicas de Silenciamento de Genes , Instabilidade Genômica , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Neoplasias Ovarianas/tratamento farmacológico , Ftalazinas/administração & dosagem , Piperazinas/administração & dosagem , Domínios e Motivos de Interação entre Proteínas , Rad51 Recombinase/química , Rad51 Recombinase/genética , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética
6.
Nat Struct Mol Biol ; 27(5): 424-437, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32398827

RESUMO

Oncogene activation during tumorigenesis generates DNA replication stress, a known driver of genome rearrangements. In response to replication stress, certain loci, such as common fragile sites and telomeres, remain under-replicated during interphase and subsequently complete locus duplication in mitosis in a process known as 'MiDAS'. Here, we demonstrate that RTEL1 (regulator of telomere elongation helicase 1) has a genome-wide role in MiDAS at loci prone to form G-quadruplex-associated R-loops, in a process that is dependent on its helicase function. We reveal that SLX4 is required for the timely recruitment of RTEL1 to the affected loci, which in turn facilitates recruitment of other proteins required for MiDAS, including RAD52 and POLD3. Our findings demonstrate that RTEL1 is required for MiDAS and suggest that RTEL1 maintains genome stability by resolving conflicts that can arise between the replication and transcription machineries.


Assuntos
DNA Helicases/genética , DNA Helicases/metabolismo , Quadruplex G , Genoma Humano/genética , Mitose , Animais , Linhagem Celular , DNA Helicases/química , DNA Polimerase III/genética , DNA Polimerase III/metabolismo , Instabilidade Genômica , Humanos , Imunoprecipitação , Camundongos , Enzimas Multifuncionais/genética , Enzimas Multifuncionais/metabolismo , Conformação de Ácido Nucleico , RNA Helicases/genética , RNA Helicases/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo
7.
Nucleic Acids Res ; 48(12): 6640-6653, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32449930

RESUMO

G-quadruplex (G4) DNA structures can form physical barriers within the genome that must be unwound to ensure cellular genomic integrity. Here, we report unanticipated roles for the Escherichia coli Rep helicase and RecA recombinase in tolerating toxicity induced by G4-stabilizing ligands in vivo. We demonstrate that Rep and Rep-X (an enhanced version of Rep) display G4 unwinding activities in vitro that are significantly higher than the closely related UvrD helicase. G4 unwinding mediated by Rep involves repetitive cycles of G4 unfolding and refolding fueled by ATP hydrolysis. Rep-X and Rep also dislodge G4-stabilizing ligands, in agreement with our in vivo G4-ligand sensitivity result. We further demonstrate that RecA filaments disrupt G4 structures and remove G4 ligands in vitro, consistent with its role in countering cellular toxicity of G4-stabilizing ligands. Together, our study reveals novel genome caretaking functions for Rep and RecA in resolving deleterious G4 structures.


Assuntos
DNA Helicases/química , Replicação do DNA/genética , Proteínas de Ligação a DNA/química , Proteínas de Escherichia coli/química , Quadruplex G , Recombinases Rec A/química , Trifosfato de Adenosina/química , DNA Helicases/genética , Proteínas de Ligação a DNA/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Ligantes , Conformação de Ácido Nucleico , Recombinases Rec A/genética
8.
Nucleic Acids Res ; 48(12): 6980-6995, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32453425

RESUMO

DNA unwinding in eukaryotic replication is performed by the Cdc45-MCM-GINS (CMG) helicase. Although the CMG architecture has been elucidated, its mechanism of DNA unwinding and replisome interactions remain poorly understood. Here we report the cryoEM structure at 3.3 Å of human CMG bound to fork DNA and the ATP-analogue ATPγS. Eleven nucleotides of single-stranded (ss) DNA are bound within the C-tier of MCM2-7 AAA+ ATPase domains. All MCM subunits contact DNA, from MCM2 at the 5'-end to MCM5 at the 3'-end of the DNA spiral, but only MCM6, 4, 7 and 3 make a full set of interactions. DNA binding correlates with nucleotide occupancy: five MCM subunits are bound to either ATPγS or ADP, whereas the apo MCM2-5 interface remains open. We further report the cryoEM structure of human CMG bound to the replisome hub AND-1 (CMGA). The AND-1 trimer uses one ß-propeller domain of its trimerisation region to dock onto the side of the helicase assembly formed by Cdc45 and GINS. In the resulting CMGA architecture, the AND-1 trimer is closely positioned to the fork DNA while its CIP (Ctf4-interacting peptide)-binding helical domains remain available to recruit partner proteins.


Assuntos
Proteínas de Ciclo Celular/ultraestrutura , DNA/ultraestrutura , Proteínas de Manutenção de Minicromossomo/ultraestrutura , Complexos Multiproteicos/ultraestrutura , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Microscopia Crioeletrônica , Cristalografia por Raios X , DNA Helicases/química , DNA Helicases/genética , DNA Helicases/ultraestrutura , Replicação do DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/ultraestrutura , Humanos , Proteínas de Manutenção de Minicromossomo/química , Proteínas de Manutenção de Minicromossomo/genética , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Conformação de Ácido Nucleico , Conformação Proteica
9.
Nucleic Acids Res ; 48(11): 6326-6339, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32374860

RESUMO

Nucleotide excision repair (NER) is a major DNA repair pathway for a variety of DNA lesions. XPB plays a key role in DNA opening at damage sites and coordinating damage incision by nucleases. XPB is conserved from archaea to human. In archaea, XPB is associated with a nuclease Bax1. Here we report crystal structures of XPB in complex with Bax1 from Archaeoglobus fulgidus (Af) and Sulfolobus tokodaii (St). These structures reveal for the first time four domains in Bax1, which interacts with XPB mainly through its N-terminal domain. A Cas2-like domain likely helps to position Bax1 at the forked DNA allowing the nuclease domain to incise one arm of the fork. Bax1 exists in monomer or homodimer but forms a heterodimer exclusively with XPB. StBax1 keeps StXPB in a closed conformation and stimulates ATP hydrolysis by XPB while AfBax1 maintains AfXPB in the open conformation and reduces its ATPase activity. Bax1 contains two distinguished nuclease active sites to presumably incise DNA damage. Our results demonstrate that protein-protein interactions regulate the activities of XPB ATPase and Bax1 nuclease. These structures provide a platform to understand the XPB-nuclease interactions important for the coordination of DNA unwinding and damage incision in eukaryotic NER.


Assuntos
Proteínas Arqueais/química , Proteínas Arqueais/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Reparo do DNA , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Adenosina Trifosfatases/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Archaeoglobus fulgidus/química , Domínio Catalítico , Cristalografia por Raios X , DNA/metabolismo , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/metabolismo , Soluções , Eletricidade Estática , Sulfolobus/química
10.
Science ; 367(6480): 875-881, 2020 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-32001526

RESUMO

Mammalian SWI/SNF family chromatin remodelers, BRG1/BRM-associated factor (BAF) and polybromo-associated BAF (PBAF), regulate chromatin structure and transcription, and their mutations are linked to cancers. The 3.7-angstrom-resolution cryo-electron microscopy structure of human BAF bound to the nucleosome reveals that the nucleosome is sandwiched by the base and the adenosine triphosphatase (ATPase) modules, which are bridged by the actin-related protein (ARP) module. The ATPase motor is positioned proximal to nucleosomal DNA and, upon ATP hydrolysis, engages with and pumps DNA along the nucleosome. The C-terminal α helix of SMARCB1, enriched in positively charged residues frequently mutated in cancers, mediates interactions with an acidic patch of the nucleosome. AT-rich interactive domain-containing protein 1A (ARID1A) and the SWI/SNF complex subunit SMARCC serve as a structural core and scaffold in the base module organization, respectively. Our study provides structural insights into subunit organization and nucleosome recognition of human BAF complex.


Assuntos
Montagem e Desmontagem da Cromatina , Proteínas Cromossômicas não Histona/química , DNA Helicases/química , Proteínas Nucleares/química , Nucleossomos/química , Proteína SMARCB1/química , Fatores de Transcrição/química , Adenosina Trifosfatases/química , Trifosfato de Adenosina/metabolismo , Proteínas Cromossômicas não Histona/genética , Microscopia Crioeletrônica , Proteínas de Ligação a DNA , Humanos , Hidrólise , Mutação , Neoplasias/genética , Conformação Proteica em alfa-Hélice , Fatores de Transcrição/genética
11.
Acta Pharm ; 70(2): 145-159, 2020 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-31955138

RESUMO

Middle East respiratory syndrome coronavirus (MERS-CoV) had emerged and spread because of the worldwide travel and inefficient healthcare provided for the infected patients in several countries. Herein we investigated the anti-MERS-CoV activity of newly synthesized sixteen halogenated triazole compounds through the inhibition of helicase activity using the FRET assay. All new compounds underwent justification for their target structures via microanalytical and spectral data. SAR studies were performed. Biological results revealed that the most potent compounds were 4-(cyclopent-1-en-3-ylamino)-5-(2-(4-iodophenyl)hydrazinyl)-4H-1,2,4-triazole-3-thiol (16) and 4-(cyclopent-1-en-3-ylamino)-5-[2-(4-chlorophenyl)hydrazinyl]-4H-1,2,4-triazole-3-thiol (12). In silico molecular docking of the most potent compounds was performed to the active binding site of MERS-CoV helicase nsp13. Molecular docking results are in agreement with experimental findings.


Assuntos
Antivirais/farmacologia , Desenho Assistido por Computador , Infecções por Coronavirus/tratamento farmacológico , DNA Helicases/antagonistas & inibidores , Desenho de Fármacos , Inibidores Enzimáticos/farmacologia , Coronavírus da Síndrome Respiratória do Oriente Médio/efeitos dos fármacos , Simulação de Acoplamento Molecular , Triazóis/farmacologia , Proteínas Virais/antagonistas & inibidores , Animais , Antivirais/síntese química , Antivirais/metabolismo , Infecções por Coronavirus/virologia , DNA Helicases/química , DNA Helicases/metabolismo , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/metabolismo , Humanos , Coronavírus da Síndrome Respiratória do Oriente Médio/enzimologia , Coronavírus da Síndrome Respiratória do Oriente Médio/crescimento & desenvolvimento , Estrutura Molecular , Ligação Proteica , Relação Estrutura-Atividade , Triazóis/síntese química , Triazóis/metabolismo , Proteínas Virais/química , Proteínas Virais/metabolismo , Replicação Viral/efeitos dos fármacos
12.
Proc Natl Acad Sci U S A ; 116(49): 24533-24541, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31744872

RESUMO

Members of the Pif1 family of helicases function in multiple pathways that involve DNA synthesis: DNA replication across G-quadruplexes; break-induced replication; and processing of long flaps during Okazaki fragment maturation. Furthermore, Pif1 increases strand-displacement DNA synthesis by DNA polymerase δ and allows DNA replication across arrays of proteins tightly bound to DNA. This is a surprising feat since DNA rewinding or annealing activities limit the amount of single-stranded DNA product that Pif1 can generate, leading to an apparently poorly processive helicase. In this work, using single-molecule Förster resonance energy transfer approaches, we show that 2 members of the Pif1 family of helicases, Pif1 from Saccharomyces cerevisiae and Pfh1 from Schizosaccharomyces pombe, unwind double-stranded DNA by a branched mechanism with 2 modes of activity. In the dominant mode, only short stretches of DNA can be processively and repetitively opened, with reclosure of the DNA occurring by mechanisms other than strand-switching. In the other less frequent mode, longer stretches of DNA are unwound via a path that is separate from the one leading to repetitive unwinding. Analysis of the kinetic partitioning between the 2 different modes suggests that the branching point in the mechanism is established by conformational selection, controlled by the interaction of the helicase with the 3' nontranslocating strand. The data suggest that the dominant and repetitive mode of DNA opening of the helicase can be used to allow efficient DNA replication, with DNA synthesis on the nontranslocating strand rectifying the DNA unwinding activity.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/metabolismo , Trifosfato de Adenosina/metabolismo , DNA/química , DNA/metabolismo , Transferência Ressonante de Energia de Fluorescência , Cinética , Saccharomyces cerevisiae/química , Schizosaccharomyces/química
13.
Nat Commun ; 10(1): 5375, 2019 11 26.
Artigo em Inglês | MEDLINE | ID: mdl-31772234

RESUMO

Pif1 plays multiple roles in maintaining genome stability and preferentially unwinds forked dsDNA, but the mechanism by which Pif1 unwinds forked dsDNA remains elusive. Here we report the structure of Bacteroides sp Pif1 (BaPif1) in complex with a symmetrical double forked dsDNA. Two interacting BaPif1 molecules are bound to each fork of the partially unwound dsDNA, and interact with the 5' arm and 3' ss/dsDNA respectively. Each of the two BaPif1 molecules is an active helicase and their interaction may regulate their helicase activities. The binding of BaPif1 to the 5' arm causes a sharp bend in the 5' ss/dsDNA junction, consequently breaking the first base-pair. BaPif1 bound to the 3' ss/dsDNA junction impacts duplex unwinding by stabilizing the unpaired first base-pair and engaging the second base-pair poised for breaking. Our results provide an unprecedented insight into how two BaPif1 coordinate with each other to unwind the forked dsDNA.


Assuntos
DNA Helicases/química , DNA Helicases/metabolismo , DNA/química , DNA/metabolismo , Difosfato de Adenosina/química , Difosfato de Adenosina/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Bacteroides fragilis/enzimologia , Pareamento de Bases , Cristalografia por Raios X , DNA Helicases/genética , Transferência Ressonante de Energia de Fluorescência , Mutagênese , Conformação de Ácido Nucleico , Conformação Proteica , Imagem Individual de Molécula
14.
BMC Cancer ; 19(1): 1009, 2019 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-31660888

RESUMO

BACKGROUND: The high expression of BLM (Bloom syndrome) helicase in tumors involves its strong association with cell expansion. Bisbenzylisoquinoline alkaloids own an antitumor property and have developed as candidates for anticancer drugs. This paper aimed to screen potential antiproliferative small molecules from 12 small molecules (the derivatives of bisbenzylisoquinoline alkaloids tetrandrine and fangchinoline) by targeting BLM642-1290 helicase. Then we explore the inhibitory mechanism of those small molecules on proliferation of MDA-MB-435 breast cancer cells. METHODS: Fluorescence polarization technique was used to screen small molecules which inhibited the DNA binding and unwinding of BLM642-1290 helicase. The effects of positive small molecules on the ATPase and conformation of BLM642-1290 helicase were studied by the malachite green-phosphate ammonium molybdate colorimetry and ultraviolet spectral scanning, respectively. The effects of positive small molecules on growth of MDA-MB-435 cells were studied by MTT method, colony formation and cell counting method. The mRNA and protein levels of BLM helicase in the MDA-MB-435 cells after positive small molecule treatments were examined by RT-PCR and ELISA, respectively. RESULTS: The compound HJNO (a tetrandrine derivative) was screened out which inhibited the DNA binding, unwinding and ATPase of BLM642-1290 helicase. That HJNO could bind BLM642-1290helicase to change its conformationcontribute to inhibiting the DNA binding, ATPase and DNA unwinding of BLM642-1290 helicase. In addition, HJNO showed its inhibiting the growth of MDA-MB-435 cells. The values of IC50 after drug treatments for 24 h, 48 h and 72 h were 19.9 µmol/L, 4.1 µmol/L and 10.9 µmol/L, respectively. The mRNA and protein levels of BLM helicase in MDA-MB-435 cells increased after HJNO treatment. Those showed a significant difference (P < 0.05) compared with negative control when the concentrations of HJNO were 5 µmol/L and 10 µmol/L, which might contribute to HJNO inhibiting the DNA binding, ATPase and DNA unwinding of BLM helicase. CONCLUSION: The small molecule HJNO was screened out by targeting BLM642-1290 helicase. And it showed an inhibition on MDA-MB-435 breast cancer cells expansion.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Benzilisoquinolinas/farmacologia , Neoplasias da Mama/tratamento farmacológico , RecQ Helicases/genética , RecQ Helicases/metabolismo , Antineoplásicos Fitogênicos/química , Antineoplásicos Fitogênicos/metabolismo , Antineoplásicos Fitogênicos/uso terapêutico , Benzilisoquinolinas/química , Benzilisoquinolinas/metabolismo , Benzilisoquinolinas/uso terapêutico , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , DNA/química , DNA/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Escherichia coli/enzimologia , Feminino , Células Endoteliais da Veia Umbilical Humana , Humanos , Concentração Inibidora 50 , Ligação Proteica , Transdução de Sinais/efeitos dos fármacos
15.
PLoS One ; 14(10): e0223875, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31626633

RESUMO

Several pathways exist to bypass DNA damage during replication. One such pathway is template switching. The Rad5 protein plays two important roles in template switching: it is an E3 ubiquitin ligase that catalyzes PCNA poly-ubiquitylation and it is a helicase that converts replication forks to chicken foot structures. To understand the structure, conformational flexibility, and mechanism of Rad5, we used a full-ensemble hybrid method combining Langevin dynamics simulations and small-angle X-ray scattering. From these studies, we generated the first experimentally validated, high-resolution structural model of Rad5. We found that Rad5 is more compact and less extended than is suggested by its large amount of predicted intrinsic disorder. Thus, Rad5 likely has a novel intra-molecular interaction that limits the range of conformational space it can sample. We provide evidence for a novel interaction between the HIRAN and the helicase domains of Rad5, and we discuss the biological and mechanistic implications of this.


Assuntos
Dano ao DNA , DNA Helicases/química , Simulação de Dinâmica Molecular , Proteínas de Saccharomyces cerevisiae/química , DNA Helicases/genética , DNA Helicases/metabolismo , Replicação do DNA , Conformação Proteica , Domínios Proteicos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Espalhamento a Baixo Ângulo , Difração de Raios X
16.
Enzymes ; 45: 225-256, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31627878

RESUMO

Despite their relative simplicity, iron-sulfur clusters have been omnipresent as cofactors in myriad cellular processes such as oxidative phosphorylation and other respiratory pathways. Recent research advances confirm the presence of different clusters in enzymes involved in nucleic acid metabolism. Iron-sulfur clusters can therefore be considered hallmarks of cellular metabolism. Helicases, nucleases, glycosylases, DNA polymerases and transcription factors, among others, incorporate various types of clusters that serve differing roles. In this chapter, we review our current understanding of the identity and functions of iron-sulfur clusters in DNA and RNA metabolizing enzymes, highlighting their importance as regulators of cellular function.


Assuntos
Coenzimas/metabolismo , Proteínas com Ferro-Enxofre/química , Proteínas com Ferro-Enxofre/metabolismo , Ácidos Nucleicos/metabolismo , Coenzimas/química , DNA Glicosilases/química , DNA Glicosilases/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , DNA Polimerase Dirigida por DNA/química , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo
17.
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
18.
Molecules ; 24(19)2019 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-31546714

RESUMO

G-quadruplexes are four-stranded guanine-rich structures that have been demonstrated to occur across the genome in humans and other organisms. They provide regulatory functions during transcription, translation and immunoglobulin gene rearrangement, but there is also a large amount of evidence that they can present a potent barrier to the DNA replication machinery. This mini-review will summarize recent advances in understanding the many strategies nature has evolved to overcome G-quadruplex-mediated replication blockage, including removal of the structure by helicases or nucleases, or circumventing the deleterious effects on the genome through homologous recombination, alternative end-joining or synthesis re-priming. Paradoxically, G-quadruplexes have also recently been demonstrated to provide a positive role in stimulating the initiation of DNA replication. These recent studies have not only illuminated the many roles and consequences of G-quadruplexes, but have also provided fundamental insights into the general mechanisms of DNA replication and its links with genetic and epigenetic stability.


Assuntos
Replicação do DNA/fisiologia , Quadruplex G , DNA Helicases/química , DNA Helicases/metabolismo , Replicação do DNA/genética , Desoxirribonucleases/química , Desoxirribonucleases/metabolismo , Humanos
19.
Nucleic Acids Res ; 47(20): 10678-10692, 2019 11 18.
Artigo em Inglês | MEDLINE | ID: mdl-31501894

RESUMO

CSB, a member of the SWI2/SNF2 superfamily, has been implicated in evicting histones to promote the DSB pathway choice towards homologous recombination (HR) repair. However, how CSB promotes HR repair remains poorly characterized. Here we demonstrate that CSB interacts with both MRE11/RAD50/NBS1 (MRN) and BRCA1 in a cell cycle regulated manner, with the former requiring its WHD and occurring predominantly in early S phase. CSB interacts with the BRCT domain of BRCA1 and this interaction is regulated by CDK-dependent phosphorylation of CSB on S1276. The CSB-BRCA1 interaction, which peaks in late S/G2 phase, is responsible for mediating the interaction of CSB with the BRCA1-C complex consisting of BRCA1, MRN and CtIP. While dispensable for histone eviction at DSBs, CSB phosphorylation on S1276 is necessary to promote efficient MRN- and CtIP-mediated DNA end resection, thereby restricting NHEJ and enforcing the DSB repair pathway choice to HR. CSB phosphorylation on S1276 is also necessary to support cell survival in response to DNA damage-inducing agents. These results altogether suggest that CSB interacts with BRCA1 to promote DNA end resection for HR repair and that although prerequisite, CSB-mediated histone eviction alone is insufficient to promote the pathway choice towards HR.


Assuntos
Proteína BRCA1/metabolismo , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/metabolismo , Fase G2 , Complexos Multiproteicos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Fase S , Proteína BRCA1/química , Camptotecina/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Montagem e Desmontagem da Cromatina/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , DNA Helicases/química , Reparo do DNA/efeitos dos fármacos , Enzimas Reparadoras do DNA/química , Fase G2/efeitos dos fármacos , Humanos , Fosforilação/efeitos dos fármacos , Fosfosserina/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Proteínas de Ligação a Poli-ADP-Ribose/química , Ligação Proteica/efeitos dos fármacos , Domínios Proteicos , Fase S/efeitos dos fármacos , Proteínas de Ligação a Telômeros/metabolismo
20.
Nucleic Acids Res ; 47(19): 10313-10326, 2019 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-31529052

RESUMO

In Eukaryotes, tRNAs, 5S RNA and U6 RNA are transcribed by RNA polymerase (Pol) III. Human Pol III is composed of 17 subunits. Three specific Pol III subunits form a stable ternary subcomplex (RPC62-RPC39-RPC32α/ß) being involved in pre-initiation complex formation. No paralogues for subunits of this subcomplex subunits have been found in Pols I or II, but hRPC62 was shown to be structurally related to the general Pol II transcription factor hTFIIEα. Here we show that these structural homologies extend to functional similarities. hRPC62 as well as hTFIIEα possess intrinsic ATP-dependent 3'-5' DNA unwinding activity. The ATPase activities of both proteins are stimulated by single-stranded DNA. Moreover, the eWH domain of hTFIIEα can replace the first eWH (eWH1) domain of hRPC62 in ATPase and DNA unwinding assays. Our results identify intrinsic enzymatic activities in hRPC62 and hTFIIEα.


Assuntos
RNA Polimerase III/química , Fatores de Transcrição TFII/genética , Transcrição Genética , Trifosfato de Adenosina , DNA Helicases/química , DNA Helicases/genética , Humanos , Subunidades Proteicas/química , Subunidades Proteicas/genética , RNA Polimerase III/genética , Fatores de Transcrição TFII/química
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