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1.
Nat Commun ; 12(1): 4126, 2021 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-34226554

RESUMO

Double stranded DNA Breaks (DSB) that occur in highly transcribed regions of the genome are preferentially repaired by homologous recombination repair (HR). However, the mechanisms that link transcription with HR are unknown. Here we identify a critical role for DHX9, a RNA helicase involved in the processing of pre-mRNA during transcription, in the initiation of HR. Cells that are deficient in DHX9 are impaired in the recruitment of RPA and RAD51 to sites of DNA damage and fail to repair DSB by HR. Consequently, these cells are hypersensitive to treatment with agents such as camptothecin and Olaparib that block transcription and generate DSB that specifically require HR for their repair. We show that DHX9 plays a critical role in HR by promoting the recruitment of BRCA1 to RNA as part of the RNA Polymerase II transcription complex, where it facilitates the resection of DSB. Moreover, defects in DHX9 also lead to impaired ATR-mediated damage signalling and an inability to restart DNA replication at camptothecin-induced DSB. Together, our data reveal a previously unknown role for DHX9 in the DNA Damage Response that provides a critical link between RNA, RNA Pol II and the repair of DNA damage by homologous recombination.


Assuntos
Proteína BRCA1/metabolismo , RNA Helicases DEAD-box/metabolismo , DNA , Recombinação Homóloga , Proteínas de Neoplasias/metabolismo , RNA , Proteína BRCA1/genética , RNA Helicases DEAD-box/genética , Dano ao DNA , DNA Helicases , Reparo do DNA , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Humanos , Ftalazinas , Piperazinas , RNA Helicases , RNA Mensageiro , Rad51 Recombinase , Reparo de DNA por Recombinação
2.
Nat Commun ; 12(1): 4255, 2021 07 12.
Artigo em Inglês | MEDLINE | ID: mdl-34253720

RESUMO

Homology-directed repair (HDR), a critical DNA repair pathway in mammalian cells, is complex, leading to multiple outcomes with different impacts on genomic integrity. However, the factors that control these different outcomes are often not well understood. Here we show that SWS1-SWSAP1-SPIDR controls distinct types of HDR. Despite their requirement for stable assembly of RAD51 recombinase at DNA damage sites, these proteins are not essential for intra-chromosomal HDR, providing insight into why patients and mice with mutations are viable. However, SWS1-SWSAP1-SPIDR is critical for inter-homolog HDR, the first mitotic factor identified specifically for this function. Furthermore, SWS1-SWSAP1-SPIDR drives the high level of sister-chromatid exchange, promotes long-range loss of heterozygosity often involved with cancer initiation, and impels the poor growth of BLM helicase-deficient cells. The relevance of these genetic interactions is evident as SWSAP1 loss prolongs Blm-mutant embryo survival, suggesting a possible druggable target for the treatment of Bloom syndrome.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga/genética , Complexos Multiproteicos/metabolismo , Animais , Síndrome de Bloom/genética , Síndrome de Bloom/patologia , Proliferação de Células , Células HEK293 , Humanos , Meiose , Camundongos , Mitose , Células-Tronco Embrionárias Murinas/metabolismo , Mutação/genética , Fenótipo , Rad51 Recombinase/metabolismo , Troca de Cromátide Irmã , Análise de Sobrevida
3.
Int J Mol Sci ; 22(13)2021 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209661

RESUMO

Self-incompatibility (SI) is conserved among members of the Brassicaceae plant family. This trait is controlled epigenetically by the dominance hierarchy of the male determinant alleles. We previously demonstrated that a single small RNA (sRNA) gene is sufficient to control the linear dominance hierarchy in Brassica rapa and proposed a model in which a homology-based interaction between sRNAs and target sites controls the complicated dominance hierarchy of male SI determinants. In Arabidopsis halleri, male dominance hierarchy is reported to have arisen from multiple networks of sRNA target gains and losses. Despite these findings, it remains unknown whether the molecular mechanism underlying the dominance hierarchy is conserved among Brassicaceae. Here, we identified sRNAs and their target sites that can explain the linear dominance hierarchy of Arabidopsis lyrata, a species closely related to A. halleri. We tested the model that we established in Brassica to explain the linear dominance hierarchy in A. lyrata. Our results suggest that the dominance hierarchy of A. lyrata is also controlled by a homology-based interaction between sRNAs and their targets.


Assuntos
Arabidopsis/genética , Epistasia Genética , Regulação da Expressão Gênica de Plantas , Genes Dominantes , Recombinação Homóloga , RNA de Plantas , Predomínio Social , Alelos , Genótipo , Haplótipos , Conformação de Ácido Nucleico
4.
Int J Mol Sci ; 22(11)2021 May 27.
Artigo em Inglês | MEDLINE | ID: mdl-34071949

RESUMO

Exposing cells to DNA damaging agents, such as ionizing radiation (IR) or cytotoxic chemicals, can cause DNA double-strand breaks (DSBs), which are crucial to repair to maintain genetic integrity. O-linked ß-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification (PTM), which has been reported to be involved in the DNA damage response (DDR) and chromatin remodeling. Here, we investigated the impact of O-GlcNAcylation on the DDR, DSB repair and chromatin status in more detail. We also applied charged particle irradiation to analyze differences of O-GlcNAcylation and its impact on DSB repair in respect of spatial dose deposition and radiation quality. Various techniques were used, such as the γH2AX foci assay, live cell microscopy and Fluorescence Lifetime Microscopy (FLIM) to detect DSB rejoining, protein accumulation and chromatin states after treating the cells with O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) inhibitors. We confirmed that O-GlcNAcylation of MDC1 is increased upon irradiation and identified additional repair factors related to Homologous Recombination (HR), CtIP and BRCA1, which were increasingly O-GlcNAcyated upon irradiation. This is consistent with our findings that the function of HR is affected by OGT inhibition. Besides, we found that OGT and OGA activity modulate chromatin compaction states, providing a potential additional level of DNA-repair regulation.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Cromatina/genética , Cromatina/metabolismo , Montagem e Desmontagem da Cromatina/genética , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Proteínas de Ligação a DNA/metabolismo , Células HeLa , Recombinação Homóloga , Humanos , Transferência Linear de Energia , N-Acetilglucosaminiltransferases/metabolismo , Processamento de Proteína Pós-Traducional , Radiação Ionizante
5.
Cell Mol Life Sci ; 78(14): 5587-5604, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34100981

RESUMO

To clarify whether differential compartmentalization of Survivin impacts temozolomide (TMZ)-triggered end points, we established a well-defined glioblastoma cell model in vitro (LN229 and A172) and in vivo, distinguishing between its nuclear and cytoplasmic localization. Expression of nuclear export sequence (NES)-mutated Survivin (SurvNESmut-GFP) led to impaired colony formation upon TMZ. This was not due to enhanced cell death but rather due to increased senescence. Nuclear-trapped Survivin reduced homologous recombination (HR)-mediated double-strand break (DSB) repair, as evaluated by γH2AX foci formation and qPCR-based HR assay leading to pronounced induction of chromosome aberrations. Opposite, clones, expressing free-shuttling cytoplasmic but not nuclear-trapped Survivin, could repair TMZ-induced DSBs and evaded senescence. Mass spectrometry-based interactomics revealed, however, no direct interaction of Survivin with any of the repair factors. The improved TMZ-triggered HR activity in Surv-GFP was associated with enhanced mRNA and stabilized RAD51 protein expression, opposite to diminished RAD51 expression in SurvNESmut cells. Notably, cytoplasmic Survivin could significantly compensate for the viability under RAD51 knockdown. Differential Survivin localization also resulted in distinctive TMZ-triggered transcriptional pathways, associated with senescence and chromosome instability as shown by global transcriptome analysis. Orthotopic LN229 xenografts, expressing SurvNESmut exhibited diminished growth and increased DNA damage upon TMZ, as manifested by PCNA and γH2AX foci expression, respectively, in brain tissue sections. Consequently, those mice lived longer. Although tumors of high-grade glioma patients expressed majorly nuclear Survivin, they exhibited rarely NES mutations which did not correlate with survival. Based on our in vitro and xenograft data, Survivin nuclear trapping would facilitate glioma response to TMZ.


Assuntos
Núcleo Celular/metabolismo , Senescência Celular , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/tratamento farmacológico , Recombinação Homóloga , Survivina/metabolismo , Temozolomida/farmacologia , Animais , Antineoplásicos Alquilantes/farmacologia , Apoptose , Biomarcadores Tumorais , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patologia , Núcleo Celular/genética , Proliferação de Células , Dano ao DNA , Reparo do DNA , Feminino , Regulação Neoplásica da Expressão Gênica , Glioblastoma/metabolismo , Glioblastoma/patologia , Humanos , Camundongos , Camundongos Nus , Survivina/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Nat Commun ; 12(1): 3636, 2021 06 17.
Artigo em Inglês | MEDLINE | ID: mdl-34140467

RESUMO

To identify approaches to target DNA repair vulnerabilities in cancer, we discovered nanomolar potent, selective, low molecular weight (MW), allosteric inhibitors of the polymerase function of DNA polymerase Polθ, including ART558. ART558 inhibits the major Polθ-mediated DNA repair process, Theta-Mediated End Joining, without targeting Non-Homologous End Joining. In addition, ART558 elicits DNA damage and synthetic lethality in BRCA1- or BRCA2-mutant tumour cells and enhances the effects of a PARP inhibitor. Genetic perturbation screening revealed that defects in the 53BP1/Shieldin complex, which cause PARP inhibitor resistance, result in in vitro and in vivo sensitivity to small molecule Polθ polymerase inhibitors. Mechanistically, ART558 increases biomarkers of single-stranded DNA and synthetic lethality in 53BP1-defective cells whilst the inhibition of DNA nucleases that promote end-resection reversed these effects, implicating these in the synthetic lethal mechanism-of-action. Taken together, these observations describe a drug class that elicits BRCA-gene synthetic lethality and PARP inhibitor synergy, as well as targeting a biomarker-defined mechanism of PARPi-resistance.


Assuntos
Proteína BRCA1/genética , Proteína BRCA2/genética , Reparo do DNA/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Inibidores da Síntese de Ácido Nucleico/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Mutações Sintéticas Letais/efeitos dos fármacos , Regulação Alostérica , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos da radiação , Dano ao DNA/efeitos dos fármacos , Proteínas de Ligação a DNA/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Desoxirribonucleases/antagonistas & inibidores , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Recombinação Homóloga/efeitos dos fármacos , Humanos , Concentração Inibidora 50 , Camundongos , Organoides/efeitos dos fármacos , Neoplasias Ovarianas/genética , Ratos , Mutações Sintéticas Letais/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/deficiência , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
7.
Pathologe ; 42(4): 391-398, 2021 Jul.
Artigo em Alemão | MEDLINE | ID: mdl-34152463

RESUMO

BACKGROUND: DNA double-strand breaks may evoke cell death or cancer. Cells have developed two fundamentally different mechanisms of DNA double-strand repair: the accurate mechanism of homologous recombination repair and the error-prone nonhomologous end joining. The deficiency of homologous recombination repair (HRD) is a frequent feature of several solid tumor entities and is associated with sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitor therapy. OBJECTIVES: Among other biomarkers, HRD testing provides an opportunity to guide PARP inhibitor therapy in solid tumors, but the basic principles are complex and the use and benefits of the different methodologies remain controversial. Knowledge of the underlying mechanisms at the molecular level will advance our understanding and will pave the way to introduce the testing of new biomarkers. METHODS: An overview of the fundamental mechanisms of DNA repair is provided. Important terms like HRR, HRD, and BRCAness are defined, and analysis methods are described, especially with regard to their integration in molecular pathology routine diagnostics. RESULTS: Currently, at least testing of the BRCA mutation status and genomic instability using a composite HRD score should be implemented in laboratories to identify subgroups of patients who might benefit from PARP inhibitor therapies. A broad range of testing methods is available with pros and cons for introduction in the clinical setting. They have to be validated carefully to reliably inform treatment selection. CONCLUSIONS: Biomarkers to identify current homologous recombination deficiency status are needed to predict the benefit from PARP inhibitors and stratify their use in clinical management. Besides commercial assays, different tests might be used for the analysis of HRD. The application depends, among other things, on the local situation and has to be extensively validated.


Assuntos
Neoplasias Ovarianas , Reparo de DNA por Recombinação , Biomarcadores , Reparo do DNA , Feminino , Recombinação Homóloga , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/uso terapêutico
8.
J Vis Exp ; (170)2021 04 28.
Artigo em Inglês | MEDLINE | ID: mdl-33999036

RESUMO

The study of the DNA damage response (DDR) is a complex and essential field, which has only become more important due to the use of DDR-targeting drugs for cancer treatment. These targets are poly(ADP-ribose) polymerases (PARPs), which initiate various forms of DNA repair. Inhibiting these enzymes using PARP inhibitors (PARPi) achieves synthetic lethality by conferring a therapeutic vulnerability in homologous recombination (HR)-deficient cells due to mutations in breast cancer type 1 (BRCA1), BRCA2, or partner and localizer of BRCA2 (PALB2). Cells treated with PARPi accumulate DNA double-strand breaks (DSBs). These breaks are processed by the DNA end resection machinery, leading to the formation of single-stranded (ss) DNA and subsequent DNA repair. In a BRCA1-deficient context, reinvigorating DNA resection through mutations in DNA resection inhibitors, such as 53BP1 and DYNLL1, causes PARPi resistance. Therefore, being able to monitor DNA resection in cellulo is critical for a clearer understanding of the DNA repair pathways and the development of new strategies to overcome PARPi resistance. Immunofluorescence (IF)-based techniques allow for monitoring of global DNA resection after DNA damage. This strategy requires long-pulse genomic DNA labeling with 5-bromo-2'-deoxyuridine (BrdU). Following DNA damage and DNA end resection, the resulting single-stranded DNA is specifically detected by an anti-BrdU antibody under native conditions. Moreover, DNA resection can also be studied using cell cycle markers to differentiate between various phases of the cell cycle. Cells in the S/G2 phase allow the study of end resection within HR, whereas G1 cells can be used to study non-homologous end joining (NHEJ). A detailed protocol for this IF method coupled to cell cycle discrimination is described in this paper.


Assuntos
Bromodesoxiuridina/química , Ciclo Celular , DNA/genética , Ciclo Celular/efeitos dos fármacos , DNA/análise , DNA/química , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Recombinação Homóloga , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia
9.
Methods Mol Biol ; 2296: 303-330, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33977456

RESUMO

Streptomyces rimosus is used for production of the broad-spectrum antibiotic oxytetracycline (OTC). S. rimosus belongs to Actinomyces species, a large group of microorganisms that produce diverse set of natural metabolites of high importance in many aspects of our life. In this chapter, we describe specific molecular biology methods and a classical homologous recombination approach for targeted in-frame deletion of a target gene or entire operon in S. rimosus genome. The presented protocols will guide you through the design of experiment and construction of homology arms and their cloning into appropriate vectors, which are suitable for gene-engineering work with S. rimosus. Furthermore, two different protocols for S. rimosus transformation are described including detailed procedure for targeted gene replacement via double crossover recombination event. Gene deletion is confirmed by colony PCR, and colonies are further characterized by cultivation and metabolite analysis. As the final step, we present in trans complementation of the deleted gene, to confirm functionality of the engineering approach achieved by gene disruption. A number of methodological steps and protocols are optimized for S. rimosus strains including the use of the selected reporter genes. Protocols described in this chapter can be applied for studying function of any individual gene product in diverse OTC-producing Streptomyces rimosus strains.


Assuntos
Oxitetraciclina/biossíntese , Streptomyces rimosus/genética , Streptomyces rimosus/metabolismo , Antibacterianos/biossíntese , Clonagem Molecular/métodos , Deleção de Genes , Genoma Bacteriano/genética , Recombinação Homóloga/genética , Biologia Molecular
10.
Am J Hum Genet ; 108(6): 1026-1039, 2021 06 03.
Artigo em Inglês | MEDLINE | ID: mdl-34004138

RESUMO

Revertant mosaicism, or "natural gene therapy," refers to the spontaneous in vivo reversion of an inherited mutation in a somatic cell. Only approximately 50 human genetic disorders exhibit revertant mosaicism, implicating a distinctive role played by mutant proteins in somatic correction of a pathogenic germline mutation. However, the process by which mutant proteins induce somatic genetic reversion in these diseases remains unknown. Here we show that heterozygous pathogenic CARD14 mutations causing autoinflammatory skin diseases, including psoriasis and pityriasis rubra pilaris, are repaired mainly via homologous recombination. Rather than altering the DNA damage response to exogenous stimuli, such as X-irradiation or etoposide treatment, mutant CARD14 increased DNA double-strand breaks under conditions of replication stress. Furthermore, mutant CARD14 suppressed new origin firings without promoting crossover events in the replication stress state. Together, these results suggest that mutant CARD14 alters the replication stress response and preferentially drives break-induced replication (BIR), which is generally suppressed in eukaryotes. Our results highlight the involvement of BIR in reversion events, thus revealing a previously undescribed role of BIR that could potentially be exploited to develop therapeutics for currently intractable genetic diseases.


Assuntos
Proteínas Adaptadoras de Sinalização CARD/genética , Replicação do DNA , Guanilato Ciclase/genética , Recombinação Homóloga , Proteínas de Membrana/genética , Mosaicismo , Mutação , Pitiríase Rubra Pilar/patologia , Psoríase/patologia , Estresse Fisiológico , Ciclo Celular , Humanos , Pitiríase Rubra Pilar/genética , Psoríase/genética
11.
Nucleic Acids Res ; 49(10): 5588-5604, 2021 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-33978741

RESUMO

Cancer-causing missense mutations in the 3418 amino acid BRCA2 breast and ovarian cancer suppressor protein frequently affect a short (∼340 residue) segment in its carboxyl-terminal domain (DBD). Here, we identify a shared molecular mechanism underlying their pathogenicity. Pathogenic BRCA2 missense mutations cluster in the DBD's helical domain (HD) and OB1-fold motifs, which engage the partner protein DSS1. Pathogenic - but not benign - DBD mutations weaken or abolish DSS1-BRCA2 assembly, provoking mutant BRCA2 oligomers that are excluded from the cell nucleus, and disable DNA repair by homologous DNA recombination (HDR). DSS1 inhibits the intracellular oligomerization of wildtype, but not mutant, forms of BRCA2. Remarkably, DSS1 expression corrects defective HDR in cells bearing pathogenic BRCA2 missense mutants with weakened, but not absent, DSS1 binding. Our findings identify a DSS1-mediated intracellular protein assembly mechanism that is disrupted by cancer-causing BRCA2 missense mutations, and suggest an approach for its therapeutic correction.


Assuntos
Proteína BRCA2 , Neoplasias da Mama/genética , Reparo do DNA , Neoplasias Ovarianas/genética , Complexo de Endopeptidases do Proteassoma/fisiologia , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Feminino , Células HEK293 , Células HeLa , Recombinação Homóloga , Humanos , Mutação de Sentido Incorreto , Ligação Proteica
12.
Aging (Albany NY) ; 13(10): 14277-14288, 2021 05 19.
Artigo em Inglês | MEDLINE | ID: mdl-34016790

RESUMO

UCHL3 belongs to the UCH family and is involved in multiple biological processes. However, the biological functions and underlying mechanisms of action of UCHL3 in radio-sensitivity of non-small cell lung cancer (NSCLC) remain unknown. Here, we reported that the expression of UCHL3 was significantly up-regulated in NSCLC tissues and cell lines, and associated with poor prognosis of NSCLC patients. The expression of UCHL3 of NSCLC cells was increased after exposure to ionizing radiation (IR). Moreover, we found that knockdown of UCHL3 enhanced the radio-sensitivity of NSCLC cells both in vitro and in vivo. Furthermore, γH2AX foci staining and Western blot analysis showed that knockdown of UCHL3 increased IR-induced DNA damage. Knockdown of UCHL3 in NSCLC cells decreased homologous recombination (HR) repair efficiency and RAD51 foci formation. Collectively, our study revealed that knockdown of UCHL3 enhanced the radio-sensitivity of NSCLC cells and increased IR-induced DNA damage via impairing HR repair.


Assuntos
Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/radioterapia , Reparo do DNA/genética , Inativação Gênica , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/radioterapia , Tolerância a Radiação/genética , Ubiquitina Tiolesterase/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/genética , Proliferação de Células/efeitos da radiação , Dano ao DNA/genética , Regulação Neoplásica da Expressão Gênica , Recombinação Homóloga/genética , Humanos , Camundongos Nus , Prognóstico , Radiação Ionizante , Ubiquitina Tiolesterase/metabolismo , Regulação para Cima/genética
13.
BMC Plant Biol ; 21(1): 167, 2021 Apr 06.
Artigo em Inglês | MEDLINE | ID: mdl-33823797

RESUMO

BACKGROUND: DNA sequence composition affects meiotic recombination. However, the correlation between tandem repeat composition and meiotic recombination in common wheat (Triticum aestivum L.) is unclear. RESULTS: Non-denaturing fluorescent in situ hybridization (ND-FISH) with oligonucleotide (oligo) probes derived from tandem repeats and single-copy FISH were used to investigate recombination in three kinds of the long arm of wheat 5A chromosome (5AL). 5AL535-18/275 arm carries the tandem repeats pTa-535, Oligo-18, and pTa-275, 5AL119.2-18/275 arm carries the tandem repeats pSc119.2, Oligo-18 and pTa-275, and 5AL119.2 arm carries the tandem repeats pSc119.2. In the progeny of 5AL535-18/275 × 5AL119.2, double recombination occurred between pSc119.2 and pTa-535 clusters (119-535 interval), and between pTa-535 and Oligo-18/pTa-275 clusters (535-18 interval). The recombination rate in the 119-535 interval in the progeny of 5AL535-18/275 × 5AL119.2-18/275 was higher than that in the progeny of 5AL535-18/275 × 5AL119.2. Recombination in the 119-535 interval produced 5AL119 + 535 segments with pTa-535 and pSc119.2 tandem repeats and 5ALNo segments without these repeats. The 5AL119 + 535 and 5ALNo segments were localized between the signal sites of the single-copy probes SC5A-479 and SC5A-527. The segment between SC5A-479 and SC5A-527 in the metaphase 5ALNo was significantly longer than that in the metaphase 5AL119 + 535. CONCLUSION: The structural variations caused by tandem repeats might be one of the factors affecting meiotic recombination in wheat. Meiotic recombination aggregated two kinds of tandemly repeated clusters into the same chromosome, making the metaphase chromosome more condensed. To conclude, our study provides a robust tool to measure meiotic recombination and select parents for wheat breeding programs.


Assuntos
Cromossomos de Plantas , Recombinação Homóloga , Meiose , Triticum/genética , Hibridização in Situ Fluorescente , Sondas de Oligonucleotídeos
14.
Int J Mol Sci ; 22(9)2021 Apr 23.
Artigo em Inglês | MEDLINE | ID: mdl-33922657

RESUMO

BRCA mutation, one of the most common types of mutations in breast and ovarian cancer, has been suggested to be synthetically lethal with depletion of RAD52. Pharmacologically inhibiting RAD52 specifically eradicates BRCA-deficient cancer cells. In this study, we demonstrated that curcumin, a plant polyphenol, sensitizes BRCA2-deficient cells to CPT-11 by impairing RAD52 recombinase in MCF7 cells. More specifically, in MCF7-siBRCA2 cells, curcumin reduced homologous recombination, resulting in tumor growth suppression. Furthermore, a BRCA2-deficient cell line, Capan1, became resistant to CPT-11 when BRCA2 was reintroduced. In vivo, xenograft model studies showed that curcumin combined with CPT-11 reduced the growth of BRCA2-knockout MCF7 tumors but not MCF7 tumors. In conclusion, our data indicate that curcumin, which has RAD52 inhibitor activity, is a promising candidate for sensitizing BRCA2-deficient cells to DNA damage-based cancer therapies.


Assuntos
Proteína BRCA2/deficiência , Neoplasias da Mama/tratamento farmacológico , Curcumina/farmacologia , Dano ao DNA , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Recombinação Homóloga , Proteína Rad52 de Recombinação e Reparo de DNA/antagonistas & inibidores , Animais , Antineoplásicos/farmacologia , Apoptose , Proteína BRCA2/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Proliferação de Células , Reparo do DNA , Feminino , Humanos , Irinotecano/farmacologia , Camundongos , Camundongos Nus , Mutação , Inibidores da Topoisomerase I/farmacologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Int J Mol Sci ; 22(8)2021 Apr 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916959

RESUMO

The recessive form of dystrophic epidermolysis bullosa (RDEB) is a crippling disease caused by impairments in the junctions of the dermis and the basement membrane of the epidermis. Using ectopic expression of hTERT/hTERT + BMI-1 in primary cells, we developed expansible cultures of RDEB fibroblasts and keratinocytes. We showed that they display the properties of their founders, including morphology, contraction ability and expression of the respective specific markers including reduced secretion of type VII collagen (C7). The immortalized keratinocytes retained normal stratification in 3D skin equivalents. The comparison of secreted protein patterns from immortalized RDEB and healthy keratinocytes revealed the differences in the contents of the extracellular matrix that were earlier observed specifically for RDEB. We demonstrated the possibility to reverse the genotype of immortalized cells to the state closer to the progenitors by the Cre-dependent hTERT switch off. Increased ß-galactosidase activity and reduced proliferation of fibroblasts were shown after splitting out of transgenes. We anticipate our cell lines to be tractable models for studying RDEB from the level of single-cell changes to the evaluation of 3D skin equivalents. Our approach permits the creation of standardized and expandable models of RDEB that can be compared with the models based on primary cell cultures.


Assuntos
Fibroblastos/metabolismo , Recombinação Homóloga , Integrases/metabolismo , Queratinócitos/metabolismo , Telomerase/genética , Transgenes , Adolescente , Adulto , Biomarcadores , Linhagem Celular Transformada , Proliferação de Células , Senescência Celular/genética , Criança , Epidermólise Bolhosa Distrófica/etiologia , Epidermólise Bolhosa Distrófica/metabolismo , Feminino , Fibroblastos/patologia , Imunofluorescência , Técnicas de Silenciamento de Genes , Ordem dos Genes , Vetores Genéticos/genética , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Mutação , Complexo Repressor Polycomb 1/genética , Complexo Repressor Polycomb 1/metabolismo , Cultura Primária de Células , Proteômica/métodos , Telomerase/metabolismo , Adulto Jovem
17.
Methods Mol Biol ; 2281: 135-149, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847956

RESUMO

Bacterial RecA and eukaryotic Rad51 are recombinases indispensable for DNA homologous recombination and repair of double-stranded DNA breaks. Understanding the functions and biophysical properties of the DNA recombinases benefits the research in human medicine such as cancer biology. Single-molecule techniques provide the mechanistic details of complex biological reactions. Tethered particle motion (TPM) experiment is a simple and multiplex single-molecule tool to monitor DNA-protein interactions. We have developed a single-molecule TPM assay to study DNA recombinase filament assembly and disassembly on individual DNA molecules in real time. Characterization of the temporal change of the Brownian motion of DNA tethers during recombinase assembly and disassembly in real time allows the determination of multiple kinetic parameters of nucleation rate, extension rate, dissociation rate, and length of the recombinase-DNA filament.


Assuntos
DNA/metabolismo , Recombinases/metabolismo , Imagem Individual de Molécula/métodos , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga
18.
Methods Mol Biol ; 2281: 193-207, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33847959

RESUMO

Homologous recombination (HR) is a highly conserved DNA repair pathway required for the accurate repair of DNA double-stranded breaks. DNA recombination is catalyzed by the RecA/Rad51 family of proteins, which are conserved from bacteria to humans. The key intermediate catalyzing DNA recombination is the presynaptic complex (PSC), which is a helical filament comprised of Rad51-bound single-stranded DNA (ssDNA). Multiple cellular factors either promote or downregulate PSC activity, and a fine balance between such regulators is required for the proper regulation of HR and maintenance of genomic integrity. However, dissecting the complex mechanisms regulating PSC activity has been a challenge using traditional ensemble methods due to the transient and dynamic nature of recombination intermediates. We have developed a single-molecule assay called ssDNA curtains that allows us to visualize individual DNA intermediates in real-time, using total internal reflection microscopy (TIRFM). This assay has allowed us to study many aspects of HR regulation that involve complex and heterogenous reaction intermediates. Here we describe the procedure for a basic ssDNA curtain assay to study PSC filament dynamics, and explain how to process and analyze the resulting data.


Assuntos
DNA de Cadeia Simples/metabolismo , Rad51 Recombinase/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula/métodos , DNA Fúngico/metabolismo , Regulação da Expressão Gênica , Recombinação Homóloga , Microscopia de Interferência , Proteínas de Saccharomyces cerevisiae/genética
19.
Biotechnol Adv ; 49: 107748, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-33823269

RESUMO

Rhodococcus spp. are a group of non-model gram-positive bacteria with diverse catabolic activities and strong adaptive capabilities, which enable their wide application in whole-cell biocatalysis, environmental bioremediation, and lignocellulosic biomass conversion. Compared with model microorganisms, the engineering of Rhodococcus is challenging because of the lack of universal molecular tools, high genome GC content (61% ~ 71%), and low transformation and recombination efficiencies. Nevertheless, because of the high interest in Rhodococcus species for bioproduction, various genetic elements and engineering tools have been recently developed for Rhodococcus spp., including R. opacus, R. jostii, R. ruber, and R. erythropolis, leading to the expansion of the genetic toolkits for Rhodococcus engineering. In this article, we provide a comprehensive review of the important developed genetic elements for Rhodococcus, including shuttle vectors, promoters, antibiotic markers, ribosome binding sites, and reporter genes. In addition, we also summarize gene transfer techniques and strategies to improve transformation efficiency, as well as random and precise genome editing tools available for Rhodococcus, including transposition, homologous recombination, recombineering, and CRISPR/Cas9. We conclude by discussing future trends in Rhodococcus engineering. We expect that more synthetic and systems biology tools (such as multiplex genome editing, dynamic regulation, and genome-scale metabolic models) will be adapted and optimized for Rhodococcus.


Assuntos
Rhodococcus , Sistemas CRISPR-Cas , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Edição de Genes , Engenharia Genética , Recombinação Homóloga , Regiões Promotoras Genéticas , Rhodococcus/genética
20.
Stem Cell Res ; 53: 102332, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-33857832

RESUMO

When studying patient specific induced pluripotent stem cells (iPS cells) as a disease model, the ideal control is an isogenic line that has corrected the point mutation, instead of iPS cells from siblings or other healthy subjects. However, repairing a point mutation in iPS cells even with the newly developed CRISPR-Cas9 technique remains difficult and time-consuming. Here we report a strategy that makes the Cas9 "knock-in" methodology both hassle-free and error-free. Instead of selecting a Cas9 recognition site close to the point mutation, we chose a site located in the nearest intron. We constructed a donor template with the fragment containing the corrected point mutation as one of the homologous recombination arms flanking a PGK-PuroR cassette. After selection with puromycin, positive clones were identified and further transfected with a CRE vector to remove the PGK-PuroR cassette. Using this methodology, we successfully repaired the point mutation G2019S of the LRRK2 gene in a Parkinson Disease (PD) patient iPS line and the point mutation R329H of the AARS1 gene in a Charcot-Marie-Tooth disease (CMT) patient iPS line. These isogenic iPS lines are ideal as a control in future studies.


Assuntos
Células-Tronco Pluripotentes Induzidas , Sistemas CRISPR-Cas/genética , Células Clonais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Recombinação Homóloga , Humanos , Mutação , Mutação Puntual
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