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1.
Appl Microbiol Biotechnol ; 104(19): 8427-8437, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32813067

RESUMO

Infectious bronchitis virus (IBV) is a member of genus gamma-coronavirus in the family Coronaviridae, causing serious economic losses to the poultry industry. Reverse genetics is a common technique to study the biological characteristics of viruses. So far, there is no BAC reverse genetic system available for rescue of IBV infectious clone. In the present study, a new strategy for the construction of IBV infectious cDNA clone was established. The full-length genomic cDNA of IBV vaccine strain H120 was constructed in pBAC vector from four IBV fragment subcloning vectors by homologous recombination, which contained the CMV promoter at the 5' end and the hepatitis D virus ribozyme (HDVR) sequence and bovine growth hormone polyadenylation (BGH) sequence after the polyA tail at the 3' end of the full-length cDNA. Subsequently, using the same technique, another plasmid pBAC-H120/SCS1 was also constructed, in which S1 gene from IBV H120 strain was replaced with that of a virulent SC021202 strain. Recombinant virus rH120 and rH120/SCS1 were rescued by transfecting the plasmids into BHK cells and passaged in embryonated chicken eggs. Finally, the pathogenicity of both the recombinant virus strains rH120 and rH120/SCS1 was evaluated in SPF chickens. The results showed that the chimeric rH120/SCS1 strain was not pathogenic compared with the wild-type IBV SC021202 strain and the chickens inoculated with rH120/SCS1 could resist challenge infection by IBV SC021202. Taken together, our results indicate that BAC reverse genetic system could be used to rescue IBV in vitro and IBV S1 protein alone might not be the key factor for IBV pathogenicity. KEY POINTS: • BAC vector was used to construct IBV full-length cDNA by homologous recombination. • Based on four subcloning vectors, a recombinant chimeric IBV H120/SCS1 was constructed and rescued. • Pathogenicity of H120/SCS1 was similar to that of H120, but different to that of SC021202.


Assuntos
Vírus da Bronquite Infecciosa/genética , Vírus da Bronquite Infecciosa/patogenicidade , Proteínas Virais/genética , Animais , Embrião de Galinha , Galinhas , Infecções por Coronavirus/veterinária , DNA Complementar , Recombinação Homóloga , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes/genética , Vacinas Virais/genética , Vacinas Virais/imunologia , Virulência/genética
2.
Methods Mol Biol ; 2203: 147-165, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833211

RESUMO

We have developed a reverse genetics system for the avian coronavirus infectious bronchitis virus (IBV) in which a full-length cDNA corresponding to the IBV genome is inserted into the vaccinia virus genome under the control of a T7 promoter sequence. Vaccinia virus as a vector for the full-length IBV cDNA has the advantage that modifications can be introduced into the IBV cDNA using homologous recombination, a method frequently used to insert and delete sequences from the vaccinia virus genome. Here, we describe the use of transient dominant selection as a method for introducing modifications into the IBV cDNA that has been successfully used for the substitution of specific nucleotides, deletion of genomic regions, and the exchange of complete genes. Infectious recombinant IBVs are generated in situ following the transfection of vaccinia virus DNA, containing the modified IBV cDNA, into cells infected with a recombinant fowlpox virus expressing T7 DNA-dependent RNA polymerase.


Assuntos
Vírus da Bronquite Infecciosa/genética , Transfecção/métodos , Vírus Vaccinia/genética , Animais , Bacteriófagos/genética , Chlorocebus aethiops , DNA Polimerase Dirigida por DNA/metabolismo , Vírus da Varíola das Aves Domésticas/genética , Recombinação Homóloga , Microrganismos Geneticamente Modificados , Vírus Vaccinia/isolamento & purificação , Células Vero
3.
Methods Mol Biol ; 2203: 167-184, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32833212

RESUMO

The Escherichia coli and vaccinia virus-based reverse genetics systems have been widely applied for the manipulation and engineering of coronavirus genomes. These systems, however, present several limitations and are sometimes difficult to establish in a timely manner for (re-)emerging viruses. In this chapter, we present a new universal reverse genetics platform for the assembly and engineering of infectious full-length cDNAs using yeast-based transformation-associated recombination cloning. This novel assembly method not only results in stable coronavirus infectious full-length cDNAs cloned in the yeast Saccharomyces cerevisiae but also fosters and accelerates the manipulation of their genomes. Such a platform is widely applicable for the scientific community, as it requires no specific equipment and can be performed in a standard laboratory setting. The protocol described can be easily adapted to virtually all known or emerging coronaviruses, such as Middle East respiratory syndrome coronavirus (MERS-CoV).


Assuntos
Coronavirus/genética , DNA Complementar/genética , Genômica/métodos , Saccharomyces cerevisiae/genética , Animais , Linhagem Celular , Coronavirus/patogenicidade , Recombinação Homóloga , Coronavírus da Síndrome Respiratória do Oriente Médio/genética , Coronavírus da Síndrome Respiratória do Oriente Médio/patogenicidade
5.
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
6.
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
7.
Nat Commun ; 11(1): 3531, 2020 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-32669601

RESUMO

Homologous recombination (HR) factors were recently implicated in DNA replication fork remodeling and protection. While maintaining genome stability, HR-mediated fork remodeling promotes cancer chemoresistance, by as-yet elusive mechanisms. Five HR cofactors - the RAD51 paralogs RAD51B, RAD51C, RAD51D, XRCC2 and XRCC3 - recently emerged as crucial tumor suppressors. Albeit extensively characterized in DNA repair, their role in replication has not been addressed systematically. Here, we identify all RAD51 paralogs while screening for modulators of RAD51 recombinase upon replication stress. Single-molecule analysis of fork progression and architecture in isogenic cellular systems shows that the BCDX2 subcomplex restrains fork progression upon stress, promoting fork reversal. Accordingly, BCDX2 primes unscheduled degradation of reversed forks in BRCA2-defective cells, boosting genomic instability. Conversely, the CX3 subcomplex is dispensable for fork reversal, but mediates efficient restart of reversed forks. We propose that RAD51 paralogs sequentially orchestrate clinically relevant transactions at replication forks, cooperatively promoting fork remodeling and restart.


Assuntos
Replicação do DNA , Rad51 Recombinase/metabolismo , Proteína BRCA2/metabolismo , Linhagem Celular Tumoral , Estruturas Cromossômicas/metabolismo , Cromossomos/ultraestrutura , Dano ao DNA , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Instabilidade Genômica , Recombinação Homóloga , Humanos , Microscopia , Mutagênicos , Mutação , Osteossarcoma/metabolismo , RNA Interferente Pequeno/metabolismo
8.
Proc Natl Acad Sci U S A ; 117(30): 17785-17795, 2020 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-32651270

RESUMO

Poly(ADP ribose) polymerase inhibitors (PARPi) have efficacy in triple negative breast (TNBC) and ovarian cancers (OCs) harboring BRCA mutations, generating homologous recombination deficiencies (HRDs). DNA methyltransferase inhibitors (DNMTi) increase PARP trapping and reprogram the DNA damage response to generate HRD, sensitizing BRCA-proficient cancers to PARPi. We now define the mechanisms through which HRD is induced in BRCA-proficient TNBC and OC. DNMTi in combination with PARPi up-regulate broad innate immune and inflammasome-like signaling events, driven in part by stimulator of interferon genes (STING), to unexpectedly directly generate HRD. This inverse relationship between inflammation and DNA repair is critical, not only for the induced phenotype, but also appears as a widespread occurrence in The Cancer Genome Atlas datasets and cancer subtypes. These discerned interactions between inflammation signaling and DNA repair mechanisms now elucidate how epigenetic therapy enhances PARPi efficacy in the setting of BRCA-proficient cancer. This paradigm will be tested in a phase I/II TNBC clinical trial.


Assuntos
Recombinação Homóloga/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , Biologia Computacional , Metilases de Modificação do DNA/antagonistas & inibidores , Reparo do DNA/efeitos dos fármacos , Anemia de Fanconi/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Interferons/metabolismo , Proteínas de Membrana/metabolismo , Modelos Biológicos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia , Fator de Necrose Tumoral alfa/metabolismo
9.
Proc Natl Acad Sci U S A ; 117(32): 19415-19424, 2020 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-32719125

RESUMO

Synthetic lethality strategies for cancer therapy exploit cancer-specific genetic defects to identify targets that are uniquely essential to the survival of tumor cells. Here we show RAD27/FEN1, which encodes flap endonuclease 1 (FEN1), a structure-specific nuclease with roles in DNA replication and repair, and has the greatest number of synthetic lethal interactions with Saccharomyces cerevisiae genome instability genes, is a druggable target for an inhibitor-based approach to kill cancers with defects in homologous recombination (HR). The vulnerability of cancers with HR defects to FEN1 loss was validated by studies showing that small-molecule FEN1 inhibitors and FEN1 small interfering RNAs (siRNAs) selectively killed BRCA1- and BRCA2-defective human cell lines. Furthermore, the differential sensitivity to FEN1 inhibition was recapitulated in mice, where a small-molecule FEN1 inhibitor reduced the growth of tumors established from drug-sensitive but not drug-resistant cancer cell lines. FEN1 inhibition induced a DNA damage response in both sensitive and resistant cell lines; however, sensitive cell lines were unable to recover and replicate DNA even when the inhibitor was removed. Although FEN1 inhibition activated caspase to higher levels in sensitive cells, this apoptotic response occurred in p53-defective cells and cell killing was not blocked by a pan-caspase inhibitor. These results suggest that FEN1 inhibitors have the potential for therapeutically targeting HR-defective cancers such as those resulting from BRCA1 and BRCA2 mutations, and other genetic defects.


Assuntos
Antineoplásicos/farmacologia , Endonucleases Flap/antagonistas & inibidores , Recombinação Homóloga/efeitos dos fármacos , Neoplasias/genética , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/deficiência , Proteína BRCA2/genética , Linhagem Celular Tumoral , Dano ao DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Replicação do DNA/efeitos dos fármacos , Endonucleases Flap/genética , Instabilidade Genômica/genética , Humanos , Camundongos , Neoplasias/tratamento farmacológico , RNA Interferente Pequeno/farmacologia , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/genética , Bibliotecas de Moléculas Pequenas/farmacologia , Mutações Sintéticas Letais , Ensaios Antitumorais Modelo de Xenoenxerto
10.
Nat Commun ; 11(1): 3181, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576832

RESUMO

The DNA damage checkpoint (DDC) is often robustly activated during the homologous recombination (HR) repair of DNA double strand breaks (DSBs). DDC activation controls several HR repair factors by phosphorylation, preventing premature segregation of entangled chromosomes formed during HR repair. The DDC mediator 53BP1/Rad9 limits the nucleolytic processing (resection) of a DSB, controlling the formation of the 3' single-stranded DNA (ssDNA) filament needed for recombination, from yeast to human. Here we show that Rad9 promotes stable annealing between the recombinogenic filament and the donor template in yeast, limiting strand rejection by the Sgs1 and Mph1 helicases. This regulation allows repair by long tract gene conversion, crossover recombination and break-induced replication (BIR), only after DDC activation. These findings shed light on how cells couple DDC with the choice and effectiveness of HR sub-pathways, with implications for genome instability and cancer.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Reparo do DNA/genética , Reparo do DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo , Proteínas de Ciclo Celular/genética , Sobrevivência Celular , RNA Helicases DEAD-box/metabolismo , Quebras de DNA de Cadeia Dupla , DNA de Cadeia Simples , Proteínas de Ligação a DNA/metabolismo , Conversão Gênica , Instabilidade Genômica , Recombinação Homóloga , Humanos , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , RecQ Helicases/metabolismo , Reparo de DNA por Recombinação , Proteínas de Saccharomyces cerevisiae/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
11.
Nature ; 582(7810): 124-128, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494071

RESUMO

In most species, homologous chromosomes must recombine in order to segregate accurately during meiosis1. Because small chromosomes would be at risk of missegregation if recombination were randomly distributed, the double-strand breaks (DSBs) that initiate recombination are not located arbitrarily2. How the nonrandomness of DSB distributions is controlled is not understood, although several pathways are known to regulate the timing, location and number of DSBs. Meiotic DSBs are generated by Spo11 and accessory DSB proteins, including Rec114 and Mer2, which assemble on chromosomes3-7 and are nearly universal in eukaryotes8-11. Here we demonstrate how Saccharomyces cerevisiae integrates multiple temporally distinct pathways to regulate the binding of Rec114 and Mer2 to chromosomes, thereby controlling the duration of a DSB-competent state. The engagement of homologous chromosomes with each other regulates the dissociation of Rec114 and Mer2 later in prophase I, whereas the timing of replication and the proximity to centromeres or telomeres influence the accumulation of Rec114 and Mer2 early in prophase I. Another early mechanism enhances the binding of Rec114 and Mer2 specifically on the shortest chromosomes, and is subject to selection pressure to maintain the hyperrecombinogenic properties of these chromosomes. Thus, the karyotype of an organism and its risk of meiotic missegregation influence the shape and evolution of its recombination landscape. Our results provide a cohesive view of a multifaceted and evolutionarily constrained system that allocates DSBs to all pairs of homologous chromosomes.


Assuntos
Cromossomos Fúngicos/genética , Recombinação Homóloga , Meiose , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Centrômero/genética , Segregação de Cromossomos , Cromossomos Fúngicos/metabolismo , Quebras de DNA de Cadeia Dupla , Período de Replicação do DNA , Meiose/genética , Prófase Meiótica I/genética , Recombinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Telômero/genética , Fatores de Tempo
12.
PLoS One ; 15(6): e0234440, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32530971

RESUMO

Research for biotechnological applications of cyanobacteria focuses on synthetic pathways and bioreactor design, while little effort is devoted to introduce new, promising organisms in the field. Applications are most often based on recombinant work, and the establishment of transformation can be a risky, time-consuming procedure. In this work we demonstrate the natural transformation of the filamentous cyanobacterium Phormidium lacuna and insertion of a selection marker into the genome by homologous recombination. This is the first example for natural transformation filamentous non-heterocystous cyanobacterium. We found that Phormidium lacuna is polyploid, each cell has about 20-90 chromosomes. Transformed filaments were resistant against up to 14 mg/ml of kanamycin. Formerly, natural transformation in cyanobacteria has been considered a rare and exclusive feature of a few unicellular species. Our finding suggests that natural competence is more distributed among cyanobacteria than previously thought. This is supported by bioinformatic analyses which show that all protein factors for natural transformation are present in the majority of the analyzed cyanobacteria.


Assuntos
Cianobactérias/genética , Farmacorresistência Bacteriana/genética , Genoma Bacteriano/genética , Recombinação Homóloga , Transformação Bacteriana , Cromossomos Bacterianos/genética , Biologia Computacional , Canamicina/farmacologia , Poliploidia
13.
BMC Bioinformatics ; 21(1): 264, 2020 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-32580695

RESUMO

BACKGROUND: Prokaryotes are asexual, but these organisms frequently engage in homologous recombination, a process that differs from meiotic recombination in sexual organisms. Most tools developed to simulate genome evolution either assume sexual reproduction or the complete absence of DNA flux in the population. As a result, very few simulators are adapted to model prokaryotic genome evolution while accounting for recombination. Moreover, many simulators are based on the coalescent, which assumes a neutral model of genomic evolution, and those are best suited for organisms evolving under weak selective pressures, such as animals and plants. In contrast, prokaryotes are thought to be evolving under much stronger selective pressures, suggesting that forward-in-time simulators are better suited for these organisms. RESULTS: Here, I present CoreSimul, a forward-in-time simulator of core genome evolution for prokaryotes modeling homologous recombination. Simulations are guided by a phylogenetic tree and incorporate different substitution models, including models of codon selection. CONCLUSIONS: CoreSimul is a flexible forward-in-time simulator that constitutes a significant addition to the limited list of available simulators applicable to prokaryote genome evolution.


Assuntos
Evolução Molecular , Genoma Bacteriano , Recombinação Homóloga , Modelos Genéticos , Códon , Genoma Arqueal , Genômica , Filogenia , Software
14.
Nat Commun ; 11(1): 3088, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32555206

RESUMO

DNA double-strand break repair by homologous recombination begins with nucleolytic resection of the 5' DNA strand at the break ends. Long-range resection is catalyzed by EXO1 and BLM-DNA2, which likely have to navigate through ribonucleotides and damaged bases. Here, we show that a short stretch of ribonucleotides at the 5' terminus stimulates resection by EXO1. Ribonucleotides within a 5' flap are resistant to cleavage by DNA2, and extended RNA:DNA hybrids inhibit both strand separation by BLM and resection by EXO1. Moreover, 8-oxo-guanine impedes EXO1 but enhances resection by BLM-DNA2, and an apurinic/apyrimidinic site stimulates resection by BLM-DNA2 and DNA strand unwinding by BLM. Accordingly, depletion of OGG1 or APE1 leads to greater dependence of DNA resection on DNA2. Importantly, RNase H2A deficiency impairs resection overall, which we attribute to the accumulation of long RNA:DNA hybrids at DNA ends. Our results help explain why eukaryotic cells possess multiple resection nucleases.


Assuntos
Quebras de DNA de Cadeia Dupla , Ribonucleotídeos/genética , Ribonucleotídeos/metabolismo , Western Blotting , Linhagem Celular Tumoral , DNA Glicosilases/genética , Enzimas Reparadoras do DNA/genética , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Exodesoxirribonucleases/genética , Imunofluorescência , Recombinação Homóloga/genética , Humanos , RecQ Helicases/genética , Recombinação Genética/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo
15.
Nat Commun ; 11(1): 3101, 2020 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-32555348

RESUMO

Orderly chromosome segregation is enabled by crossovers between homologous chromosomes in the first meiotic division. Crossovers arise from recombination-mediated repair of programmed DNA double-strand breaks (DSBs). Multiple DSBs initiate recombination, and most are repaired without crossover formation, although one or more generate crossovers on each chromosome. Although the underlying mechanisms are ill-defined, the differentiation and maturation of crossover-specific recombination intermediates requires the cyclin-like CNTD1. Here, we identify PRR19 as a partner of CNTD1. We find that, like CNTD1, PRR19 is required for timely DSB repair and the formation of crossover-specific recombination complexes. PRR19 and CNTD1 co-localise at crossover sites, physically interact, and are interdependent for accumulation, indicating a PRR19-CNTD1 partnership in crossing over. Further, we show that CNTD1 interacts with a cyclin-dependent kinase, CDK2, which also accumulates in crossover-specific recombination complexes. Thus, the PRR19-CNTD1 complex may enable crossover differentiation by regulating CDK2.


Assuntos
Troca Genética/genética , Ciclinas/genética , Quebras de DNA de Cadeia Dupla , Meiose/genética , Animais , Cromossomos/genética , Quinase 2 Dependente de Ciclina/genética , Dano ao DNA/genética , Reparo do DNA/genética , Feminino , Recombinação Homóloga/genética , Masculino , Camundongos
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.
Nature ; 582(7813): 586-591, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32494005

RESUMO

Deregulation of metabolism and disruption of genome integrity are hallmarks of cancer1. Increased levels of the metabolites 2-hydroxyglutarate, succinate and fumarate occur in human malignancies owing to somatic mutations in the isocitrate dehydrogenase-1 or -2 (IDH1 or IDH2) genes, or germline mutations in the fumarate hydratase (FH) and succinate dehydrogenase genes (SDHA, SDHB, SDHC and SDHD), respectively2-4. Recent work has made an unexpected connection between these metabolites and DNA repair by showing that they suppress the pathway of homology-dependent repair (HDR)5,6 and confer an exquisite sensitivity to inhibitors of poly (ADP-ribose) polymerase (PARP) that are being tested in clinical trials. However, the mechanism by which these oncometabolites inhibit HDR remains poorly understood. Here we determine the pathway by which these metabolites disrupt DNA repair. We show that oncometabolite-induced inhibition of the lysine demethylase KDM4B results in aberrant hypermethylation of histone 3 lysine 9 (H3K9) at loci surrounding DNA breaks, masking a local H3K9 trimethylation signal that is essential for the proper execution of HDR. Consequently, recruitment of TIP60 and ATM, two key proximal HDR factors, is substantially impaired at DNA breaks, with reduced end resection and diminished recruitment of downstream repair factors. These findings provide a mechanistic basis for oncometabolite-induced HDR suppression and may guide effective strategies to exploit these defects for therapeutic gain.


Assuntos
Cromatina/metabolismo , Reparo do DNA , Recombinação Homóloga , Neoplasias/metabolismo , Transdução de Sinais , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Linhagem Celular Tumoral , Cromatina/efeitos dos fármacos , Quebras de DNA/efeitos dos fármacos , Reparo do DNA/efeitos dos fármacos , Recombinação Homóloga/efeitos dos fármacos , Humanos , Histona Desmetilases com o Domínio Jumonji/antagonistas & inibidores , Lisina Acetiltransferase 5/metabolismo , Metilação/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Transdução de Sinais/efeitos dos fármacos
18.
Nat Commun ; 11(1): 2834, 2020 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-32503981

RESUMO

Recruitment of DNA repair proteins to DNA damage sites is a critical step for DNA repair. Post-translational modifications of proteins at DNA damage sites serve as DNA damage codes to recruit specific DNA repair factors. Here, we show that mRNA is locally modified by m5C at sites of DNA damage. The RNA methyltransferase TRDMT1 is recruited to DNA damage sites to promote m5C induction. Loss of TRDMT1 compromises homologous recombination (HR) and increases cellular sensitivity to DNA double-strand breaks (DSBs). In the absence of TRDMT1, RAD51 and RAD52 fail to localize to sites of reactive oxygen species (ROS)-induced DNA damage. In vitro, RAD52 displays an increased affinity for DNA:RNA hybrids containing m5C-modified RNA. Loss of TRDMT1 in cancer cells confers sensitivity to PARP inhibitors in vitro and in vivo. These results reveal an unexpected TRDMT1-m5C axis that promotes HR, suggesting that post-transcriptional modifications of RNA can also serve as DNA damage codes to regulate DNA repair.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Processamento Pós-Transcricional do RNA/genética , RNA Mensageiro/metabolismo , Animais , Linhagem Celular Tumoral , Citosina/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , Resistencia a Medicamentos Antineoplásicos/genética , Técnicas de Silenciamento de Genes , Humanos , Metilação , Camundongos , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/patologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
19.
J Vis Exp ; (159)2020 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-32478746

RESUMO

The M42 aminopeptidases form functionally active complexes made of 12 subunits. Their assembly process appears to be regulated by their metal ion cofactors triggering a dimer-dodecamer transition. Upon metal ion binding, several structural modifications occur in the active site and at the interaction interface, shaping dimers to promote the self-assembly. To observe such modifications, stable oligomers must be isolated prior to structural study. Reported here is a method that allows the purification of stable dodecamers and dimers of TmPep1050, an M42 aminopeptidase of T. maritima, and their structure determination by X-ray crystallography. Dimers were prepared from dodecamers by removing metal ions with a chelating agent. Without their cofactor, dodecamers became less stable and were fully dissociated upon heating. The oligomeric structures were solved by the straightforward molecular replacement approach. To illustrate the methodology, the structure of a TmPep1050 variant, totally impaired in metal ion binding, is presented showing no further breakdown of dimers to monomers.


Assuntos
Aminopeptidases/química , Cristalografia por Raios X , Multimerização Proteica , Thermotoga maritima/enzimologia , Sequência de Aminoácidos , Aminopeptidases/isolamento & purificação , Aminopeptidases/metabolismo , Cromatografia em Gel , Cristalização , Recombinação Homóloga , Modelos Moleculares
20.
Toxicology ; 440: 152441, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32433928

RESUMO

4-Aminobiphenyl (4-ABP), a well-known human carcinogen, has been shown to cause oxidative DNA damage and induce miR-630 expression in HepG2 cells treated with 18.75 µM-300 µM for 24 h. However, the underlying mechanism regarding the epigenetic regulation of miR-630 on DNA damage repair in liver cells is still not understood and needs to be investigated. In present study, our results showed that miR-630 was upregulated, resulting in mediating a decrease of DNA homologous recombination (HR) repair in L-02, HepG2 or Hep3B cells. Results from a luciferase reporting experiment showed that RAD18 and MCM8 were the potential targets of miR-630 during DNA damage induction. The downregulation of RAD18 or MCM8 by miR-630 was accompanied by inhibition of HR repair. Conversely, inhibiting miR-630 enhanced the expression of RAD18 and MCM8, and rescued HR repair. Additionally, we proved that the transcription factor CREB was related to miR-630 biogenesis in liver cells. Moreover, the levels of CREB, miR-630 expression, and double-strand breaks (DSBs) were attenuated by 5 mM N-acetyl-L-cysteine (NAC) pretreatment, indicating that reactive oxygen species (ROS)-dependent CREB-miR-630 was involved in DSB repair. These findings indicated that the ROS/CREB/-miR-630 axis plays a relevant role in the regulation of RAD18 and MCM8 in HR repair, which may facilitate our understanding of molecular mechanisms regarding the role of miR-630 downregulating DNA damage repair in liver cells.


Assuntos
Compostos de Aminobifenil/farmacologia , Proteínas de Ligação a DNA/antagonistas & inibidores , Fígado/metabolismo , MicroRNAs/metabolismo , Proteínas de Manutenção de Minicromossomo/antagonistas & inibidores , Reparo de DNA por Recombinação/efeitos dos fármacos , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Acetilcisteína/farmacologia , Linhagem Celular , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/biossíntese , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Depuradores de Radicais Livres/farmacologia , Recombinação Homóloga , Humanos , Fígado/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo
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