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1.
Nucleic Acids Res ; 49(1): 285-305, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33332547

RESUMO

RECQ5 is one of five RecQ helicases found in humans and is thought to participate in homologous DNA recombination by acting as a negative regulator of the recombinase protein RAD51. Here, we use kinetic and single molecule imaging methods to monitor RECQ5 behavior on various nucleoprotein complexes. Our data demonstrate that RECQ5 can act as an ATP-dependent single-stranded DNA (ssDNA) motor protein and can translocate on ssDNA that is bound by replication protein A (RPA). RECQ5 can also translocate on RAD51-coated ssDNA and readily dismantles RAD51-ssDNA filaments. RECQ5 interacts with RAD51 through protein-protein contacts, and disruption of this interface through a RECQ5-F666A mutation reduces translocation velocity by ∼50%. However, RECQ5 readily removes the ATP hydrolysis-deficient mutant RAD51-K133R from ssDNA, suggesting that filament disruption is not coupled to the RAD51 ATP hydrolysis cycle. RECQ5 also readily removes RAD51-I287T, a RAD51 mutant with enhanced ssDNA-binding activity, from ssDNA. Surprisingly, RECQ5 can bind to double-stranded DNA (dsDNA), but it is unable to translocate. Similarly, RECQ5 cannot dismantle RAD51-bound heteroduplex joint molecules. Our results suggest that the roles of RECQ5 in genome maintenance may be regulated in part at the level of substrate specificity.


Assuntos
DNA de Cadeia Simples/metabolismo , Recombinação Homóloga , Proteínas Motores Moleculares/metabolismo , RecQ Helicases/metabolismo , Imagem Individual de Molécula , Trifosfato de Adenosina/metabolismo , DNA de Cadeia Simples/ultraestrutura , Humanos , Hidrólise , Cinética , Microscopia de Força Atômica , Proteínas Motores Moleculares/ultraestrutura , Mutação de Sentido Incorreto , Mutação Puntual , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/genética , RecQ Helicases/ultraestrutura , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes/metabolismo , Proteína de Replicação A/metabolismo , Especificidade por Substrato
2.
PLoS Genet ; 16(12): e1008603, 2020 12.
Artigo em Inglês | MEDLINE | ID: mdl-33370275

RESUMO

Telomeres have the ability to adopt a lariat conformation and hence, engage in long and short distance intra-chromosome interactions. Budding yeast telomeres were proposed to fold back into subtelomeric regions, but a robust assay to quantitatively characterize this structure has been lacking. Therefore, it is not well understood how the interactions between telomeres and non-telomeric regions are established and regulated. We employ a telomere chromosome conformation capture (Telo-3C) approach to directly analyze telomere folding and its maintenance in S. cerevisiae. We identify the histone modifiers Sir2, Sin3 and Set2 as critical regulators for telomere folding, which suggests that a distinct telomeric chromatin environment is a major requirement for the folding of yeast telomeres. We demonstrate that telomeres are not folded when cells enter replicative senescence, which occurs independently of short telomere length. Indeed, Sir2, Sin3 and Set2 protein levels are decreased during senescence and their absence may thereby prevent telomere folding. Additionally, we show that the homologous recombination machinery, including the Rad51 and Rad52 proteins, as well as the checkpoint component Rad53 are essential for establishing the telomere fold-back structure. This study outlines a method to interrogate telomere-subtelomere interactions at a single unmodified yeast telomere. Using this method, we provide insights into how the spatial arrangement of the chromosome end structure is established and demonstrate that telomere folding is compromised throughout replicative senescence.


Assuntos
Replicação do DNA , Histona Desacetilases/metabolismo , Metiltransferases/metabolismo , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/metabolismo , Sirtuína 2/metabolismo , Telômero/genética , Histona Desacetilases/genética , Metiltransferases/genética , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas Repressoras/genética , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Proteínas Reguladoras de Informação Silenciosa de Saccharomyces cerevisiae/genética , Sirtuína 2/genética , Telômero/química , Homeostase do Telômero
3.
Proc Natl Acad Sci U S A ; 117(40): 24947-24956, 2020 10 06.
Artigo em Inglês | MEDLINE | ID: mdl-32968016

RESUMO

The acquisition of mutations plays critical roles in adaptation, evolution, senescence, and tumorigenesis. Massive genome sequencing has allowed extraction of specific features of many mutational landscapes but it remains difficult to retrospectively determine the mechanistic origin(s), selective forces, and trajectories of transient or persistent mutations and genome rearrangements. Here, we conducted a prospective reciprocal approach to inactivate 13 single or multiple evolutionary conserved genes involved in distinct genome maintenance processes and characterize de novo mutations in 274 diploid Saccharomyces cerevisiae mutation accumulation lines. This approach revealed the diversity, complexity, and ultimate uniqueness of mutational landscapes, differently composed of base substitutions, small insertions/deletions (InDels), structural variants, and/or ploidy variations. Several landscapes parallel the repertoire of mutational signatures in human cancers while others are either novel or composites of subsignatures resulting from distinct DNA damage lesions. Notably, the increase of base substitutions in the homologous recombination-deficient Rad51 mutant, specifically dependent on the Polζ translesion polymerase, yields COSMIC signature 3 observed in BRCA1/BRCA2-mutant breast cancer tumors. Furthermore, "mutome" analyses in highly polymorphic diploids and single-cell bottleneck lineages revealed a diverse spectrum of loss-of-heterozygosity (LOH) signatures characterized by interstitial and terminal chromosomal events resulting from interhomolog mitotic cross-overs. Following the appearance of heterozygous mutations, the strong stimulation of LOHs in the rad27/FEN1 and tsa1/PRDX1 backgrounds leads to fixation of homozygous mutations or their loss along the lineage. Overall, these mutomes and their trajectories provide a mechanistic framework to understand the origin and dynamics of genome variations that accumulate during clonal evolution.


Assuntos
Neoplasias da Mama/genética , Carcinogênese/genética , Mutação/genética , Saccharomyces cerevisiae/genética , Acetiltransferases/genética , Proteína BRCA1/genética , Proteína BRCA2/genética , Neoplasias da Mama/patologia , Dano ao DNA/genética , DNA Polimerase Dirigida por DNA , Diploide , Feminino , Endonucleases Flap/genética , Genoma Fúngico/genética , Humanos , Perda de Heterozigosidade/genética , Proteínas de Membrana/genética , Peroxirredoxinas/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Sequenciamento Completo do Genoma
4.
PLoS Pathog ; 16(8): e1008772, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32866214

RESUMO

The tick-borne apicomplexan parasite, Babesia bovis, a highly persistent bovine pathogen, expresses VESA1 proteins on the infected erythrocyte surface to mediate cytoadhesion. The cytoadhesion ligand, VESA1, which protects the parasite from splenic passage, is itself protected from a host immune response by rapid antigenic variation. B. bovis relies upon segmental gene conversion (SGC) as a major mechanism to vary VESA1 structure. Gene conversion has been considered a form of homologous recombination (HR), a process for which Rad51 proteins are considered pivotal components. This could make BbRad51 a choice target for development of inhibitors that both interfere with parasite genome integrity and disrupt HR-dependent antigenic variation. Previously, we knocked out the Bbrad51 gene from the B. bovis haploid genome, resulting in a phenotype of sensitivity to methylmethane sulfonate (MMS) and apparent loss of HR-dependent integration of exogenous DNA. In a further characterization of BbRad51, we demonstrate here that ΔBbrad51 parasites are not more sensitive than wild-type to DNA damage induced by γ-irradiation, and repair their genome with similar kinetics. To assess the need for BbRad51 in SGC, RT-PCR was used to observe alterations to a highly variant region of ves1α transcripts over time. Mapping of these amplicons to the genome revealed a significant reduction of in situ transcriptional switching (isTS) among ves loci, but not cessation. By combining existing pipelines for analysis of the amplicons, we demonstrate that SGC continues unabated in ΔBbrad51 parasites, albeit at an overall reduced rate, and a reduction in SGC tract lengths was observed. By contrast, no differences were observed in the lengths of homologous sequences at which recombination occurred. These results indicate that, whereas BbRad51 is not essential to babesial antigenic variation, it influences epigenetic control of ves loci, and its absence significantly reduces successful variation. These results necessitate a reconsideration of the likely enzymatic mechanism(s) underlying SGC and suggest the existence of additional targets for development of small molecule inhibitors.


Assuntos
Antígenos de Protozoários , Babesia bovis , Conversão Gênica/imunologia , Genoma de Protozoário/imunologia , Proteínas de Protozoários , Rad51 Recombinase , Antígenos de Protozoários/genética , Antígenos de Protozoários/imunologia , Babesia bovis/genética , Babesia bovis/imunologia , DNA de Protozoário/genética , DNA de Protozoário/imunologia , Haploidia , Proteínas de Protozoários/genética , Proteínas de Protozoários/imunologia , Rad51 Recombinase/genética , Rad51 Recombinase/imunologia
5.
Nucleic Acids Res ; 48(17): 9649-9659, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32785644

RESUMO

BRCA2 is a key breast cancer associated protein that is predicted to have interspersed regions of intrinsic disorder. Intrinsic disorder coupled with large size likely allows BRCA2 to sample a broad range of conformational space. We expect that the resulting dynamic arrangements of BRCA2 domains are a functionally important aspect of its role in homologous recombination DNA repair. To determine the architectural organization and the associated conformational landscape of BRCA2, we used scanning force microscopy based single molecule analyses to map the flexible regions of the protein and characterize which regions influence oligomerization. We show that the N- and the C-terminal regions are the main flexible regions. Both of these regions also influence BRCA2 oligomerization and interaction with RAD51. In the central Brc repeat region, Brc 1-4 and Brc 5-8 contribute synergistically to BRCA2 interaction with RAD51. We also analysed several single amino acid changes that are potentially clinically relevant and found one, the variant of F1524V, which disrupts key interactions and alters the conformational landscape of the protein. We describe the overall conformation spectrum of BRCA2, which suggests that dynamic structural transitions are key features of its biological function, maintaining genomic stability.


Assuntos
Proteína BRCA2/química , Proteína BRCA2/metabolismo , Rad51 Recombinase/metabolismo , Proteína BRCA2/genética , Humanos , Microscopia de Força Atômica , Mutação de Sentido Incorreto , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Rad51 Recombinase/genética
6.
Nucleic Acids Res ; 48(16): 9135-9146, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32735676

RESUMO

Microcephalin 1 (MCPH1) was identified from genetic mutations in patients with primary autosomal recessive microcephaly. In response to DNA double-strand breaks (DSBs), MCPH1 forms damage-induced foci and recruits BRCA2-RAD51 complex, a key component of the DSB repair machinery for homologous recombination (HR), to damage sites. Accordingly, the efficiency of HR is significantly attenuated upon depletion of MCPH1. The biochemical characteristics of MCPH1 and its functional interaction with the HR machinery had remained unclear due to lack of highly purified MCPH1 recombinant protein for functional study. Here, we established a mammalian expression system to express and purify MCPH1 protein. We show that MCPH1 is a bona fide DNA-binding protein and provide direct biochemical analysis of this MCPH family protein. Furthermore, we reveal that MCPH1 directly interacts with RAD51 at multiple contact points, providing evidence for how MCPH1 physically engages with the HR machinery. Importantly, we demonstrate that MCPH1 enhances the stability of RAD51 on single-strand DNA, a prerequisite step for RAD51-mediated recombination. Single-molecule tethered particle motion analysis showed a ∼2-fold increase in the lifetime of RAD51-ssDNA filaments in the presence of MCPH1. Thus, our study demonstrates direct crosstalk between microcephaly protein MCPH1 and the recombination component RAD51 for DSB repair.


Assuntos
Proteína BRCA2/genética , Proteínas de Ciclo Celular/genética , Proteínas do Citoesqueleto/genética , Microcefalia/genética , Rad51 Recombinase/genética , Citoesqueleto/genética , Quebras de DNA de Cadeia Dupla , Dano ao DNA/genética , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Proteínas de Ligação a DNA/genética , Instabilidade Genômica/genética , Recombinação Homóloga/genética , Humanos , Microcefalia/patologia , Nucleoproteínas/genética
7.
Nucleic Acids Res ; 48(16): 9161-9180, 2020 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-32797166

RESUMO

FANCJ, a DNA helicase and interacting partner of the tumor suppressor BRCA1, is crucial for the repair of DNA interstrand crosslinks (ICL), a highly toxic lesion that leads to chromosomal instability and perturbs normal transcription. In diploid cells, FANCJ is believed to operate in homologous recombination (HR) repair of DNA double-strand breaks (DSB); however, its precise role and molecular mechanism is poorly understood. Moreover, compensatory mechanisms of ICL resistance when FANCJ is deficient have not been explored. In this work, we conducted a siRNA screen to identify genes of the DNA damage response/DNA repair regime that when acutely depleted sensitize FANCJ CRISPR knockout cells to a low concentration of the DNA cross-linking agent mitomycin C (MMC). One of the top hits from the screen was RAP80, a protein that recruits repair machinery to broken DNA ends and regulates DNA end-processing. Concomitant loss of FANCJ and RAP80 not only accentuates DNA damage levels in human cells but also adversely affects the cell cycle checkpoint, resulting in profound chromosomal instability. Genetic complementation experiments demonstrated that both FANCJ's catalytic activity and interaction with BRCA1 are important for ICL resistance when RAP80 is deficient. The elevated RPA and RAD51 foci in cells co-deficient of FANCJ and RAP80 exposed to MMC are attributed to single-stranded DNA created by Mre11 and CtIP nucleases. Altogether, our cell-based findings together with biochemical studies suggest a critical function of FANCJ to suppress incompletely processed and toxic joint DNA molecules during repair of ICL-induced DNA damage.


Assuntos
Proteína BRCA1/genética , Proteínas de Ligação a DNA/genética , Proteínas de Grupos de Complementação da Anemia de Fanconi/genética , Instabilidade Genômica/genética , Chaperonas de Histonas/genética , RNA Helicases/genética , Rad51 Recombinase/genética , Instabilidade Cromossômica/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/deficiência , Técnicas de Inativação de Genes , Células HeLa , Chaperonas de Histonas/deficiência , Humanos , Mitomicina/farmacologia , Reparo de DNA por Recombinação/genética
8.
Nucleic Acids Res ; 48(17): e100, 2020 09 25.
Artigo em Inglês | MEDLINE | ID: mdl-32797168

RESUMO

Tracking DNA double strand break (DSB) repair is paramount for the understanding and therapeutic development of various diseases including cancers. Herein, we describe a multiplexed bioluminescent repair reporter (BLRR) for non-invasive monitoring of DSB repair pathways in living cells and animals. The BLRR approach employs secreted Gaussia and Vargula luciferases to simultaneously detect homology-directed repair (HDR) and non-homologous end joining (NHEJ), respectively. BLRR data are consistent with next-generation sequencing results for reporting HDR (R2 = 0.9722) and NHEJ (R2 = 0.919) events. Moreover, BLRR analysis allows longitudinal tracking of HDR and NHEJ activities in cells, and enables detection of DSB repairs in xenografted tumours in vivo. Using the BLRR system, we observed a significant difference in the efficiency of CRISPR/Cas9-mediated editing with guide RNAs only 1-10 bp apart. Moreover, BLRR analysis detected altered dynamics for DSB repair induced by small-molecule modulators. Finally, we discovered HDR-suppressing functions of anticancer cardiac glycosides in human glioblastomas and glioma cancer stem-like cells via inhibition of DNA repair protein RAD51 homolog 1 (RAD51). The BLRR method provides a highly sensitive platform to simultaneously and longitudinally track HDR and NHEJ dynamics that is sufficiently versatile for elucidating the physiology and therapeutic development of DSB repair.


Assuntos
Genes Reporter , Luciferases/genética , Reparo de DNA por Recombinação , Animais , Sistemas CRISPR-Cas , Linhagem Celular Tumoral , Copépodes/enzimologia , Reparo do DNA por Junção de Extremidades , Feminino , Células HEK293 , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Luciferases/metabolismo , Camundongos , Camundongos Nus , Reação em Cadeia da Polimerase Multiplex/métodos , Imagem Óptica/métodos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Análise de Sequência de DNA/métodos
9.
Ecotoxicol Environ Saf ; 202: 110962, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32800233

RESUMO

Chronic exposure to fluoride (F) beyond the permissible limit (1.5 ppm) is known to cause detrimental health effects by induction of oxidative stress-mediated DNA damage overpowering the DNA repair machinery. In the present study, we assessed F induced oxidative stress through monitoring biochemical parameters and looked into the effect of chronic F exposure on two crucial DNA repair genes Ogg1 and Rad51 having important role against ROS induced DNA damages. To address this issue, we exposed Swiss albino mice to an environmentally relevant concentration of fluoride (15 ppm NaF) for 8 months. Results revealed histoarchitectural damages in liver, brain, kidney and spleen. Depletion of GSH, increase in lipid peroxidation and catalase activity in liver and brain confirmed the generation of oxidative stress. qRT-PCR result showed that expressions of Ogg1 and Rad51 were altered after F exposure in the affected organs. Promoter hypermethylation was associated with the downregulation of Rad51. F-induced DNA damage and the compromised DNA repair machinery triggered intrinsic pathway of apoptosis in liver and brain. The present study indicates the possible association of epigenetic regulation with F induced neurotoxicity.


Assuntos
Dano ao DNA , DNA Glicosilases/genética , Reparo do DNA , Epigênese Genética/efeitos dos fármacos , Fluoretos/toxicidade , Rad51 Recombinase/genética , Animais , Apoptose/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Relação Dose-Resposta a Droga , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Masculino , Camundongos , Estresse Oxidativo/efeitos dos fármacos
10.
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
11.
Mol Cell ; 79(3): 425-442.e7, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32615088

RESUMO

Double-strand breaks (DSBs) are the most deleterious DNA lesions, which, if left unrepaired, may lead to genome instability or cell death. Here, we report that, in response to DSBs, the RNA methyltransferase METTL3 is activated by ATM-mediated phosphorylation at S43. Phosphorylated METTL3 is then localized to DNA damage sites, where it methylates the N6 position of adenosine (m6A) in DNA damage-associated RNAs, which recruits the m6A reader protein YTHDC1 for protection. In this way, the METTL3-m6A-YTHDC1 axis modulates accumulation of DNA-RNA hybrids at DSBs sites, which then recruit RAD51 and BRCA1 for homologous recombination (HR)-mediated repair. METTL3-deficient cells display defective HR, accumulation of unrepaired DSBs, and genome instability. Accordingly, depletion of METTL3 significantly enhances the sensitivity of cancer cells and murine xenografts to DNA damage-based therapy. These findings uncover the function of METTL3 and YTHDC1 in HR-mediated DSB repair, which may have implications for cancer therapy.


Assuntos
Adenosina/análogos & derivados , Neoplasias de Cabeça e Pescoço/genética , Metiltransferases/genética , Proteínas do Tecido Nervoso/genética , Fatores de Processamento de RNA/genética , Reparo de DNA por Recombinação/efeitos dos fármacos , Carcinoma de Células Escamosas de Cabeça e Pescoço/genética , Adenosina/metabolismo , Animais , Antibióticos Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Bleomicina/farmacologia , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Células HEK293 , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/mortalidade , Neoplasias de Cabeça e Pescoço/patologia , Humanos , Metiltransferases/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Proteínas do Tecido Nervoso/metabolismo , Hibridização de Ácido Nucleico , Osteoblastos/efeitos dos fármacos , Osteoblastos/metabolismo , Osteoblastos/patologia , Fosforilação , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Fatores de Processamento de RNA/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Ribonuclease H/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Análise de Sobrevida , Ensaios Antitumorais Modelo de Xenoenxerto
12.
Nucleic Acids Res ; 48(15): 8474-8489, 2020 09 04.
Artigo em Inglês | MEDLINE | ID: mdl-32652040

RESUMO

Highly toxic DNA double-strand breaks (DSBs) readily trigger the DNA damage response (DDR) in cells, which delays cell cycle progression to ensure proper DSB repair. In Saccharomyces cerevisiae, mitotic S phase (20-30 min) is lengthened upon DNA damage. During meiosis, Spo11-induced DSB onset and repair lasts up to 5 h. We report that the NH2-terminal domain (NTD; residues 1-66) of Rad51 has dual functions for repairing DSBs during vegetative growth and meiosis. Firstly, Rad51-NTD exhibits autonomous expression-enhancing activity for high-level production of native Rad51 and when fused to exogenous ß-galactosidase in vivo. Secondly, Rad51-NTD is an S/T-Q cluster domain (SCD) harboring three putative Mec1/Tel1 target sites. Mec1/Tel1-dependent phosphorylation antagonizes the proteasomal degradation pathway, increasing the half-life of Rad51 from ∼30 min to ≥180 min. Our results evidence a direct link between homologous recombination and DDR modulated by Rad51 homeostasis.


Assuntos
Quebras de DNA de Cadeia Dupla , Endodesoxirribonucleases/genética , Meiose/genética , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Dano ao DNA/genética , Reparo do DNA/genética , Proteínas de Ligação a DNA/genética , Regulação Fúngica da Expressão Gênica/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Fosforilação/genética , Complexo de Endopeptidases do Proteassoma/genética , Domínios Proteicos/genética , Proteínas Serina-Treonina Quinases/genética , Proteólise , Saccharomyces cerevisiae/genética , beta-Galactosidase/genética
13.
PLoS Genet ; 16(7): e1008828, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32609721

RESUMO

Homologous recombination (HR) has an intimate relationship with genome replication, both during repair of DNA lesions that might prevent DNA synthesis and in tackling stalls to the replication fork. Recent studies led us to ask if HR might have a more central role in replicating the genome of Leishmania, a eukaryotic parasite. Conflicting evidence has emerged regarding whether or not HR genes are essential, and genome-wide mapping has provided evidence for an unorthodox organisation of DNA replication initiation sites, termed origins. To answer this question, we have employed a combined CRISPR/Cas9 and DiCre approach to rapidly generate and assess the effect of conditional ablation of RAD51 and three RAD51-related proteins in Leishmania major. Using this approach, we demonstrate that loss of any of these HR factors is not immediately lethal but in each case growth slows with time and leads to DNA damage and accumulation of cells with aberrant DNA content. Despite these similarities, we show that only loss of RAD51 or RAD51-3 impairs DNA synthesis and causes elevated levels of genome-wide mutation. Furthermore, we show that these two HR factors act in distinct ways, since ablation of RAD51, but not RAD51-3, has a profound effect on DNA replication, causing loss of initiation at the major origins and increased DNA synthesis at subtelomeres. Our work clarifies questions regarding the importance of HR to survival of Leishmania and reveals an unanticipated, central role for RAD51 in the programme of genome replication in a microbial eukaryote.


Assuntos
Recombinação Homóloga/genética , Leishmania major/genética , Leishmaniose Cutânea/genética , Rad51 Recombinase/genética , Sistemas CRISPR-Cas/genética , Dano ao DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Técnicas de Inativação de Genes , Genoma/genética , Humanos , Leishmania major/patogenicidade , Leishmaniose Cutânea/parasitologia
14.
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
15.
Proc Natl Acad Sci U S A ; 117(28): 16527-16536, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601218

RESUMO

Folate deprivation drives the instability of a group of rare fragile sites (RFSs) characterized by CGG trinucleotide repeat (TNR) sequences. Pathological expansion of the TNR within the FRAXA locus perturbs DNA replication and is the major causative factor for fragile X syndrome, a sex-linked disorder associated with cognitive impairment. Although folate-sensitive RFSs share many features with common fragile sites (CFSs; which are found in all individuals), they are induced by different stresses and share no sequence similarity. It is known that a pathway (termed MiDAS) is employed to complete the replication of CFSs in early mitosis. This process requires RAD52 and is implicated in generating translocations and copy number changes at CFSs in cancers. However, it is unclear whether RFSs also utilize MiDAS and to what extent the fragility of CFSs and RFSs arises by shared or distinct mechanisms. Here, we demonstrate that MiDAS does occur at FRAXA following folate deprivation but proceeds via a pathway that shows some mechanistic differences from that at CFSs, being dependent on RAD51, SLX1, and POLD3. A failure to complete MiDAS at FRAXA leads to severe locus instability and missegregation in mitosis. We propose that break-induced DNA replication is required for the replication of FRAXA under folate stress and define a cellular function for human SLX1. These findings provide insights into how folate deprivation drives instability in the human genome.


Assuntos
Endodesoxirribonucleases/metabolismo , Ácido Fólico/metabolismo , Síndrome do Cromossomo X Frágil/metabolismo , Mitose , Rad51 Recombinase/metabolismo , DNA/genética , DNA/metabolismo , Reparo do DNA , Endodesoxirribonucleases/genética , Síndrome do Cromossomo X Frágil/genética , Síndrome do Cromossomo X Frágil/fisiopatologia , Humanos , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Recombinases/genética , Recombinases/metabolismo
16.
Nat Commun ; 11(1): 2950, 2020 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-32528002

RESUMO

During homologous recombination, Rad51 forms a nucleoprotein filament on single-stranded DNA to promote DNA strand exchange. This filament binds to double-stranded DNA (dsDNA), searches for homology, and promotes transfer of the complementary strand, producing a new heteroduplex. Strand exchange proceeds via two distinct three-strand intermediates, C1 and C2. C1 contains the intact donor dsDNA whereas C2 contains newly formed heteroduplex DNA. Here, we show that the conserved DNA binding motifs, loop 1 (L1) and loop 2 (L2) in site I of Rad51, play distinct roles in this process. L1 is involved in formation of the C1 complex whereas L2 mediates the C1-C2 transition, producing the heteroduplex. Another DNA binding motif, site II, serves as the DNA entry position for initial Rad51 filament formation, as well as for donor dsDNA incorporation. Our study provides a comprehensive molecular model for the catalytic process of strand exchange mediated by eukaryotic RecA-family recombinases.


Assuntos
DNA/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Trifosfato de Adenosina/metabolismo , Sítios de Ligação/genética , DNA/genética , Dano ao DNA/genética , Dano ao DNA/fisiologia , Reparo do DNA/genética , Reparo do DNA/fisiologia , DNA de Cadeia Simples/genética , Recombinação Homóloga/genética , Recombinação Homóloga/fisiologia , Humanos , Mutação/genética , Ácidos Nucleicos Heteroduplexes/genética , Ácidos Nucleicos Heteroduplexes/metabolismo , Estrutura Secundária de Proteína , Rad51 Recombinase/genética , Saccharomyces cerevisiae/genética , Schizosaccharomyces/genética
17.
Nucleic Acids Res ; 48(12): 6624-6639, 2020 07 09.
Artigo em Inglês | MEDLINE | ID: mdl-32463460

RESUMO

Repair of DNA double-strand breaks (DSBs) with homologous chromosomes is a hallmark of meiosis that is mediated by recombination 'bridges' between homolog axes. This process requires cooperation of DMC1 and RAD51 to promote homology search and strand exchange. The mechanism(s) regulating DMC1/RAD51-ssDNA nucleoprotein filament and the components of 'bridges' remain to be investigated. Here we show that MEIOK21 is a newly identified component of meiotic recombination bridges and is required for efficient formation of DMC1/RAD51 foci. MEIOK21 dynamically localizes on chromosomes from on-axis foci to 'hanging foci', then to 'bridges', and finally to 'fused foci' between homolog axes. Its chromosome localization depends on DSBs. Knockout of Meiok21 decreases the numbers of HSF2BP and DMC1/RAD51 foci, disrupting DSB repair, synapsis and crossover recombination and finally causing male infertility. Therefore, MEIOK21 is a novel recombination factor and probably mediates DMC1/RAD51 recruitment to ssDNA or their stability on chromosomes through physical interaction with HSF2BP.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Recombinação Homóloga/genética , Infertilidade Masculina/genética , Espermatogênese/genética , Animais , Proteínas de Transporte/genética , Pareamento Cromossômico/genética , Cromossomos/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA/genética , DNA de Cadeia Simples/genética , Técnicas de Inativação de Genes , Proteínas de Choque Térmico/genética , Masculino , Meiose/genética , Camundongos , Camundongos Knockout , Proteínas de Ligação a Fosfato/genética , Rad51 Recombinase/genética
18.
Mol Cell ; 78(5): 951-959.e6, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32359443

RESUMO

BRCA1 promotes the DNA end resection and RAD51 loading steps of homologous recombination (HR). Whether these functions can be uncoupled, and whether mutant proteins retaining partial activity can complement one another, is unclear and could affect the severity of BRCA1-associated Fanconi anemia (FA). Here we generated a Brca1CC mouse with a coiled-coil (CC) domain deletion. Brca1CC/CC mice are born at low frequencies, and post-natal mice have FA-like abnormalities, including bone marrow failure. Intercrossing with Brca1Δ11, which is homozygous lethal, generated Brca1CC/Δ11 mice at Mendelian frequencies that were indistinguishable from Brca1+/+ mice. Brca1CC and Brca1Δ11 proteins were individually responsible for counteracting 53BP1-RIF1-Shieldin activity and promoting RAD51 loading, respectively. Thus, Brca1CC and Brca1Δ11 alleles represent separation-of-function mutations that combine to provide a level of HR sufficient for normal development and hematopoiesis. Because BRCA1 activities can be genetically separated, compound heterozygosity for functional complementary mutations may protect individuals from FA.


Assuntos
Proteína BRCA1/genética , Recombinação Homóloga/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Proteína BRCA1/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Éxons , Anemia de Fanconi/genética , Anemia de Fanconi/metabolismo , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C3H , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
19.
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
20.
J Pharmacol Sci ; 143(3): 219-225, 2020 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-32418739

RESUMO

OBJECTIVE: Chemoresistance is one of the major barriers in chemotherapy-based hepatocellular carcinoma (HCC) intervention. 5-Fluorouracil (5-Fu) is a widely used as an anticancer drug. Liver cancer stem cells (LCSCs) are considered the origin of tumor recurrence and resistance. CCND1 (Cyclin D1) plays an important role in tumorigenesis and metastasis in multiple cancers including HCC. Herein, this study was designed to explore the role of CCND1 in regulating LCSCs differentiation and 5-Fu resistance in HCC cells. METHODS: The CCND1 mRNA level was examined by qRT-PCR. The protein levels of γ-H2AX (a DNA damage marker) and RAD51 (a DNA repair protein) were examined by Western blot. CD133 was used as a LCSC marker and CD133+ cell percentage in HCC cells was detected by flow cytometry. RESULTS: CCND1 silencing decreased CD133+ cell percentage in HepG2 and SMMC-7721 cells. Furthermore, CCND1 silencing significantly increased protein level of γ-H2AX and decreased that of RAD51 under 5-Fu exposure. Moreover, CCND1 silencing enhanced the sensitivity of HepG2 and SMMC-7721 cells to 5-Fu, which was effectively abrogated by RAD51 upregulation. CONCLUSION: Collectively, CCND1 silencing suppresses LCSCs differentiation and overcomes 5-Fu resistance in HCC.


Assuntos
Antineoplásicos/farmacologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Diferenciação Celular/genética , Ciclina D1/genética , Ciclina D1/fisiologia , Resistencia a Medicamentos Antineoplásicos/genética , Fluoruracila/farmacologia , Inativação Gênica , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Células-Tronco Neoplásicas/patologia , Técnicas de Cultura de Células , Células Hep G2 , Humanos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Regulação para Cima/genética
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