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1.
Oncol Rep ; 47(2)2022 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-34935059

RESUMO

Although gemcitabine (GEM) has been used to treat bladder cancer (BC) for a number of years, severe adverse events or drug resistance frequently develops. A series of drugs have been proved to sensitize patients to GEM and reduce the side effects. The aim of the present study was to evaluate the potential effects of berberine (BER) on GEM­induced cytotoxicity in BC and to explore the possible underlying mechanisms. T24 and 5637 human BC cell lines were treated with GEM and/or BER before cell proliferation, apoptosis and migration were studied. Oncomine databases and Gene Expression Profiling Interactive Analysis (GEPIA) were used to retrieve RAD51 recombinase (Rad51) mRNA expression. Overexpression plasmid or specific Rad51 small interfering RNA were used to examine the role of Rad51 in drug­treated BC cells. BC model mice were administered with GEM and/or BER before changes in tumor volume, size and Ki67 expression were assessed. BER enhanced GEM­induced cytotoxicity, apoptosis and inhibition of migration, whilst attenuating the GEM­induced upregulation of phosphorylated Akt and Rad51 expression. According to Oncomine and GEPIA analyses, Rad51 was found to be significantly upregulated in BC tissues compared with that in normal tissues, where there was a weak positive correlation between Rad51 and Akt1 expression. Knockdown of Rad51 enhanced GEM­induced cytotoxicity, whilst overexpression of Rad51 reversed the suppressed cell viability induced by BER and GEM. Inactivation of the PI3K/Akt pathway by LY294002 or BER enhanced GEM­induced cytotoxicity and downregulated Rad51 expression, whilst overexpression of constitutively active Akt restored Rad51 expression and cell viability that was previously decreased by BER and GEM. BER additively inhibited tumor growth and Ki67 expression when combined with GEM in vivo. These results suggest that BER can enhance GEM­induced cytotoxicity in BC by downregulating Rad51 expression through inactivating the PI3K/Akt pathway, which may represent a novel therapeutic target for BC treatment.


Assuntos
Berberina/farmacologia , Desoxicitidina/análogos & derivados , Sinergismo Farmacológico , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Rad51 Recombinase/metabolismo , Neoplasias da Bexiga Urinária/tratamento farmacológico , Animais , Antimetabólitos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Proteínas de Transporte , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Regulação para Baixo , Feminino , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Regulação para Cima , Neoplasias da Bexiga Urinária/genética
2.
Nat Commun ; 12(1): 5748, 2021 09 30.
Artigo em Inglês | MEDLINE | ID: mdl-34593815

RESUMO

Mutations in the tumour suppressor gene BRCA2 are associated with predisposition to breast and ovarian cancers. BRCA2 has a central role in maintaining genome integrity by facilitating the repair of toxic DNA double-strand breaks (DSBs) by homologous recombination (HR). BRCA2 acts by controlling RAD51 nucleoprotein filament formation on resected single-stranded DNA, but how BRCA2 activity is regulated during HR is not fully understood. Here, we delineate a pathway where ATM and ATR kinases phosphorylate a highly conserved region in BRCA2 in response to DSBs. These phosphorylations stimulate the binding of the protein phosphatase PP2A-B56 to BRCA2 through a conserved binding motif. We show that the phosphorylation-dependent formation of the BRCA2-PP2A-B56 complex is required for efficient RAD51 filament formation at sites of DNA damage and HR-mediated DNA repair. Moreover, we find that several cancer-associated mutations in BRCA2 deregulate the BRCA2-PP2A-B56 interaction and sensitize cells to PARP inhibition. Collectively, our work uncovers PP2A-B56 as a positive regulator of BRCA2 function in HR with clinical implications for BRCA2 and PP2A-B56 mutated cancers.


Assuntos
Proteína BRCA2/metabolismo , Neoplasias da Mama/genética , Neoplasias Ovarianas/genética , Proteína Fosfatase 2/metabolismo , Reparo de DNA por Recombinação , Proteína BRCA2/genética , Quebras de DNA de Cadeia Dupla , Feminino , Predisposição Genética para Doença , Células HeLa , Humanos , Mutação , Fosforilação/genética , Ligação Proteica/genética , Proteína Fosfatase 2/genética , Rad51 Recombinase/metabolismo
3.
Int J Mol Sci ; 22(18)2021 Sep 09.
Artigo em Inglês | MEDLINE | ID: mdl-34575923

RESUMO

Molecules involved in DNA damage response (DDR) are often overexpressed in cancer cells, resulting in poor responses to chemotherapy and radiotherapy. Although treatment efficacy can be improved with the concomitant use of DNA repair inhibitors, the accompanying side effects can compromise the quality of life of patients. Therefore, in this study, we identified a natural compound that could inhibit DDR, using the single-strand annealing yeast-cell analysis system, and explored its mechanisms of action and potential as a chemotherapy adjuvant in hepatocellular carcinoma (HCC) cell lines using comet assay, flow cytometry, Western blotting, immunofluorescence staining, and functional analyses. We developed a mouse model to verify the in vitro findings. We found that hydroxygenkwanin (HGK) inhibited the expression of RAD51 and progression of homologous recombination, thereby suppressing the ability of the HCC cell lines to repair DNA damage and enhancing their sensitivity to doxorubicin. HGK inhibited the phosphorylation of DNA damage checkpoint proteins, leading to apoptosis in the HCC cell lines. In the mouse xenograft model, HGK enhanced the sensitivity of liver cancer cells to doxorubicin without any physiological toxicity. Thus, HGK can inhibit DDR in liver cancer cells and mouse models, making it suitable for use as a chemotherapy adjuvant.


Assuntos
Antineoplásicos Fitogênicos/farmacologia , Dano ao DNA/efeitos dos fármacos , Flavonoides/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/genética , Linhagem Celular Tumoral , Reparo do DNA/efeitos dos fármacos , Modelos Animais de Doenças , Sinergismo Farmacológico , Medicamentos de Ervas Chinesas , Regulação da Expressão Gênica , Recombinação Homóloga/efeitos dos fármacos , Humanos , Camundongos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , Leveduras/efeitos dos fármacos , Leveduras/genética , Leveduras/metabolismo
4.
Molecules ; 26(18)2021 Sep 08.
Artigo em Inglês | MEDLINE | ID: mdl-34576930

RESUMO

RAD51 is the central protein in DNA repair by homologous recombination (HR), involved in several steps of this process. It is shown that overexpression of the RAD51 protein is correlated with increased survival of cancer cells to cancer treatments. For the past decade, RAD51 overexpression-mediated resistance has justified the development of targeted inhibitors. One of the first molecules described to inhibit RAD51 was the 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) molecule. This small molecule is effective in inhibiting different functions of RAD51, however its mode of action and the chemical functions involved in this inhibition have not been identified. In this work, we used several commercial molecules derived from DIDS to characterize the structural determinants involved in modulating the activity of RAD51. By combining biochemical and biophysical approaches, we have shown that DIDS and two analogs were able to inhibit the binding of RAD51 to ssDNA and prevent the formation of D-loop by RAD51. Both isothiocyanate substituents of DIDS appear to be essential in the inhibition of RAD51. These results open the way to the synthesis of new molecules derived from DIDS that should be greater modulators of RAD51 and more efficient for HR inhibition.


Assuntos
Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/análogos & derivados , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/administração & dosagem , Ácido 4,4'-Di-Isotiocianoestilbeno-2,2'-Dissulfônico/farmacologia , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/administração & dosagem , Ácido 4-Acetamido-4'-isotiocianatostilbeno-2,2'-dissulfônico/farmacologia , DNA de Cadeia Simples/metabolismo , Relação Dose-Resposta a Droga , Rad51 Recombinase/antagonistas & inibidores
5.
Nat Commun ; 12(1): 5617, 2021 09 23.
Artigo em Inglês | MEDLINE | ID: mdl-34556671

RESUMO

Although prime editing is a promising genome editing method, the efficiency of prime editor 2 (PE2) is often insufficient. Here we generate a more efficient variant of PE2, named hyPE2, by adding the Rad51 DNA-binding domain. When tested at endogenous sites, hyPE2 shows a median of 1.5- or 1.4- fold (range, 0.99- to 2.6-fold) higher efficiencies than PE2; furthermore, at sites where PE2-induced prime editing is very inefficient (efficiency < 1%), hyPE2 enables prime editing with efficiencies ranging from 1.1% to 2.9% at up to 34% of target sequences, potentially facilitating prime editing applications.


Assuntos
Algoritmos , Sistemas CRISPR-Cas , DNA/metabolismo , Edição de Genes/métodos , Modelos Genéticos , Rad51 Recombinase/metabolismo , Sequência de Aminoácidos , Sítios de Ligação/genética , DNA/genética , Células HCT116 , Células HEK293 , Humanos , Rad51 Recombinase/genética , Reprodutibilidade dos Testes
6.
Nat Commun ; 12(1): 5380, 2021 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-34508092

RESUMO

The RAD51 recombinase plays critical roles in safeguarding genome integrity, which is fundamentally important for all living cells. While interphase functions of RAD51 in maintaining genome stability are well-characterised, its role in mitosis remains contentious. In this study, we show that RAD51 protects under-replicated DNA in mitotic human cells and, in this way, promotes mitotic DNA synthesis (MiDAS) and successful chromosome segregation. In cells experiencing mild replication stress, MiDAS was detected irrespective of mitotically generated DNA damage. MiDAS broadly required de novo RAD51 recruitment to single-stranded DNA, which was supported by the phosphorylation of RAD51 by the key mitotic regulator Polo-like kinase 1. Importantly, acute inhibition of MiDAS delayed anaphase onset and induced centromere fragility, suggesting a mechanism that prevents the satisfaction of the spindle assembly checkpoint while chromosomal replication remains incomplete. This study hence identifies an unexpected function of RAD51 in promoting genomic stability in mitosis.


Assuntos
Anáfase/genética , Cromatina/metabolismo , Reparo do DNA , Pontos de Checagem da Fase M do Ciclo Celular/genética , Rad51 Recombinase/metabolismo , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Segregação de Cromossomos , DNA/biossíntese , Dano ao DNA , Replicação do DNA , Instabilidade Genômica , Humanos , Microscopia Intravital , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo
7.
Int J Mol Sci ; 22(17)2021 Aug 28.
Artigo em Inglês | MEDLINE | ID: mdl-34502253

RESUMO

In the past, major findings in meiosis have been achieved, but questions towards the global understanding of meiosis remain concealed. In plants, one of these questions covers the need for two diverse meiotic active SPO11 proteins. In Arabidopsis and other plants, both meiotic SPO11 are indispensable in a functional form for double strand break induction during meiotic prophase I. This stands in contrast to mammals and fungi, where a single SPO11 is present and sufficient. We aimed to investigate the specific function and evolution of both meiotic SPO11 paralogs in land plants. By performing immunostaining of both SPO11-1 and -2, an investigation of the spatiotemporal localization of each SPO11 during meiosis was achieved. We further exchanged SPO11-1 and -2 in Arabidopsis and could show a species-specific function of the respective SPO11. By additional changes of regions between SPO11-1 and -2, a sequence-specific function for both the SPO11 proteins was revealed. Furthermore, the previous findings about the aberrant splicing of each SPO11 were refined by narrowing them down to a specific developmental phase. These findings let us suggest that the function of both SPO11 paralogs is highly sequence specific and that the orthologs are species specific.


Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Endodesoxirribonucleases/genética , Splicing de RNA/genética , Arabidopsis/química , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/fisiologia , Quebras de DNA de Cadeia Dupla , Proteínas de Ligação a DNA/metabolismo , Endodesoxirribonucleases/química , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/fisiologia , Meiose/fisiologia , Rad51 Recombinase/metabolismo , Recombinação Genética , Especificidade da Espécie
8.
Elife ; 102021 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-34477552

RESUMO

DNA double-strand break (DSB) repair by homologous recombination (HR) is thought to be restricted to the S- and G2- phases of the cell cycle in part due to 53BP1 antagonizing DNA end resection in G1-phase and non-cycling quiescent (G0) cells. Here, we show that LIN37, a component of the DREAM transcriptional repressor, functions in a 53BP1-independent manner to prevent DNA end resection and HR in G0 cells. Loss of LIN37 leads to the expression of HR proteins, including BRCA1, BRCA2, PALB2, and RAD51, and promotes DNA end resection in G0 cells even in the presence of 53BP1. In contrast to 53BP1-deficiency, DNA end resection in LIN37-deficient G0 cells depends on BRCA1 and leads to RAD51 filament formation and HR. LIN37 is not required to protect DNA ends in cycling cells at G1-phase. Thus, LIN37 regulates a novel 53BP1-independent cell phase-specific DNA end protection pathway that functions uniquely in quiescent cells.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , Rad51 Recombinase/metabolismo , Transativadores/metabolismo , Proteína BRCA1/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Replicação do DNA , Fase G1 , Fase G2 , Recombinação Homóloga , Humanos , Fase S , Transativadores/genética , Proteína 1 de Ligação à Proteína Supressora de Tumor p53/metabolismo
9.
Nat Commun ; 12(1): 5514, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34535663

RESUMO

Human shelterin components POT1 and TPP1 form a stable heterodimer that protects telomere ends from ATR-dependent DNA damage responses and regulates telomerase-dependent telomere extension. Mice possess two functionally distinct POT1 proteins. POT1a represses ATR/CHK1 DNA damage responses and the alternative non-homologous end-joining DNA repair pathway while POT1b regulates C-strand resection and recruits the CTC1-STN1-TEN1 (CST) complex to telomeres to mediate C-strand fill-in synthesis. Whether POT1a and POT1b are involved in regulating the length of the telomeric G-strand is unclear. Here we demonstrate that POT1b, independent of its CST function, enhances recruitment of telomerase to telomeres through three amino acids in its TPP1 interacting C-terminus. POT1b thus coordinates the synthesis of both telomeric G- and C-strands. In contrast, POT1a negatively regulates telomere length by inhibiting telomerase recruitment to telomeres. The identification of unique amino acids between POT1a and POT1b helps us understand mechanistically how human POT1 switches between end protective functions and promoting telomerase recruitment.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Telomerase/metabolismo , Proteínas de Ligação a Telômeros/metabolismo , Telômero/metabolismo , Animais , Sistemas CRISPR-Cas/genética , Análise Mutacional de DNA , Camundongos , Ligação Proteica , Rad51 Recombinase/metabolismo , Sarcoma/patologia
10.
Nat Commun ; 12(1): 5545, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545070

RESUMO

The RAD51 recombinase assembles as helical nucleoprotein filaments on single-stranded DNA (ssDNA) and mediates invasion and strand exchange with homologous duplex DNA (dsDNA) during homologous recombination (HR), as well as protection and restart of stalled replication forks. Strand invasion by RAD51-ssDNA complexes depends on ATP binding. However, RAD51 can bind ssDNA in non-productive ADP-bound or nucleotide-free states, and ATP-RAD51-ssDNA complexes hydrolyse ATP over time. Here, we define unappreciated mechanisms by which the RAD51 paralog complex RFS-1/RIP-1 limits the accumulation of RAD-51-ssDNA complexes with unfavorable nucleotide content. We find RAD51 paralogs promote the turnover of ADP-bound RAD-51 from ssDNA, in striking contrast to their ability to stabilize productive ATP-bound RAD-51 nucleoprotein filaments. In addition, RFS-1/RIP-1 inhibits binding of nucleotide-free RAD-51 to ssDNA. We propose that 'nucleotide proofreading' activities of RAD51 paralogs co-operate to ensure the enrichment of active, ATP-bound RAD-51 filaments on ssDNA to promote HR.


Assuntos
Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/metabolismo , Nucleotídeos/metabolismo , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo , Homologia de Sequência de Aminoácidos , Difosfato de Adenosina/farmacologia , Trifosfato de Adenosina/farmacologia , Animais , DNA de Cadeia Simples/metabolismo , Fluorescência , Interferometria , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Especificidade da Espécie
11.
Biomolecules ; 11(7)2021 07 03.
Artigo em Inglês | MEDLINE | ID: mdl-34356606

RESUMO

The genetic principle of synthetic lethality has most successfully been exploited in therapies engaging Poly-ADP-ribose-polymerase (PARP) inhibitors to treat patients with homologous recombination (HR)-defective tumors. In this work, we went a step further following the idea of a local molecular cooperation and designed hybrid compounds M1-M3. The drug conjugates M1-M3 combine Olaparib, the first PARP inhibitor approved for clinical use, with Cpd 1, an inhibitor of RAD51 that blocks its HR functions and yet permits RAD51 nucleoprotein filament formation on single-stranded DNA. While in M2 and M3, the parental drugs are linked by -CO-(CH2)n-CO-spacers (n = 2 and 4, respectively), they are directly merged omitting the piperazine ring of Olaparib in M1. Monitoring anti-survival effects of M1-M3 in six breast cancer cell lines of different molecular subtypes showed that in each cell line, at least one of the drug conjugates decreased viability by one to two orders of magnitude compared with parental drugs. While triple-negative breast cancer (TNBC) cells with frequent BRCA1 pathway dysfunction were sensitive to spacer-linked hybrid compounds M1 and M2 regardless of their HR capacities, non-TNBC cells were responsive to the merged drug conjugate M1 only, suggesting different spatial requirements for dual inhibition in these two groups of cell lines. These results demonstrate that, depending on chemical linkage, dual PARP1-RAD51 inhibitory drugs can either sensitize non-TNBC and re-sensitize TNBC cells, or discriminate between these groups of cells.


Assuntos
Antineoplásicos , Proteínas de Neoplasias/antagonistas & inibidores , Ftalazinas , Piperazinas , Inibidores de Poli(ADP-Ribose) Polimerases , Rad51 Recombinase/antagonistas & inibidores , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Feminino , Humanos , Células MCF-7 , Proteínas de Neoplasias/metabolismo , Ftalazinas/química , Ftalazinas/farmacologia , Piperazinas/química , Piperazinas/farmacologia , Inibidores de Poli(ADP-Ribose) Polimerases/síntese química , Inibidores de Poli(ADP-Ribose) Polimerases/química , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Poli(ADP-Ribose) Polimerases/metabolismo , Rad51 Recombinase/metabolismo , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologia
12.
Nat Commun ; 12(1): 5016, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408138

RESUMO

DNA damage prompts a diverse range of alterations to the chromatin landscape. The RNF168 E3 ubiquitin ligase catalyzes the mono-ubiquitination of histone H2A at lysine (K)13/15 (mUb-H2A), forming a binding module for DNA repair proteins. BRCA1 promotes homologous recombination (HR), in part, through its interaction with PALB2, and the formation of a larger BRCA1-PALB2-BRCA2-RAD51 (BRCA1-P) complex. The mechanism by which BRCA1-P is recruited to chromatin surrounding DNA breaks is unclear. In this study, we reveal that an RNF168-governed signaling pathway is responsible for localizing the BRCA1-P complex to DNA damage. Using mice harboring a Brca1CC (coiled coil) mutation that blocks the Brca1-Palb2 interaction, we uncovered an epistatic relationship between Rnf168- and Brca1CC alleles, which disrupted development, and reduced the efficiency of Palb2-Rad51 localization. Mechanistically, we show that RNF168-generated mUb-H2A recruits BARD1 through a BRCT domain ubiquitin-dependent recruitment motif (BUDR). Subsequently, BARD1-BRCA1 accumulate PALB2-RAD51 at DNA breaks via the CC domain-mediated BRCA1-PALB2 interaction. Together, these findings establish a series of molecular interactions that connect the DNA damage signaling and HR repair machinery.


Assuntos
Proteína BRCA1/metabolismo , Dano ao DNA , Proteína do Grupo de Complementação N da Anemia de Fanconi/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Animais , Proteína BRCA1/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , DNA/genética , DNA/metabolismo , Proteína do Grupo de Complementação N da Anemia de Fanconi/genética , Histonas/genética , Histonas/metabolismo , Humanos , Camundongos , Ligação Proteica , Transporte Proteico , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Reparo de DNA por Recombinação , Proteínas Supressoras de Tumor/genética , Ubiquitina-Proteína Ligases/genética , Ubiquitinação
13.
Elife ; 102021 08 24.
Artigo em Inglês | MEDLINE | ID: mdl-34427184

RESUMO

Meiosis is conserved across eukaryotes yet varies in the details of its execution. Here we describe a new comparative model system for molecular analysis of meiosis, the nematode Pristionchus pacificus, a distant relative of the widely studied model organism Caenorhabditis elegans. P. pacificus shares many anatomical and other features that facilitate analysis of meiosis in C. elegans. However, while C. elegans has lost the meiosis-specific recombinase Dmc1 and evolved a recombination-independent mechanism to synapse its chromosomes, P. pacificus expresses both DMC-1 and RAD-51. We find that SPO-11 and DMC-1 are required for stable homolog pairing, synapsis, and crossover formation, while RAD-51 is dispensable for these key meiotic processes. RAD-51 and DMC-1 localize sequentially to chromosomes during meiotic prophase and show nonoverlapping functions. We also present a new genetic map for P. pacificus that reveals a crossover landscape very similar to that of C. elegans, despite marked divergence in the regulation of synapsis and crossing-over between these lineages.


Assuntos
Pareamento Cromossômico , Segregação de Cromossomos , Troca Genética , Rabditídios/genética , Animais , Endodesoxirribonucleases/genética , Endodesoxirribonucleases/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Modelos Genéticos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Rabditídios/metabolismo
14.
Genes (Basel) ; 12(6)2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34208492

RESUMO

Targeting DNA repair proteins with small-molecule inhibitors became a proven anti-cancer strategy. Previously, we identified an inhibitor of a major protein of homologous recombination (HR) RAD51, named B02. B02 inhibited HR in human cells and sensitized them to chemotherapeutic drugs in vitro and in vivo. Here, using a medicinal chemistry approach, we aimed to improve the potency of B02. We identified the B02 analog, B02-isomer, which inhibits HR in human cells with significantly higher efficiency. We also show that B02-iso sensitizes triple-negative breast cancer MDA-MB-231 cells to the PARP inhibitor (PARPi) olaparib.


Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Recombinação Homóloga/efeitos dos fármacos , Quinazolinonas/farmacologia , Rad51 Recombinase/antagonistas & inibidores , Antineoplásicos/química , Sítios de Ligação , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Inibidores Enzimáticos/química , Humanos , Simulação de Acoplamento Molecular , Ftalazinas/farmacologia , Piperazinas/farmacologia , Ligação Proteica , Quinazolinonas/química , Rad51 Recombinase/química , Rad51 Recombinase/metabolismo
15.
Theriogenology ; 172: 239-254, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34298284

RESUMO

Meiotic recombination is key to the repair of DNA double-strand break damage, provide a link between homologs for proper chromosome segregation as well as ensure genetic diversity in organisms. Defects in recombination often lead to sterility. The ubiquitously expressed Rad51 and the meiosis-specific DMC1 are two closely related recombinases that catalyze the key strand invasion and exchange step of meiotic recombination. This study cloned and sequenced the coding region of cattle-yak Rad51 and determined its mRNA and protein expression levels, evaluated its molecular and evolutionary relationship as well as evaluated the histo-morphological structure of testes in the yellow cattle, yak and the sterile cattle-yak hybrid. The Rad51 gene was amplified using PCR, cloned and sequenced using testicular cDNA from yak and cattle-yak. Real-time PCR was used to examine the expression levels of Rad51/DMC1 mRNA in the cattle, yak and cattle-yak testis while western blotting, immunofluorescence and immunohistochemistry were used to assess the protein expression and localization of Rad51/DMC1 protein in the testicular tissue sections. The results revealed that the mRNA and protein expression of Rad51 and DMC1 are extremely low in the male cattle-yak testis with a corresponding higher incidence of germ cell apoptosis. There was also thinning of the germinal epithelium possibly due to the depletion of the germ cells leading to the widening of the lumen area of the cattle-yak seminiferous tubule. Our findings provide support for the hypothesis that the low expression of Rad51 and DMC1 may contribute to the male hybrid sterility in the cattle-yak.


Assuntos
Reparo do DNA , Testículo , Animais , Bovinos/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Recombinação Homóloga , Masculino , Meiose , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Testículo/metabolismo
16.
Exp Cell Res ; 406(1): 112742, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34302857

RESUMO

BACKGROUND: Mutations at sites crucial for the interaction between RAD51 and BRC domains impair the ability of BRCA2 homologous recombination. We aimed to clarify whether BRCA2 BRC domain-associated mutation correlates with sensibility of platinum-based chemotherapy and survival in high-grade serous ovarian cancer (HGSOC). METHODS: We identified BRCA2 BRC domain mutations by sequencing PCR-amplified amplicons of genomic DNA isolated from tumor tissues and peripheral blood leukocytes (PBL)in 113 patients with advanced EOC, and assessed platinum-free interval (PFI), progression-free survival (PFS) and overall survival (OS). RESULTS: 21.23% (24 of 113) cases with somatic missense mutation but not germline mutation were identified. Among 24 cases with mutation, 33.3% (8 of 24) cases with nonsense mutation (C-terminal truncation) significantly prolonged median PFI (37 vs 8 months,P = 0.000), PFS (43 vs 14 months, p = 0.000) and OS (56 vs 31 months, P = 0.002); 66.7% (16 of 24) cases with missense mutation also prolonged median PFI (15 vs 8 months, P = 0.044), PFS (21 vs 14 months, P = 0.049) and OS (38 vs 31 months, P = 0.037), compared to those without any mutation. CONCLUSIONS: Somatic mutations in BRCA2 BRC domain confer a higher sensitivity to platinum-based therapy and are associated with a favourable survival in HGSOC.


Assuntos
Proteína BRCA2/genética , Cisplatino/uso terapêutico , Cistadenocarcinoma Seroso/genética , Mutação , Neoplasias Ovarianas/genética , Rad51 Recombinase/genética , Adulto , Sequência de Aminoácidos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Proteína BRCA2/metabolismo , Sequência de Bases , Cistadenocarcinoma Seroso/tratamento farmacológico , Cistadenocarcinoma Seroso/mortalidade , Cistadenocarcinoma Seroso/patologia , Resistencia a Medicamentos Antineoplásicos/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Metástase Linfática , Pessoa de Meia-Idade , Gradação de Tumores , Estadiamento de Neoplasias , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/mortalidade , Neoplasias Ovarianas/patologia , Rad51 Recombinase/metabolismo , Transdução de Sinais , Análise de Sobrevida
17.
Nucleic Acids Res ; 49(13): 7554-7570, 2021 07 21.
Artigo em Inglês | MEDLINE | ID: mdl-34197606

RESUMO

Polo-like kinase 1 (PLK1) is a master kinase that regulates cell cycle progression. How its enzymatic activity is regulated in response to DNA damage is not fully understood. We show that PLK1 is enriched at double strand breaks (DSBs) within seconds of UV laser irradiation in a PARP-1-dependent manner and then disperses within 10 min in a PARG-dependent manner. Poly(ADP-)ribose (PAR) chains directly bind to PLK1 in vitro and inhibit its enzymatic activity. CHK1-mediated PLK1 phosphorylation at S137 prevents its binding to PAR and recruitment to DSBs but ensures PLK1 phosphorylation at T210 and its enzymatic activity toward RAD51 at S14. This subsequent phosphorylation event at S14 primes RAD51 for CHK1-mediated phosphorylation at T309, which is essential for full RAD51 activation. This CHK1-PLK1-RAD51 axis ultimately promotes homologous recombination (HR)-mediated repair and ensures chromosome stability and cellular radiosensitivity. These findings provide biological insight for combined cancer therapy using inhibitors of PARG and CHK1.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Quinase 1 do Ponto de Checagem/metabolismo , Poli(ADP-Ribose) Polimerase-1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Reparo de DNA por Recombinação , Proteínas de Ciclo Celular/antagonistas & inibidores , Linhagem Celular , Quebras de DNA de Cadeia Dupla , Glicosídeo Hidrolases , Humanos , Fosforilação , Poli Adenosina Difosfato Ribose/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Rad51 Recombinase/metabolismo , Raios Ultravioleta
18.
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
19.
Elife ; 102021 07 13.
Artigo em Inglês | MEDLINE | ID: mdl-34254584

RESUMO

Breast cancer type two susceptibility protein (BRCA2) is an essential protein in genome maintenance, homologous recombination (HR), and replication fork protection. Its function includes multiple interaction partners and requires timely localization to relevant sites in the nucleus. We investigated the importance of the highly conserved DNA-binding domain (DBD) and C-terminal domain (CTD) of BRCA2. We generated BRCA2 variants missing one or both domains in mouse embryonic stem (ES) cells and defined their contribution in HR function and dynamic localization in the nucleus, by single-particle tracking of BRCA2 mobility. Changes in molecular architecture of BRCA2 induced by binding partners of purified BRCA2 were determined by scanning force microscopy. BRCA2 mobility and DNA-damage-induced increase in the immobile fraction were largely unaffected by C-terminal deletions. The purified proteins missing CTD and/or DBD were defective in architectural changes correlating with reduced HR function in cells. These results emphasize BRCA2 activity at sites of damage beyond promoting RAD51 delivery.


Assuntos
Proteína BRCA2/química , Proteína BRCA2/genética , Reparo do DNA , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Conformação de Ácido Nucleico , Animais , Proteína BRCA2/metabolismo , DNA/química , DNA/metabolismo , Replicação do DNA , Proteínas de Ligação a DNA/metabolismo , Recombinação Homóloga , Humanos , Camundongos , Células-Tronco Embrionárias Murinas , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Imagem Individual de Molécula
20.
Cells ; 10(6)2021 06 11.
Artigo em Inglês | MEDLINE | ID: mdl-34207997

RESUMO

Homologous recombination (HR) depends on the formation of a nucleoprotein filament of the recombinase Rad51 to scan the genome and invade the homologous sequence used as a template for DNA repair synthesis. Therefore, HR is highly accurate and crucial for genome stability. Rad51 filament formation is controlled by positive and negative factors. In Saccharomyces cerevisiae, the mediator protein Rad52 catalyzes Rad51 filament formation and stabilizes them, mostly by counteracting the disruptive activity of the translocase Srs2. Srs2 activity is essential to avoid the formation of toxic Rad51 filaments, as revealed by Srs2-deficient cells. We previously reported that Rad52 SUMOylation or mutations disrupting the Rad52-Rad51 interaction suppress Rad51 filament toxicity because they disengage Rad52 from Rad51 filaments and reduce their stability. Here, we found that mutations in Rad52 N-terminal domain also suppress the DNA damage sensitivity of Srs2-deficient cells. Structural studies showed that these mutations affect the Rad52 oligomeric ring structure. Overall, in vivo and in vitro analyzes of these mutants indicate that Rad52 ring structure is important for protecting Rad51 filaments from Srs2, but can increase Rad51 filament stability and toxicity in Srs2-deficient cells. This stabilization function is distinct from Rad52 mediator and annealing activities.


Assuntos
DNA Helicases/metabolismo , Recombinação Homóloga , Mutação , Rad51 Recombinase/química , Proteína Rad52 de Recombinação e Reparo de DNA/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Helicases/genética , Domínios Proteicos , Rad51 Recombinase/genética , Rad51 Recombinase/metabolismo , Proteína Rad52 de Recombinação e Reparo de DNA/química , Proteína Rad52 de Recombinação e Reparo de DNA/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genética
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