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1.
Nature ; 571(7766): 521-527, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31270457

RESUMO

The integrity of genomes is constantly threatened by problems encountered by the replication fork. BRCA1, BRCA2 and a subset of Fanconi anaemia proteins protect stalled replication forks from degradation by nucleases, through pathways that involve RAD51. The contribution and regulation of BRCA1 in replication fork protection, and how this role relates to its role in homologous recombination, is unclear. Here we show that BRCA1 in complex with BARD1, and not the canonical BRCA1-PALB2 interaction, is required for fork protection. BRCA1-BARD1 is regulated by a conformational change mediated by the phosphorylation-directed prolyl isomerase PIN1. PIN1 activity enhances BRCA1-BARD1 interaction with RAD51, thereby increasing the presence of RAD51 at stalled replication structures. We identify genetic variants of BRCA1-BARD1 in patients with cancer that exhibit poor protection of nascent strands but retain homologous recombination proficiency, thus defining domains of BRCA1-BARD1 that are required for fork protection and associated with cancer development. Together, these findings reveal a BRCA1-mediated pathway that governs replication fork protection.


Assuntos
Proteína BRCA1/química , Proteína BRCA1/metabolismo , Replicação do DNA , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteína BRCA1/genética , Linhagem Celular Tumoral , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Isomerismo , Mutação , Peptidilprolil Isomerase de Interação com NIMA/metabolismo , Fosforilação , Fosfosserina/metabolismo , Ligação Proteica , Rad51 Recombinase/metabolismo
2.
Cell Mol Biol Lett ; 24: 42, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31236120

RESUMO

Human bronchial epithelium (HBE)-Dp71 anti-sense(AS)cells with stably transfected Dp71 siRNA plasmids were prepared for further exploration of Dp71 biological traits in cells other than PC12. HBE-Dp71AS cells displayed increased DNA damage induced by H2O2. Apoptosis of HBE-Dp71AS cells induced by H2O2 was increased via enhancing caspase 3, caspase 8 and caspase 9. HBE-Dp71AS cells also displayed decreased proliferation and clonogenic formation. RAD51 was proved to be a new binding partner of Dp71 by co-immunoprecipitation (Ip) and immunofluorescence. Reduced RAD51 mRNA and protein levels were observed in HBE-Dp71AS cells. Decreased lamin B1, focal adhesion kinase (FAK), phosphorylated focal adhesion kinase (p-FAK) and phosphorylated protein kinase B (p-AKT) were detected in the HBE-Dp71AS cells, which functioned together with RAD51 as the molecular explanations for the character alterations of HBE-Dp71AS cells.


Assuntos
Apoptose , Dano ao DNA , Distrofina/metabolismo , Peróxido de Hidrogênio/toxicidade , Estresse Oxidativo , Rad51 Recombinase/genética , Linhagem Celular , DNA/efeitos dos fármacos , DNA/metabolismo , Reparo do DNA , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Quinase 1 de Adesão Focal/genética , Quinase 1 de Adesão Focal/metabolismo , Regulação da Expressão Gênica , Humanos , Peróxido de Hidrogênio/farmacologia , Lamina Tipo B/genética , Fosforilação , Processamento de Proteína Pós-Traducional , Rad51 Recombinase/metabolismo
3.
Nat Commun ; 10(1): 2135, 2019 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-31086179

RESUMO

The exosome is a ribonucleolytic complex that plays important roles in RNA metabolism. Here we show that the exosome is necessary for the repair of DNA double-strand breaks (DSBs) in human cells and that RNA clearance is an essential step in homologous recombination. Transcription of DSB-flanking sequences results in the production of damage-induced long non-coding RNAs (dilncRNAs) that engage in DNA-RNA hybrid formation. Depletion of EXOSC10, an exosome catalytic subunit, leads to increased dilncRNA and DNA-RNA hybrid levels. Moreover, the targeting of the ssDNA-binding protein RPA to sites of DNA damage is impaired whereas DNA end resection is hyper-stimulated in EXOSC10-depleted cells. The DNA end resection deregulation is abolished by transcription inhibitors, and RNase H1 overexpression restores the RPA recruitment defect caused by EXOSC10 depletion, which suggests that RNA clearance of newly synthesized dilncRNAs is required for RPA recruitment, controlled DNA end resection and assembly of the homologous recombination machinery.


Assuntos
Quebras de DNA de Cadeia Dupla , Exorribonucleases/metabolismo , Complexo Multienzimático de Ribonucleases do Exossomo/metabolismo , Recombinação Homóloga , Proteína de Replicação A/metabolismo , DNA/genética , Exorribonucleases/genética , Complexo Multienzimático de Ribonucleases do Exossomo/genética , Exossomos/metabolismo , Técnicas de Silenciamento de Genes , Células HeLa , Humanos , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/metabolismo , Ribonuclease H/metabolismo
4.
Nat Commun ; 10(1): 2212, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101808

RESUMO

In mammalian cells, double-stranded DNA breaks (DSBs) are preferentially repaired through end-joining processes that generally lead to mixtures of insertions and deletions (indels) or other rearrangements at the cleavage site. In the presence of homologous DNA, homology-directed repair (HDR) can generate specific mutations, albeit typically with modest efficiency and a low ratio of HDR products:indels. Here, we develop hRad51 mutants fused to Cas9(D10A) nickase (RDN) that mediate HDR while minimizing indels. We use RDN to install disease-associated point mutations in HEK293T cells with comparable or better efficiency than Cas9 nuclease and a 2.7-to-53-fold higher ratio of desired HDR product:undesired byproducts. Across five different human cell types, RDN variants generally result in higher HDR:indel ratios and lower off-target activity than Cas9 nuclease, although HDR efficiencies remain strongly site- and cell type-dependent. RDN variants provide precision editing options in cell types amenable to HDR, especially when byproducts of DSBs must be minimized.


Assuntos
Proteína 9 Associada à CRISPR/metabolismo , Engenharia Genética/métodos , Rad51 Recombinase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Reparo de DNA por Recombinação , Proteína 9 Associada à CRISPR/genética , Quebras de DNA de Cadeia Dupla , Edição de Genes/métodos , Células HEK293 , Células HeLa , Humanos , Células-Tronco Pluripotentes Induzidas , Células K562 , Rad51 Recombinase/genética , Proteínas Recombinantes de Fusão/genética , Transfecção/métodos
5.
Genes Cells ; 24(5): 377-389, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-30929290

RESUMO

In Caenorhabditis elegans, germline cells remain transcriptionally silenced during embryogenesis. The transcriptional silencing is achieved by two different mechanisms: One is the inhibition of RNA polymerase II in P2-P4 cells at the establishment stage, and another is chromatin-based silencing in two primordial germ cells (PGCs) at the maintenance stage; however, the molecular mechanism underlying chromatin-based silencing is less understood. We investigated the role of the chromodomain protein MRG-1, which is an essential maternal factor for germline development, in transcriptional silencing in PGCs. PGCs lacking maternal MRG-1 showed increased levels of two histone modifications (H3K4me2 and H4K16ac), which are epigenetic markers for active transcription, and precocious activation of germline promoters. Loss of MES-4, a H3K36 methyltransferase, also caused similar derepression of the germline genes in PGCs, suggesting that both MRG-1 and MES-4 function in chromatin-based silencing in PGCs. In addition, the mrg-1 null mutant showed abnormal chromosome structures and a decrease in homologous recombinase RAD-51 foci in PGCs, but the mes-4 null mutant did not show such phenotypes. Taken together, we propose that MRG-1 has two distinct functions: chromatin-based transcriptional silencing and preserving genomic integrity at the maintenance stage of PGCs.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Cromatina/genética , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Células Germinativas/metabolismo , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Cromatina/metabolismo , Instabilidade Genômica , Células Germinativas/citologia , Código das Histonas , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo
6.
mSphere ; 4(2)2019 03 20.
Artigo em Inglês | MEDLINE | ID: mdl-30894431

RESUMO

DNA damage-induced Rad51 focus formation is the hallmark of homologous recombination-mediated DNA repair. Earlier, we reported that Rad51 physically interacts with Hsp90, and under the condition of Hsp90 inhibition, it undergoes proteasomal degradation. Here, we show that the dynamic interaction between Rad51 and Hsp90 is crucial for the DNA damage-induced nuclear function of Rad51. Guided by a bioinformatics study, we generated a single mutant of Rad51, which resides at the N-terminal domain, outside the ATPase core domain. The mutant with an E to L change at residue 108 (Rad51E108L) was predicted to bind more strongly with Hsp90 than the wild-type (Rad51WT). A coimmunoprecipitation study demonstrated that there exists a distinct difference between the in vivo associations of Rad51WT-Hsp90 and of Rad51E108L-Hsp90. We found that upon DNA damage, the association between Rad51WT and Hsp90 was significantly reduced compared to that in the undamaged condition. However, the mutant Rad51E108L remained tightly associated with Hsp90 even after DNA damage. Consequently, the recruitment of Rad51E108L to the double-stranded broken ends was reduced significantly. The E108L-rad51 strain manifested severe sensitivity toward methyl methanesulfonate (MMS) and a complete loss of gene conversion efficiency, a phenotype similar to that of the Δrad51 strain. Previously, some of the N-terminal domain mutants of Rad51 were identified in a screen for a Rad51 interaction-deficient mutant; however, our study shows that Rad51E108L is not defective either in the self-interaction or its interaction with the members of the Rad52 epistatic group. Our study thus identifies a novel mutant of Rad51 which, owing to its greater association with Hsp90, exhibits a severe DNA repair defect.IMPORTANCE Rad51-mediated homologous recombination is the major mechanism for repairing DNA double-strand break (DSB) repair in cancer cells. Thus, regulating Rad51 activity could be an attractive target. The sequential assembly and disassembly of Rad51 to the broken DNA ends depend on reversible protein-protein interactions. Here, we discovered that a dynamic interaction with molecular chaperone Hsp90 is one such regulatory event that governs the recruitment of Rad51 onto the damaged DNA. We uncovered that Rad51 associates with Hsp90, and upon DNA damage, this complex dissociates to facilitate the loading of Rad51 onto broken DNA. In a mutant where such dissociation is incomplete, the occupancy of Rad51 at the broken DNA is partial, which results in inefficient DNA repair. Thus, it is reasonable to propose that any small molecule that may alter the dynamics of the Rad51-Hsp90 interaction is likely to impact DSB repair in cancer cells.


Assuntos
Dano ao DNA , Reparo do DNA , Proteínas de Choque Térmico HSP90/metabolismo , Rad51 Recombinase/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Animais , Biologia Computacional , DNA Fúngico/genética , Proteínas de Ligação a DNA/genética , Proteínas de Choque Térmico HSP90/genética , Humanos , Metanossulfonato de Metila/farmacologia , Camundongos , Mutação , Ligação Proteica , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/efeitos dos fármacos
7.
Nat Commun ; 10(1): 1407, 2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30926776

RESUMO

RAD51 assembly on single-stranded (ss)DNAs is a crucial step in the homology-dependent repair of DNA damage for genomic stability. The formation of the RAD51 filament is promoted by various RAD51-interacting proteins including RAD51 paralogues. However, the mechanisms underlying the differential control of RAD51-filament dynamics by these factors remain largely unknown. Here, we report a role for the human RAD51 paralogue, SWSAP1, as a novel regulator of RAD51 assembly. Swsap1-deficient cells show defects in DNA damage-induced RAD51 assembly during both mitosis and meiosis. Defective RAD51 assembly in SWSAP1-depleted cells is suppressed by the depletion of FIGNL1, which binds to RAD51 as well as SWSAP1. Purified FIGNL1 promotes the dissociation of RAD51 from ssDNAs. The dismantling activity of FIGNL1 does not require its ATPase but depends on RAD51-binding. Purified SWSAP1 inhibits the RAD51-dismantling activity of FIGNL1. Taken together, our data suggest that SWSAP1 protects RAD51 filaments by antagonizing the anti-recombinase, FIGNL1.


Assuntos
ATPases Associadas a Diversas Atividades Celulares/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo , Recombinases Rec A/fisiologia , Homologia de Sequência de Aminoácidos , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Linhagem Celular , Cromossomos Humanos/metabolismo , DNA/metabolismo , Dano ao DNA , Proteínas de Ligação a DNA/deficiência , Proteínas de Ligação a DNA/metabolismo , Células Germinativas/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mitose , Modelos Biológicos , Ligação Proteica , Recombinases Rec A/genética
8.
Reproduction ; 157(3): 223-234, 2019 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-30817312

RESUMO

Homologous recombination (HR) plays a critical role in facilitating replication fork progression when the polymerase complex encounters a blocking DNA lesion, and it also serves as the primary mechanism for error-free DNA repair of double-stranded breaks. DNA repair protein RAD51 homolog 1 (RAD51) plays a central role in HR. However, the role of RAD51 during porcine early embryo development is unknown. In the present study, we examined whether RAD51 is involved in the regulation of early embryonic development of porcine parthenotes. We found that inhibition of RAD51 delayed cleavage and ceased development before the blastocyst stage. Disrupting RAD51 activity with RNAi or an inhibitor induces sustained DNA damage, as demonstrated by the formation of distinct γH2AX foci in nuclei of four-cell embryos. Inhibiting RAD51 triggers a DNA damage checkpoint by activating the ataxia telangiectasia mutated (ATM)-p53-p21 pathway. Furthermore, RAD51 inhibition caused apoptosis, reactive oxygen species accumulation, abnormal mitochondrial distribution and decreased pluripotent gene expression in blastocysts. Thus, our results indicate that RAD51 is required for proper porcine parthenogenetic activation (PA) embryo development.


Assuntos
Blastocisto/efeitos dos fármacos , Desenvolvimento Embrionário/efeitos dos fármacos , Rad51 Recombinase/antagonistas & inibidores , Animais , Apoptose/efeitos dos fármacos , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Blastocisto/metabolismo , Reparo do DNA/efeitos dos fármacos , Feminino , Gravidez , Rad51 Recombinase/metabolismo , Transdução de Sinais/efeitos dos fármacos , Suínos , Proteína Supressora de Tumor p53/metabolismo
9.
Methods Mol Biol ; 1929: 447-460, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30710290

RESUMO

DNA damage possesses the capacity to threaten the genomic integrity of an organism. A multitude of proteins are involved in the detection and repair of DNA double-strand breaks (DSBs), a severe kind of DNA damage. The function of DNA repair proteins can be examined by biochemical assays in vitro as well as in cell-based studies. The Ca2+-binding protein S100A11 shows functional interactions with factors involved in the repair of DSBs by homologous recombination (HR), a high-fidelity DNA repair pathway, such as RAD51 and RAD54B. The key enzyme of the homologous recombination repair is RAD51 that catalyzes the invasion of single-stranded DNA (ssDNA) into double-stranded DNA (dsDNA) containing homologous regions and the exchange of these DNA molecules generating heteroduplex DNA (hDNA). In this chapter, we describe a protocol for the purification of S100A11 to near homogeneity. Using purified proteins, we show the ability of S100A11 to stimulate RAD51 in a DNA strand exchange assay. Additionally, we describe a protocol how S100A11 can be localized in sites of DNA repair by immunofluorescence staining. Furthermore, we present a protocol for assessment of chromosomal aberrations after depletion of S100A11 that illustrate the apparent involvement of S100A11 in genome integrity.


Assuntos
DNA/metabolismo , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Proteínas S100/metabolismo , Sítios de Ligação , Cálcio/metabolismo , Linhagem Celular Tumoral , Aberrações Cromossômicas , DNA/química , Dano ao DNA , Imunofluorescência , Humanos , Microscopia Confocal
10.
Nat Commun ; 10(1): 722, 2019 02 13.
Artigo em Inglês | MEDLINE | ID: mdl-30760716

RESUMO

Homologous recombination (HR) repairs DNA double-strand breaks (DSBs) to maintain genomic integrity. Recombinase recruited to the DSBs by the mediator protein BRCA2 catalyzes the homology-directed repair. During meiotic HR, programmed DSBs are introduced genome-wide but their repair mechanisms, including the regulation of BRCA2, have remained largely elusive. Here we identify a meiotic localizer of BRCA2, MEILB2/HSF2BP, that localizes to the site of meiotic DSBs in mice. Disruption of Meilb2 abolishes the localization of RAD51 and DMC1 recombinases in spermatocytes, leading to errors in DSB repair and male sterility. MEILB2 directly binds to BRCA2 and regulates its association to meiotic DSBs. We map the MEILB2-binding domain within BRCA2 that is distinct from the canonical DNA-binding domain but is sufficient to localize to meiotic DSBs in a MEILB2-dependent manner. We conclude that localization of BRCA2 to meiotic DSBs is mediated by MEILB2, which is an integral mechanism to repair abundant meiotic DSBs.


Assuntos
Proteína BRCA2/metabolismo , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Meiose/fisiologia , Recombinases/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Proteínas Cromossômicas não Histona/metabolismo , Reparo do DNA , Proteínas de Ligação a DNA/genética , Feminino , Técnicas de Inativação de Genes , Masculino , Camundongos , Proteínas Nucleares/metabolismo , Rad51 Recombinase/metabolismo
11.
PLoS Genet ; 15(2): e1007942, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30735491

RESUMO

NSMCE2 is an E3 SUMO ligase and a subunit of the SMC5/6 complex that associates with the replication fork and protects against genomic instability. Here, we study the fate of collapsed replication forks generated by prolonged hydroxyurea treatment in human NSMCE2-deficient cells. Double strand breaks accumulate during rescue by converging forks in normal cells but not in NSMCE2-deficient cells. Un-rescued forks persist into mitosis, leading to increased mitotic DNA damage. Excess RAD51 accumulates and persists at collapsed forks in NSMCE2-deficient cells, possibly due to lack of BLM recruitment to stalled forks. Despite failure of BLM to accumulate at stalled forks, NSMCE2-deficient cells exhibit lower levels of hydroxyurea-induced sister chromatid exchange. In cells deficient in both NSMCE2 and BLM, hydroxyurea-induced double strand breaks and sister chromatid exchange resembled levels found in NSCME2-deficient cells. We conclude that the rescue of collapsed forks by converging forks is dependent on NSMCE2.


Assuntos
Dano ao DNA , Ligases/metabolismo , Mitose , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Replicação do DNA , Epistasia Genética , Instabilidade Genômica , Células HEK293 , Células HeLa , Humanos , Hidroxiureia/farmacologia , Ligases/deficiência , Ligases/genética , Modelos Biológicos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , RecQ Helicases/deficiência , RecQ Helicases/genética , RecQ Helicases/metabolismo , Troca de Cromátide Irmã/efeitos dos fármacos , Sumoilação
12.
PLoS Genet ; 15(2): e1007952, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30716097

RESUMO

Meiotic recombination permits exchange of genetic material between homologous chromosomes. The replication protein A (RPA) complex, the predominant ssDNA-binding complex, is required for nearly all aspects of DNA metabolism, but its role in mammalian meiotic recombination remains unknown due to the embryonic lethality of RPA mutant mice. RPA is a heterotrimer of RPA1, RPA2, and RPA3. We find that loss of RPA1, the largest subunit, leads to disappearance of RPA2 and RPA3, resulting in the absence of the RPA complex. Using an inducible germline-specific inactivation strategy, we find that loss of RPA completely abrogates loading of RAD51/DMC1 recombinases to programmed meiotic DNA double strand breaks, thus blocking strand invasion required for chromosome pairing and synapsis. Surprisingly, loading of MEIOB, SPATA22, and ATR to DNA double strand breaks is RPA-independent and does not promote RAD51/DMC1 recruitment in the absence of RPA. Finally, inactivation of RPA reduces crossover formation. Our results demonstrate that RPA plays two distinct roles in meiotic recombination: an essential role in recombinase recruitment at early stages and an important role in promoting crossover formation at later stages.


Assuntos
Recombinação Homóloga , Meiose/genética , Proteína de Replicação A/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Pareamento Cromossômico , Troca Genética , Quebras de DNA de Cadeia Dupla , Replicação do DNA , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Mutação , Proteínas Nucleares/metabolismo , Estabilidade Proteica , Rad51 Recombinase/deficiência , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína de Replicação A/deficiência , Proteína de Replicação A/genética , Espermatócitos/citologia , Espermatócitos/metabolismo
13.
Eur J Med Chem ; 165: 80-92, 2019 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-30660828

RESUMO

Olaparib is a PARP inhibitor (PARPi). For patients bearing BRCA1 or BRCA2 mutations, olaparib is approved to treat ovarian cancer and in clinical trials to treat breast and pancreatic cancers. In BRCA2-defective patients, PARPi inhibits DNA single-strand break repair, while BRCA2 mutations hamper double-strand break repair. Recently, we identified a series of triazole derivatives that mimic BRCA2 mutations by disrupting the Rad51-BRCA2 interaction and thus double-strand break repair. Here, we have computationally designed, synthesized, and tested over 40 novel derivatives. Additionally, we designed and conducted novel biological assays to characterize how they disrupt the Rad51-BRCA2 interaction and inhibit double-strand break repair. These compounds synergized with olaparib to target pancreatic cancer cells with functional BRCA2. This supports the idea that small organic molecules can mimic genetic mutations to improve the profile of anticancer drugs for precision medicine. Moreover, this paradigm could be exploited in other genetic pathways to discover innovative anticancer targets and drug candidates.


Assuntos
Antineoplásicos/química , Proteína BRCA2/metabolismo , Recombinação Homóloga/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Rad51 Recombinase/metabolismo , Triazóis/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteína BRCA2/genética , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Mimetismo Molecular , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Ftalazinas/uso terapêutico , Piperazinas/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Triazóis/síntese química
14.
Nat Commun ; 10(1): 65, 2019 01 08.
Artigo em Inglês | MEDLINE | ID: mdl-30622262

RESUMO

Polyamines, often elevated in cancer cells, have been shown to promote cell growth and proliferation. Whether polyamines regulate other cell functions remains unclear. Here, we explore whether and how polyamines affect genome integrity. When DNA double-strand break (DSB) is induced in hair follicles by ionizing radiation, reduction of cellular polyamines augments dystrophic changes with delayed regeneration. Mechanistically, polyamines facilitate homologous recombination-mediated DSB repair without affecting repair via non-homologous DNA end-joining and single-strand DNA annealing. Biochemical reconstitution and functional analyses demonstrate that polyamines enhance the DNA strand exchange activity of RAD51 recombinase. The effect of polyamines on RAD51 stems from their ability to enhance the capture of homologous duplex DNA and synaptic complex formation by the RAD51-ssDNA nucleoprotein filament. Our work demonstrates a novel function of polyamines in the maintenance of genome integrity via homology-directed DNA repair.


Assuntos
Poliaminas/metabolismo , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação/fisiologia , Animais , Linhagem Celular Tumoral , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Reparo do DNA por Junção de Extremidades/efeitos dos fármacos , Reparo do DNA por Junção de Extremidades/fisiologia , DNA de Cadeia Simples/metabolismo , Feminino , Raios gama/efeitos adversos , Células HEK293 , Folículo Piloso/metabolismo , Folículo Piloso/efeitos da radiação , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Ornitina Descarboxilase/metabolismo , Ornitina Descarboxilase/farmacologia , Inibidores da Ornitina Descarboxilase , Ftalazinas/farmacologia , Piperazinas/farmacologia , Rad51 Recombinase/genética , Reparo de DNA por Recombinação/efeitos dos fármacos
15.
Biochem Biophys Res Commun ; 508(3): 722-728, 2019 01 15.
Artigo em Inglês | MEDLINE | ID: mdl-30528234

RESUMO

Topoisomerase IIß-binding protein 1 (TopBP1) is BRCT domain-containing protein that is required for DNA double-strand break (DSB) repair and DNA damage responses; however, its function during the early stage of spermatogenesis is still unclear. To investigate the physiological role of TopBP1, we have generated germ cell-specific TopBP1-depleted mouse model. TopBP1-deleted mice were infertile, showed a loss of germ cells and had meiotic defects. Conditional TopBP1 deletion resulted in reduced testis size, reduced number of epididymal sperm, increased apoptosis, and severely compromised fertility. TopBP1 deficiency caused defects in DMC1 and Rad51 foci formation, abnormal synaptonemal complexes and meiotic chromosome defects. Collectively, these results suggest that TopBP1 deficiency during spermatogenesis impairs the localization of proteins involved in early recombination at DSBs, results in meiotic chromosome defects and leads to infertility.


Assuntos
Proteínas de Transporte/metabolismo , Cromossomos de Mamíferos/metabolismo , Meiose/genética , Recombinação Genética , Espermatogênese/genética , Animais , Animais Recém-Nascidos , Apoptose , Proteínas de Ciclo Celular/metabolismo , Quebras de DNA de Cadeia Dupla , Infertilidade Masculina/genética , Infertilidade Masculina/patologia , Masculino , Camundongos Knockout , Proteínas Nucleares/metabolismo , Estágio Paquíteno , Transporte Proteico , Rad51 Recombinase/metabolismo , Túbulos Seminíferos/metabolismo , Túbulos Seminíferos/patologia , Cromossomos Sexuais/metabolismo , Espermatozoides/metabolismo , Complexo Sinaptonêmico/metabolismo
16.
Toxicol Lett ; 303: 38-47, 2019 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-30586609

RESUMO

Although many studies have investigated the toxic effects and even the reproductive toxicity of chlorothalonil, almost no studies have focused on the ovary, the organ of oocyte development. Puberty is a critical window for development of the female reproductive system. Therefore, this investigation aimed to explore the effects and underlying mechanisms of chlorothalonil at low doses on peripubertal mouse ovarian development. Chlorothalonil is frequently used in horticulture with short intervals between applications, therefore, vegetables and fruits may be potential sources of chlorothalonil contamination. For the first time, this study demonstrated that chlorothalonil inhibited ovarian development during puberty in mice, and at levels currently assumed to have no adverse health consequences for humans. Chlorothalonil exposure inhibited mouse ovarian development by increasing the number of primary follicles and decreasing the number of mature follicles. It acted by decreasing the levels of hormone production proteins, such as FSH receptor and estrogen receptor alpha, while increasing the levels of DNA repairing marker RAD51 and cell apoptosis. These results suggest that chlorothalonil may disrupt endocrine function and inhibit murine ovarian development. Therefore it may pose a potential health risk to female reproductive systems in other species, especially to the ovary.


Assuntos
Disruptores Endócrinos/toxicidade , Nitrilos/toxicidade , Oogênese/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Alanina Transaminase/sangue , Animais , Apoptose/efeitos dos fármacos , Aspartato Aminotransferases/sangue , Dano ao DNA , Reparo do DNA , Feminino , Marcadores Genéticos , Hormônios Esteroides Gonadais/sangue , Camundongos , Camundongos Endogâmicos ICR , Folículo Ovariano/citologia , Folículo Ovariano/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Receptores do FSH/genética , Receptores do FSH/metabolismo , Reprodução/efeitos dos fármacos
17.
Mol Cell ; 73(2): 224-237.e6, 2019 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-30554948

RESUMO

The BRCA1-BRCA2-RAD51 axis is essential for homologous recombination repair (HRR) and is frequently disrupted in breast cancers. PARP inhibitors (PARPis) are used clinically to treat BRCA-mutated breast tumors. Using a genetic screen, we identified EMI1 as a modulator of PARPi sensitivity in triple-negative breast cancer (TNBC) cells. This function requires the F-box domain of EMI1, through which EMI1 assembles a canonical SCF ubiquitin ligase complex that constitutively targets RAD51 for degradation. In response to genotoxic stress, CHK1-mediated phosphorylation of RAD51 counteracts EMI1-dependent degradation by enhancing RAD51's affinity for BRCA2, leading to RAD51 accumulation. Inhibition of RAD51 degradation restores HRR in BRCA1-depleted cells. Human breast cancer samples display an inverse correlation between EMI1 and RAD51 protein levels. A subset of BRCA1-deficient TNBC cells develop resistance to PARPi by downregulating EMI1 and restoring RAD51-dependent HRR. Notably, reconstitution of EMI1 expression reestablishes PARPi sensitivity both in cellular systems and in an orthotopic mouse model.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Resistencia a Medicamentos Antineoplásicos , Proteínas F-Box/metabolismo , Ftalazinas/farmacologia , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Animais , Proteína BRCA1/deficiência , Proteína BRCA1/genética , Proteína BRCA2/genética , Proteína BRCA2/metabolismo , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Quinase 1 do Ponto de Checagem/genética , Quinase 1 do Ponto de Checagem/metabolismo , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas F-Box/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Células HEK293 , Humanos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Proteólise , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Transdução de Sinais/efeitos dos fármacos , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Nat Commun ; 9(1): 5376, 2018 12 18.
Artigo em Inglês | MEDLINE | ID: mdl-30560944

RESUMO

DNA double-strand breaks (DSBs) are toxic DNA lesions, which, if not properly repaired, may lead to genomic instability, cell death and senescence. Damage-induced long non-coding RNAs (dilncRNAs) are transcribed from broken DNA ends and contribute to DNA damage response (DDR) signaling. Here we show that dilncRNAs play a role in DSB repair by homologous recombination (HR) by contributing to the recruitment of the HR proteins BRCA1, BRCA2, and RAD51, without affecting DNA-end resection. In S/G2-phase cells, dilncRNAs pair to the resected DNA ends and form DNA:RNA hybrids, which are recognized by BRCA1. We also show that BRCA2 directly interacts with RNase H2, mediates its localization to DSBs in the S/G2 cell-cycle phase, and controls DNA:RNA hybrid levels at DSBs. These results demonstrate that regulated DNA:RNA hybrid levels at DSBs contribute to HR-mediated repair.


Assuntos
Proteína BRCA1/metabolismo , Proteína BRCA2/metabolismo , RNA Longo não Codificante/metabolismo , Reparo de DNA por Recombinação , Ribonuclease H/metabolismo , Proteína BRCA1/genética , Proteína BRCA2/genética , Linhagem Celular Tumoral , DNA/genética , DNA/metabolismo , Quebras de DNA de Cadeia Dupla , Fase G2/genética , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , RNA Longo não Codificante/genética , RNA Interferente Pequeno/metabolismo , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ribonuclease H/genética , Fase S/genética
19.
Cell Physiol Biochem ; 49(6): 2111-2123, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30273928

RESUMO

BACKGROUND/AIMS: Adult T-cell leukemia-lymphoma (ATL) is an aggressive disease which is highly resistant to chemotherapy. Studies show that enhanced ability of DNA damage repair (DDR) in cancer cells plays a key role in chemotherapy resistance. Here, we suggest that defect in DDR related genes might be a promising target to destroy the genome stability of tumor cells. METHODS: Since KU70 is highly expressed in Jurkat cells, one of the most representative cell lines of ATL, we knocked down KU70 by shRNA and analyzed the impact of KU70 deficiency in Jurkat cells as well as in NOD-SCID animal models by western blot, immunofluorescence, flow cytometry and measuring DNA repair efficiency. RESULTS: It is observed that silencing of KU70 resulted in accumulated DNA damage and impaired DDR in Jurkat cells, resulting in more apoptosis, decreased cell proliferation and cell cycle arrest. DNA damage leads to DNA double-strand breaks (DSBs), which are processed by either non-homologous end joining(NHEJ) or homologous recombination(HR). In our study, both NHEJ and HR are impaired because of KU70 defect, accompanied with increased protein level of SHP-1, a dephosphorylation enzyme. In turn, SHP-1 led to dephosphorylation of SIRT1, which further impaired HR repair efficiency. Moreover, KU70 deficiency prolonged survival of Jurkat-xenografted mice. CONCLUSION: These findings suggest that targeting KU70 is a promising target for ATL and might overcome the existing difficulties in chemotherapy.


Assuntos
Reparo do DNA por Junção de Extremidades , Autoantígeno Ku/metabolismo , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Reparo de DNA por Recombinação , Sirtuína 1/metabolismo , Animais , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Pontos de Checagem do Ciclo Celular , Quebras de DNA de Cadeia Dupla , Humanos , Células Jurkat , Autoantígeno Ku/antagonistas & inibidores , Autoantígeno Ku/genética , Leucemia-Linfoma de Células T do Adulto/tratamento farmacológico , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fosforilação , Proteína Tirosina Fosfatase não Receptora Tipo 6/antagonistas & inibidores , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Rad51 Recombinase/metabolismo
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