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1.
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
2.
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
3.
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
4.
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
5.
PLoS Genet ; 15(3): e1008001, 2019 03.
Artigo em Inglês | MEDLINE | ID: mdl-30822309

RESUMO

We have used two different live-cell fluorescent protein markers to monitor the formation and localization of double-strand breaks (DSBs) in budding yeast. Using GFP derivatives of the Rad51 recombination protein or the Ddc2 checkpoint protein, we find that cells with three site-specific DSBs, on different chromosomes, usually display 2 or 3 foci that may coalesce and dissociate. This motion is independent of Rad52 and microtubules. Rad51-GFP, by itself, is unable to repair DSBs by homologous recombination in mitotic cells, but is able to form foci and allow repair when heterozygous with a wild type Rad51 protein. The kinetics of formation and disappearance of a Rad51-GFP focus parallels the completion of site-specific DSB repair. However, Rad51-GFP is proficient during meiosis when homozygous, similar to rad51 "site II" mutants that can bind single-stranded DNA but not complete strand exchange. Rad52-RFP and Rad51-GFP co-localize to the same DSB, but a significant minority of foci have Rad51-GFP without visible Rad52-RFP. We conclude that co-localization of foci in cells with 3 DSBs does not represent formation of a homologous recombination "repair center," as the same distribution of Ddc2-GFP foci was found in the absence of the Rad52 protein.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas de Ciclo Celular/genética , Quebras de DNA de Cadeia Dupla , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Proteínas de Saccharomyces cerevisiae/genética , Dano ao DNA/genética , Proteínas de Ligação a DNA/genética , Proteínas de Fluorescência Verde/genética , Recombinação Homóloga/genética , Cinética , Meiose/genética , Saccharomyces cerevisiae/genética
6.
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
7.
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
8.
PLoS Genet ; 15(1): e1007439, 2019 01.
Artigo em Inglês | MEDLINE | ID: mdl-30668564

RESUMO

Homologous recombination (HR) is the principal mechanism of DNA repair acting during meiosis and is fundamental for the segregation of chromosomes and the increase of genetic diversity. Nevertheless, non-homologous end joining (NHEJ) mechanisms can also act during meiosis, mainly in response to exogenously-induced DNA damage in late stages of first meiotic prophase. In order to better understand the relationship between these two repair pathways, we studied the response to DNA damage during male mouse meiosis after gamma radiation. We clearly discerned two types of responses immediately after treatment. From leptotene to early pachytene, exogenous damage triggered the massive presence of γH2AX throughout the nucleus, which was associated with DNA repair mediated by HR components (DMC1 and RAD51). This early pathway finished with the sequential removal of DMC1 and RAD51 and was no longer inducible at mid pachytene. However, from mid-pachytene to diplotene, γH2AX appeared as large discrete foci. This late repair pattern was mediated initially by NHEJ, involving Ku70 and XRCC4, which were constitutively present, and 53BP1, which appeared at sites of damage soon after irradiation. Nevertheless, 24 hours after irradiation, a HR pathway involving RAD51 but not DMC1 mostly replaced NHEJ. Additionally, we observed the occurrence of synaptonemal complex bridges between bivalents, most likely representing chromosome translocation events that may involve DMC1, RAD51 or 53BP1. Our results reinforce the idea that the early "meiotic" repair pathway that acts by default at the beginning of meiosis is replaced from mid-pachytene onwards by a "somatic-like" repair pattern. This shift might be important to resolve DNA damage (either endogenous or exogenous) that could not be repaired by the early meiotic mechanisms, for instance those in the sex chromosomes, which lack a homologous chromosome to repair with. This transition represents another layer of functional changes that occur in meiotic cells during mid pachytene, in addition to epigenetic reprograming, reactivation of transcription, changes in the gene expression profile and acquisition of competence to proceed to metaphase.


Assuntos
Proteínas de Ciclo Celular/genética , Reparo do DNA por Junção de Extremidades/genética , Recombinação Homóloga/genética , Proteínas Nucleares/genética , Rad51 Recombinase/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética , Animais , Cromossomos/genética , Cromossomos/efeitos da radiação , Dano ao DNA/efeitos da radiação , Reparo do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Raios gama , Histonas/genética , Autoantígeno Ku/genética , Meiose/genética , Camundongos , Estágio Paquíteno/genética , Complexo Sinaptonêmico/genética
9.
BMB Rep ; 52(2): 151-156, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30638176

RESUMO

RAD51 recombinase plays a critical role in homologous recombination and DNA damage repair. Here we showed that expression of RAD51 is frequently upregulated in lung cancer tumors compared with normal tissues and is associated with poor survival (hazard ratio (HR) = 2, P = 0.0009). Systematic investigation of lung cancer cell lines revealed higher expression of RAD51 in KRAS mutant (MT) cells compared to wildtype (WT) cells. We further showed that MT KRAS, but not WT KRAS, played a critical role in RAD51 overexpression via MYC. Moreover, our results revealed that KRAS MT cells are highly dependent on RAD51 for survival and depletion of RAD51 resulted in enhanced DNA double strand breaks, defective colony formation and cell death. Together, our results suggest that mutant KRAS promotes RAD51 expression to enhance DNA damage repair and lung cancer cell survival, suggesting that RAD51 may be an effective therapeutic target to overcome chemo/radioresistance in KRAS mutant cancers. [BMB Reports 2019; 52(2): 151-156].


Assuntos
Dano ao DNA , Reparo do DNA , Neoplasias Pulmonares/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Rad51 Recombinase/biossíntese , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Rad51 Recombinase/genética , Regulação para Cima
10.
Oncogene ; 38(17): 3274-3287, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30631149

RESUMO

High-risk human papillomaviruses (HPVs) constitutively activate ataxia telangiectasia mutated (ATM) and ataxia telangiectasia- and Rad3-related (ATR) DNA damage repair pathways for viral genome amplification. HPVs activate these pathways through the immune regulator STAT-5. For the ATR pathway, STAT-5 increases expression of the topoisomerase IIß-binding protein 1 (TopBP1), a scaffold protein that binds ATR and recruits it to sites of DNA damage. TopBP1 also acts as a transcriptional regulator, and we investigated how this activity influenced the HPV life cycle. We determined that TopBP1 levels are increased in cervical intraepithelial neoplasias as well as cervical carcinomas, consistent with studies in HPV-positive cell lines. Suppression of TopBP1 by shRNAs impairs HPV genome amplification and activation of the ATR pathway but does not affect the total levels of ATR and CHK1. In contrast, knockdown reduces the expression of other DNA damage factors such as RAD51 and Mre11 but not BRCA2 or NBS1. Interestingly, TopBP1 positively regulates the expression of E2F1, a TopBP1-binding partner, and p73 in HPV-positive cells in contrast to its effects in other cell types. TopBP1 transcriptional activity is regulated by AKT, and treatment with AKT inhibitors suppresses expression of E2F1 and p73 without interfering with ATR signaling. Importantly, the levels of p73 are elevated in HPV-positive cells and its knockdown impairs HPV genome amplification. This demonstrates that p73, like p63 and p53, is an important regulator of the HPV life cycle that is controlled by the transcriptional activating properties of the multifunctional TopBP1 protein.


Assuntos
Proteínas de Transporte/genética , Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Fator de Transcrição E2F1/genética , Células Epiteliais/patologia , Amplificação de Genes/genética , Proteínas Nucleares/genética , Infecções por Papillomavirus/genética , Proteína Tumoral p73/genética , Animais , Proteínas Mutadas de Ataxia Telangiectasia/genética , Linhagem Celular , Neoplasia Intraepitelial Cervical/genética , Neoplasia Intraepitelial Cervical/virologia , Quinase 1 do Ponto de Checagem/genética , Dano ao DNA/genética , Feminino , Regulação da Expressão Gênica/genética , Humanos , Proteína Homóloga a MRE11/genética , Camundongos , Células NIH 3T3 , Papillomaviridae/patogenicidade , Rad51 Recombinase/genética , Fator de Transcrição STAT5/genética , Transdução de Sinais/genética , Transcrição Genética/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/virologia
11.
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
12.
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
13.
Oncogene ; 38(14): 2611-2626, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30531838

RESUMO

There is increasing evidence that genomic instability is a prerequisite for cancer progression. Here we show that SIM2s, a member of the bHLH/PAS family of transcription factors, regulates DNA damage repair through enhancement of homologous recombination (HR), and prevents epithelial-mesenchymal transitions (EMT) in an Ataxia-telangiectasia mutated (ATM)-dependent manner. Mechanistically, we found that SIM2s interacts with ATM and is stabilized through ATM-dependent phosphorylation in response to IR. Once stabilized, SIM2s interacts with BRCA1 and supports RAD51 recruitment to the site of DNA damage. Loss of SIM2s through the introduction of shSIM2 or the mutation of SIM2s at one of the predicted ATM phosphorylation sites (S115) reduces HR efficiency through disruption of RAD51 recruitment, resulting in genomic instability and induction of EMT. The EMT induced by the mutation of S115 is characterized by a decrease in E-cadherin and an induction of the basal marker, K14, resulting in increased invasion and metastasis. Together, these results identify a novel player in the DNA damage repair pathway and provides a link in ductal carcinoma in situ progression to invasive ductal carcinoma through loss of SIM2s, increased genomic instability, EMT, and metastasis.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transição Epitelial-Mesenquimal/genética , Recombinação Homóloga/genética , Animais , Proteína BRCA1/genética , Caderinas/genética , Carcinoma Intraductal não Infiltrante/genética , Linhagem Celular Tumoral , Dano ao DNA/genética , Reparo do DNA/genética , Feminino , Instabilidade Genômica/genética , Humanos , Células MCF-7 , Camundongos , Camundongos Nus , Fosforilação/genética , Rad51 Recombinase/genética
14.
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
15.
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
16.
BMC Cancer ; 18(1): 1287, 2018 Dec 22.
Artigo em Inglês | MEDLINE | ID: mdl-30577817

RESUMO

BACKGROUND: Adult T-cell leukemia/lymphoma (ATLL) is a lymphoproliferative disorder of HTLV-1-host interactions in infected TCD4+ cells. In this study, the HTLV-1 proviral load (PVL) and HBZ as viral elements and AKT1, BAD, FOXP3, RORγt and IFNλ3 as the host factors were investigated. METHODS: The study was conducted in ATLLs, HTLV-1-associated myelopathy/tropical spastic paraparesis patients (HAM/TSPs) and HTLV-1-asympthomatic carriers (ACs). The DNA and mRNA from peripheral blood mononuclear cells were extracted for gene expression assessments via qRT-PCR, TaqMan assay, and then confirmed by western blotting. RESULTS: As it was expected, the HTLV-1-PVL were higher in ATLLs than ACs (P = 0.002) and HAM/TSP (P = 0.041). The HBZ expression in ATLL (101.76 ± 61.3) was radically higher than in ACs (0.12 ± 0.05) and HAM/TSP (0.01 ± 0.1) (P = 0.001). Furthermore, the AKT1 expression in ATLLs (13.52 ± 4.78) was higher than ACs (1.17 ± 0.27) (P = 0.05) and HAM/TSPs (0.72 ± 0.49) (P = 0.008). However, BAD expression in ATLL was slightly higher than ACs and HAM/TSPs and not significant. The FOXP3 in ATLLs (41.02 ± 24.2) was more than ACs (1.44 ± 1) (P = 0.007) and HAM/TSP (0.45 ± 0.15) (P = 0.01). However, RORγt in ATLLs (27.43 ± 14.8) was higher than ACs (1.05 ± 0.32) (P = 0.02) but not HAM/TSPs. Finally, the IFNλ3 expression between ATLLs (31.92 ± 26.02) and ACs (1.46 ± 0.63) (P = 0.01) and ACs and HAM/TSPs (680.62 ± 674.6) (P = 0.02) were statistically different, but not between ATLLs and HAM/TSPs. CONCLUSIONS: The present and our previous study demonstrated that HTLV-1-PVL and HBZ and host AKT1 and Rad 51 are novel candidates for molecular targeting therapy of ATLL. However, high level of RORγt may inhibit Th1 response and complicated in ATLL progressions.


Assuntos
Fatores de Transcrição de Zíper de Leucina Básica/genética , Infecções por HTLV-I/genética , Vírus Linfotrópico T Tipo 1 Humano/genética , Leucemia-Linfoma de Células T do Adulto/genética , Proteínas dos Retroviridae/genética , Adulto , Feminino , Fatores de Transcrição Forkhead/genética , Regulação Neoplásica da Expressão Gênica , Regulação Viral da Expressão Gênica/genética , Infecções por HTLV-I/sangue , Infecções por HTLV-I/patologia , Infecções por HTLV-I/virologia , Interações Hospedeiro-Patógeno/genética , Vírus Linfotrópico T Tipo 1 Humano/patogenicidade , Humanos , Leucemia-Linfoma de Células T do Adulto/sangue , Leucemia-Linfoma de Células T do Adulto/patologia , Leucemia-Linfoma de Células T do Adulto/virologia , Leucócitos Mononucleares/metabolismo , Leucócitos Mononucleares/patologia , Masculino , Pessoa de Meia-Idade , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Paraparesia Espástica Tropical/sangue , Paraparesia Espástica Tropical/genética , Paraparesia Espástica Tropical/patologia , Paraparesia Espástica Tropical/virologia , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , Rad51 Recombinase/genética , Células Th1 , Carga Viral , Proteína de Morte Celular Associada a bcl/genética
17.
Genes Dev ; 32(17-18): 1242-1251, 2018 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-30181361

RESUMO

During homologous recombination, cells must coordinate repair, DNA damage checkpoint signaling, and movement of chromosomal loci to facilitate homology search. In Saccharomyces cerevisiae, increased movement of damaged loci (local mobility) and undamaged loci (global mobility) precedes homolog pairing in mitotic cells. How cells modulate chromosome mobility in response to DNA damage remains unclear. Here, we demonstrate that global chromosome mobility is regulated by the Rad51 recombinase and its mediator, Rad52. Surprisingly, rad51Δ rad52Δ cells display checkpoint-dependent constitutively increased mobility, indicating that a regulatory circuit exists between recombination and checkpoint machineries to govern chromosomal mobility. We found that the requirement for Rad51 in this circuit is distinct from its role in recombination and that interaction with Rad52 is necessary to alleviate inhibition imposed by mediator recruitment to ssDNA. Thus, interplay between recombination factors and the checkpoint restricts increased mobility until recombination proteins are assembled at damaged sites.


Assuntos
Cromossomos Fúngicos/metabolismo , Dano ao DNA , Recombinação Homóloga , Rad51 Recombinase/fisiologia , Proteína Rad52 de Recombinação e Reparo de DNA/fisiologia , Proteínas de Saccharomyces cerevisiae/fisiologia , Mutação , Rad51 Recombinase/genética , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
18.
DNA Repair (Amst) ; 68: 34-40, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-29929045

RESUMO

Classical non-homologous end-joining (cNHEJ) is the main pathway for the repair of DNA double strand breaks (DSBs) in mammalian cells. In the absence of c-NHEJ, an alternative end-joining (A-EJ) mechanism resolves DSBs. To date, no A-EJ specific factor has been identified. Instead, this mechanism appears to co-opt proteins involved in more than one DNA repair pathway. These include components of base-excision repair (PARP1/XRCC1/LIG3), interstrand cross-link repair (BRCA1/FANCD2), and DSB response/DNA end-resection (MRE11A/RAD50/RBBP8). To clarify the contribution of these factors to A-EJ, here we examined their expression and recruitment to DSBs in correlation with surrogates of cNHEJ (53BP1) and homologous recombination (RAD51) in cells deficient for the cNHEJ end-ligation component XRCC4. This revealed XRCC4-deficient cells exhibited marked increases in the stability of A-EJ transcripts that result in correspondingly elevated levels of associated proteins, in comparison to WT cells. RAD51 was also increased while 53BP1 was unaffected. Treatment with radiomimetic DSB-inducing drug doxorubicin did not influence these activities. However, FANCD2, BRCA1 and XRCC1 foci, prominently associated with 53BP1 foci and hence DSBs resolved by cNHEJ, were only detected in doxorubicin-treated XRCC4-deficient cells. Strikingly, treatment of XRCC4-deficient cells with the PARP-specific inhibitor Niraparib enhanced A-EJ, and substantially induced 53BP1 transcripts and the numbers of A-EJ-associated 53BP1 DNA damage foci. RAD51 was severely inhibited, and upstream cNHEJ (KU70/KU80/DNA-PKCs/ARTEMIS) transcripts were substantially induced. These latter results were recapitulated in BRCA1-deficient cells, which contrastingly did not affect 53BP1 or PARP1 status irrespective of doxorubicin or Niraparib treatment. Hence A-EJ is regulated transcriptionally, reduced by a higher turnover rate in cNHEJ-proficient cells and sustained but fine-tuned by PARP1 in XRCC4-deficient cells to promote DNA repair and survival. Upstream cNHEJ components are similarly transcriptionally down-modulated by PARP1 and BRCA1 in a manner inversely correlated with HR and mechanistically distinct from A-EJ respectively in cNHEJ-deficient and cNHEJ-proficient settings.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteínas de Ligação a DNA/genética , Transdução de Sinais , Animais , Proteína BRCA1/metabolismo , Células Cultivadas , DNA/metabolismo , Reparo do DNA por Junção de Extremidades/genética , Proteínas de Ligação a DNA/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Humanos , Camundongos , Poli(ADP-Ribose) Polimerase-1/metabolismo , Rad51 Recombinase/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/genética
19.
J Cancer Res Ther ; 14(3): 614-618, 2018 Apr-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29893328

RESUMO

Background and Aim of Study: Colorectal cancer (CRC) is among the most common cancers and accounts as the second leading cause of death from cancers in the world. RAD51 plays a crucial role in double-strand breaks repair of DNA. Single nucleotide polymorphisms within this gene could influence on the potential of DNA repair and in consequence on the susceptibility to various tumors such as CRC. This is the first report about the role of RAD51 polymorphisms in Iranian CRC susceptibility. The study was conducted to evaluate the association of 135G>C polymorphism of RAD51 gene with sporadic CRC in a subset of Iranian population. Materials and Methods: The current case-control study was performed from 2013 to 2015. One hundred patients with sporadic CRC and one hundred controls were enrolled from two referral centers in Isfahan. All samples were genotyped for the RAD51 gene using polymerase chain reaction-restriction fragment length polymorphism assay. Results: The results revealed no significant association between the RAD51 135G>C and sporadic CRC (odds ratio = 0.86, 95% confidence interval = 0.464-1.595). The frequency of genotypes and also alleles of the mentioned polymorphism were not significantly different between case and control groups (P = 0.2 and 0.4, respectively). Conclusion: The results suggest that RAD51 135G>C probably has not a crucial role in Iranian CRC risk and is not an important potential risk factor in molecular diagnostics of mentioned disease among Iranian population.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Predisposição Genética para Doença , Polimorfismo de Nucleotídeo Único , Rad51 Recombinase/genética , Estudos de Casos e Controles , Neoplasias Colorretais/epidemiologia , Feminino , Seguimentos , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Fatores de Risco
20.
Medicine (Baltimore) ; 97(26): e11251, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29952992

RESUMO

AIM: Available data concerning the association between RAD51 135G/C (rs1801320) polymorphism and the risk of 3 common gynecological cancers still could not reach a consensus. Thus, we conducted a meta-analysis to explore the relationship. METHODS: Several electronic databases and bibliographies of relevant articles were screened to identify the studies up to July 2017. Then a meta-analysis was performed to evaluate the connection between 3 common gynecological tumors' susceptibility and RAD51 135G/C polymorphism in different inheritance models. Simultaneously, we did subgroup analysis and sensitivity analysis if necessary. RESULTS: A total of 11 articles including 14 studies involving 4097 cases and 5890 controls were included in this meta-analysis. Overall, RAD51 135G/C polymorphism increased the risk of 3 common gynecological tumors. The subgroup analysis stratified by cancer types- endometrial carcinoma (EC) and ovarian cancer (OC)-showed that RAD51 135G/C polymorphism increased the risk of EC: allele model (C vs G: odds ratio [OR] = 4.32, 95% confidence interval [CI] = 2.63-7.10, P < .00001), dominant model (CC + GC vs GG: OR = 2.28, 95% CI = 1.44-3.60, P = .004), recessive model (CC vs GC + GG: OR = 10.27, 95% CI = 14.71-22.38, P < .00001), and homozygous model (CC vs GG: OR = 7.26, 95% CI = 3.59-14.68, P < .00001), but there was no significant association between RAD51 135G/C polymorphism and OC. In the subgroup analysis stratified by source of controls, a significantly increased risk was observed in hospital-based studies. Nevertheless, the data showed RAD51 135G/C polymorphism had no link in population-based studies. CONCLUSIONS: This meta-analysis suggested that RAD51 135G/C polymorphism was a risk factor for the three common gynecological tumors, especially for EC among hospital-based populations.


Assuntos
Neoplasias dos Genitais Femininos/genética , Rad51 Recombinase/genética , Alelos , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Polimorfismo de Nucleotídeo Único , Fatores de Risco
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