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1.
Cell Death Dis ; 15(5): 321, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38719812

RESUMO

RAD18, an important ubiquitin E3 ligase, plays a dual role in translesion DNA synthesis (TLS) and homologous recombination (HR) repair. However, whether and how the regulatory mechanism of O-linked N-acetylglucosamine (O-GlcNAc) modification governing RAD18 and its function during these processes remains unknown. Here, we report that human RAD18, can undergo O-GlcNAcylation at Ser130/Ser164/Thr468, which is important for optimal RAD18 accumulation at DNA damage sites. Mechanistically, abrogation of RAD18 O-GlcNAcylation limits CDC7-dependent RAD18 Ser434 phosphorylation, which in turn significantly reduces damage-induced PCNA monoubiquitination, impairs Polη focus formation and enhances UV sensitivity. Moreover, the ubiquitin and RAD51C binding ability of RAD18 at DNA double-strand breaks (DSBs) is O-GlcNAcylation-dependent. O-GlcNAcylated RAD18 promotes the binding of RAD51 to damaged DNA during HR and decreases CPT hypersensitivity. Our findings demonstrate a novel role of RAD18 O-GlcNAcylation in TLS and HR regulation, establishing a new rationale to improve chemotherapeutic treatment.


Assuntos
Acetilglucosamina , Proteínas de Ligação a DNA , Antígeno Nuclear de Célula em Proliferação , Rad51 Recombinase , Reparo de DNA por Recombinação , Ubiquitina-Proteína Ligases , Humanos , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Ubiquitina-Proteína Ligases/metabolismo , Acetilglucosamina/metabolismo , Rad51 Recombinase/metabolismo , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fosforilação , Replicação do DNA , Ubiquitinação , Quebras de DNA de Cadeia Dupla , DNA Polimerase Dirigida por DNA/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Dano ao DNA , DNA/metabolismo , Células HEK293 , Raios Ultravioleta , Ligação Proteica , Glicosilação , Síntese de DNA Translesão
2.
PLoS One ; 19(5): e0288578, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38739603

RESUMO

As a versatile genome editing tool, the CRISPR-Cas9 system induces DNA double-strand breaks at targeted sites to activate mainly two DNA repair pathways: HDR which allows precise editing via recombination with a homologous template DNA, and NHEJ which connects two ends of the broken DNA, which is often accompanied by random insertions and deletions. Therefore, how to enhance HDR while suppressing NHEJ is a key to successful applications that require precise genome editing. Histones are small proteins with a lot of basic amino acids that generate electrostatic affinity to DNA. Since H2A.X is involved in DNA repair processes, we fused H2A.X to Cas9 and found that this fusion protein could improve the HDR/NHEJ ratio by suppressing NHEJ. As various post-translational modifications of H2A.X play roles in the regulation of DNA repair, we also fused H2A.X mimicry variants to replicate these post-translational modifications including phosphorylation, methylation, and acetylation. However, none of them were effective to improve the HDR/NHEJ ratio. We further fused other histone variants to Cas9 and found that H2A.1 suppressed NHEJ better than H2A.X. Thus, the fusion of histone variants to Cas9 is a promising option to enhance precise genome editing.


Assuntos
Proteína 9 Associada à CRISPR , Sistemas CRISPR-Cas , Reparo do DNA por Junção de Extremidades , Edição de Genes , Histonas , Histonas/metabolismo , Histonas/genética , Humanos , Proteína 9 Associada à CRISPR/metabolismo , Proteína 9 Associada à CRISPR/genética , Edição de Genes/métodos , Processamento de Proteína Pós-Traducional , Quebras de DNA de Cadeia Dupla , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/genética , Células HEK293 , Acetilação
3.
Sci Rep ; 14(1): 11468, 2024 05 20.
Artigo em Inglês | MEDLINE | ID: mdl-38769339

RESUMO

Diffusing alpha-emitters radiation therapy (Alpha-DaRT) is a unique method, in which interstitial sources carrying 224Ra release a chain of short-lived daughter atoms from their surface. Although DNA damage response (DDR) is crucial to inducing cell death after irradiation, how the DDR occurs during Alpha-DaRT treatment has not yet been explored. In this study, we temporo-spatially characterized DDR such as kinetics of DNA double-strand breaks (DSBs) and cell cycle, in two-dimensional (2D) culture conditions qualitatively mimicking Alpha-DaRT treatments, by employing HeLa cells expressing the Fucci cell cycle-visualizing system. The distribution of the alpha-particle pits detected by a plastic nuclear track detector, CR-39, strongly correlated with γH2AX staining, a marker of DSBs, around the 224Ra source, but the area of G2 arrested cells was more widely spread 24 h from the start of the exposure. Thereafter, close time-lapse observation revealed varying cell cycle kinetics, depending on the distance from the source. A medium containing daughter nuclides prepared from 224Ra sources allowed us to estimate the radiation dose after 24 h of exposure, and determine surviving fractions. The present experimental model revealed for the first time temporo-spatial information of DDR occurring around the source in its early stages.


Assuntos
Partículas alfa , Quebras de DNA de Cadeia Dupla , Humanos , Células HeLa , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Dano ao DNA/efeitos da radiação , Ciclo Celular/efeitos da radiação , Histonas/metabolismo , Técnicas de Cultura de Células/métodos
4.
Int J Mol Sci ; 25(9)2024 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-38731948

RESUMO

Based on the need for radiobiological databases, in this work, we mined experimental ionizing radiation data of human cells treated with X-rays, γ-rays, carbon ions, protons and α-particles, by manually searching the relevant literature in PubMed from 1980 until 2024. In order to calculate normal and tumor cell survival α and ß coefficients of the linear quadratic (LQ) established model, as well as the initial values of the double-strand breaks (DSBs) in DNA, we used WebPlotDigitizer and Python programming language. We also produced complex DNA damage results through the fast Monte Carlo code MCDS in order to complete any missing data. The calculated α/ß values are in good agreement with those valued reported in the literature, where α shows a relatively good association with linear energy transfer (LET), but not ß. In general, a positive correlation between DSBs and LET was observed as far as the experimental values are concerned. Furthermore, we developed a biophysical prediction model by using machine learning, which showed a good performance for α, while it underscored LET as the most important feature for its prediction. In this study, we designed and developed the novel radiobiological 'RadPhysBio' database for the prediction of irradiated cell survival (α and ß coefficients of the LQ model). The incorporation of machine learning and repair models increases the applicability of our results and the spectrum of potential users.


Assuntos
Sobrevivência Celular , Quebras de DNA de Cadeia Dupla , Transferência Linear de Energia , Radiação Ionizante , Radiobiologia , Humanos , Sobrevivência Celular/efeitos da radiação , Radiobiologia/métodos , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Bases de Dados Factuais , Método de Monte Carlo
5.
Proc Natl Acad Sci U S A ; 121(19): e2401386121, 2024 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-38696471

RESUMO

In the meiotic prophase, programmed DNA double-strand breaks are repaired by meiotic recombination. Recombination-defective meiocytes are eliminated to preserve genome integrity in gametes. BRCA1 is a critical protein in somatic homologous recombination, but studies have suggested that BRCA1 is dispensable for meiotic recombination. Here we show that BRCA1 is essential for meiotic recombination. Interestingly, BRCA1 also has a function in eliminating recombination-defective oocytes. Brca1 knockout (KO) rescues the survival of Dmc1 KO oocytes far more efficiently than removing CHK2, a vital component of the DNA damage checkpoint in oocytes. Mechanistically, BRCA1 activates chromosome asynapsis checkpoint by promoting ATR activity at unsynapsed chromosome axes in Dmc1 KO oocytes. Moreover, Brca1 KO also rescues the survival of asynaptic Spo11 KO oocytes. Collectively, our study not only unveils an unappreciated role of chromosome asynapsis in eliminating recombination-defective oocytes but also reveals the dual functions of BRCA1 in safeguarding oocyte genome integrity.


Assuntos
Proteína BRCA1 , Proteínas de Ciclo Celular , Camundongos Knockout , Oócitos , Oócitos/metabolismo , Animais , Proteína BRCA1/genética , Proteína BRCA1/metabolismo , Feminino , Camundongos , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Meiose/genética , Proteínas Mutadas de Ataxia Telangiectasia/genética , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteínas Mutadas de Ataxia Telangiectasia/deficiência , Quebras de DNA de Cadeia Dupla , Pareamento Cromossômico/genética , Endodesoxirribonucleases/metabolismo , Endodesoxirribonucleases/genética , Quinase do Ponto de Checagem 2/genética , Quinase do Ponto de Checagem 2/metabolismo , Proteínas de Ligação a Fosfato/metabolismo , Proteínas de Ligação a Fosfato/genética , Recombinação Genética , Recombinação Homóloga , Instabilidade Genômica
6.
J Med Virol ; 96(5): e29674, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38757834

RESUMO

Human Papillomaviruses (HPV) are a diverse family of non-enveloped dsDNA viruses that infect the skin and mucosal epithelia. Persistent HPV infections can lead to cancer frequently involving integration of the virus into the host genome, leading to sustained oncogene expression and loss of capsid and genome maintenance proteins. Microhomology-mediated double-strand break repair, a DNA double-stranded breaks repair pathway present in many organisms, was initially thought to be a backup but it's now seen as vital, especially in homologous recombination-deficient contexts. Increasing evidence has identified microhomology (MH) near HPV integration junctions, suggesting MH-mediated repair pathways drive integration. In this comprehensive review, we present a detailed summary of both the mechanisms underlying MH-mediated repair and the evidence for its involvement in HPV integration in cancer. Lastly, we highlight the involvement of these processes in the integration of other DNA viruses and the broader implications on virus lifecycles and host innate immune response.


Assuntos
Carcinogênese , Papillomaviridae , Infecções por Papillomavirus , Humanos , Papillomaviridae/patogenicidade , Papillomaviridae/genética , Papillomaviridae/fisiologia , Infecções por Papillomavirus/virologia , Infecções por Papillomavirus/complicações , Integração Viral , Reparo do DNA , Quebras de DNA de Cadeia Dupla , DNA Viral/genética
7.
Signal Transduct Target Ther ; 9(1): 135, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38760366

RESUMO

DNA double-strand break (DSB) sites that prevent the disjunction of broken DNA ends are formed through poly (ADP-ribose) (PAR) polymerase 1 (PARP1)-DNA co-condensation. The co-condensates apply mechanical forces to hold the DNA ends together and generate enzymatic activity for the synthesis of PAR. PARylation can promote the release of PARP1 from DNA ends and recruit various proteins, such as Fused in sarcoma (FUS) proteins, thereby stabilizing broken DNA ends and preventing their separation.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , DNA , Poli(ADP-Ribose) Polimerase-1 , Humanos , Poli(ADP-Ribose) Polimerase-1/genética , Poli(ADP-Ribose) Polimerase-1/metabolismo , Reparo do DNA/genética , DNA/genética , DNA/metabolismo
8.
Sci Rep ; 14(1): 10400, 2024 05 06.
Artigo em Inglês | MEDLINE | ID: mdl-38710823

RESUMO

Without the protective shielding of Earth's atmosphere, astronauts face higher doses of ionizing radiation in space, causing serious health concerns. Highly charged and high energy (HZE) particles are particularly effective in causing complex and difficult-to-repair DNA double-strand breaks compared to low linear energy transfer. Additionally, chronic cortisol exposure during spaceflight raises further concerns, although its specific impact on DNA damage and repair remains unknown. This study explorers the effect of different radiation qualities (photons, protons, carbon, and iron ions) on the DNA damage and repair of cortisol-conditioned primary human dermal fibroblasts. Besides, we introduce a new measure, the Foci-Integrated Damage Complexity Score (FIDCS), to assess DNA damage complexity by analyzing focus area and fluorescent intensity. Our results show that the FIDCS captured the DNA damage induced by different radiation qualities better than counting the number of foci, as traditionally done. Besides, using this measure, we were able to identify differences in DNA damage between cortisol-exposed cells and controls. This suggests that, besides measuring the total number of foci, considering the complexity of the DNA damage by means of the FIDCS can provide additional and, in our case, improved information when comparing different radiation qualities.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Fibroblastos , Hidrocortisona , Humanos , Fibroblastos/efeitos da radiação , Fibroblastos/metabolismo , Quebras de DNA de Cadeia Dupla/efeitos da radiação , Hidrocortisona/farmacologia , Radiação Ionizante , Células Cultivadas , Dano ao DNA
10.
Phys Med ; 121: 103367, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38701625

RESUMO

PURPOSE: Diffusing alpha-emitters radiation therapy (DaRT) is a brachytherapy technique using α-particles to treat solid tumours. The high linear energy transfer (LET) and short range of α-particles make them good candidates for the targeted treatment of cancer. Treatment planning of DaRT requires a good understanding of the dose from α-particles and the other particles released in the 224Ra decay chain. METHODS: The Geant4 Monte Carlo toolkit has been used to simulate a DaRT seed to better understand the dose contribution from all particles and simulate the DNA damage due to this treatment. RESULTS: Close to the seed α-particles deliver the majority of dose, however at radial distances greater than 4 mm, the contribution of ß-particles is greater. The RBE has been estimated as a function of number of double strand breaks (DSBs) and complex DSBs. A maximum seed spacing of 5.5 mm and 6.5 mm was found to deliver at least 20 Gy RBE weighted dose between the seeds for RBEDSB and RBEcDSB respectively. CONCLUSIONS: The DNA damage changes with radial distance from the seed and has been found to become less complex with distance, which is potentially easier for the cell to repair. Close to the seed α-particles contribute the majority of dose, however the contribution from other particles cannot be neglected and may influence the choice of seed spacing.


Assuntos
Partículas alfa , Dano ao DNA , Método de Monte Carlo , Partículas alfa/uso terapêutico , Dosagem Radioterapêutica , Doses de Radiação , Eficiência Biológica Relativa , Difusão , Braquiterapia/métodos , Humanos , Transferência Linear de Energia , Planejamento da Radioterapia Assistida por Computador/métodos , Quebras de DNA de Cadeia Dupla/efeitos da radiação
11.
Nat Commun ; 15(1): 2890, 2024 Apr 03.
Artigo em Inglês | MEDLINE | ID: mdl-38570537

RESUMO

DNA double-strand breaks (DSBs) can be repaired by several pathways. In eukaryotes, DSB repair pathway choice occurs at the level of DNA end resection and is controlled by the cell cycle. Upon cell cycle-dependent activation, cyclin-dependent kinases (CDKs) phosphorylate resection proteins and thereby stimulate end resection and repair by homologous recombination (HR). However, inability of CDK phospho-mimetic mutants to bypass this cell cycle regulation, suggests that additional cell cycle regulators may be important. Here, we identify Dbf4-dependent kinase (DDK) as a second major cell cycle regulator of DNA end resection. Using inducible genetic and chemical inhibition of DDK in budding yeast and human cells, we show that end resection and HR require activation by DDK. Mechanistically, DDK phosphorylates at least two resection nucleases in budding yeast: the Mre11 activator Sae2, which promotes resection initiation, as well as the Dna2 nuclease, which promotes resection elongation. Notably, synthetic activation of DDK allows limited resection and HR in G1 cells, suggesting that DDK is a key component of DSB repair pathway selection.


Assuntos
Quebras de DNA de Cadeia Dupla , Proteínas de Saccharomyces cerevisiae , Humanos , Ciclo Celular , Recombinação Homóloga , Divisão Celular , Endonucleases/metabolismo , Quinases Ciclina-Dependentes/genética , Quinases Ciclina-Dependentes/metabolismo , DNA , Reparo do DNA , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
12.
Nat Commun ; 15(1): 2941, 2024 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-38580643

RESUMO

Programmed DNA double-strand break (DSB) formation is a crucial feature of meiosis in most organisms. DSBs initiate recombination-mediated linking of homologous chromosomes, which enables correct chromosome segregation in meiosis. DSBs are generated on chromosome axes by heterooligomeric focal clusters of DSB-factors. Whereas DNA-driven protein condensation is thought to assemble the DSB-machinery, its targeting to chromosome axes is poorly understood. We uncover in mice that efficient biogenesis of DSB-machinery clusters requires seeding by axial IHO1 platforms. Both IHO1 phosphorylation and formation of axial IHO1 platforms are diminished by chemical inhibition of DBF4-dependent kinase (DDK), suggesting that DDK contributes to the control of the axial DSB-machinery. Furthermore, we show that axial IHO1 platforms are based on an interaction between IHO1 and the chromosomal axis component HORMAD1. IHO1-HORMAD1-mediated seeding of the DSB-machinery on axes ensures sufficiency of DSBs for efficient pairing of homologous chromosomes. Without IHO1-HORMAD1 interaction, residual DSBs depend on ANKRD31, which enhances both the seeding and the growth of DSB-machinery clusters. Thus, recombination initiation is ensured by complementary pathways that differentially support seeding and growth of DSB-machinery clusters, thereby synergistically enabling DSB-machinery condensation on chromosomal axes.


Assuntos
Proteínas de Ciclo Celular , Quebras de DNA de Cadeia Dupla , Camundongos , Animais , Proteínas de Ciclo Celular/metabolismo , DNA , Meiose/genética , Complexo Sinaptonêmico/metabolismo , Recombinação Genética , Recombinação Homóloga
13.
Sci Rep ; 14(1): 8797, 2024 04 16.
Artigo em Inglês | MEDLINE | ID: mdl-38627415

RESUMO

Deletions of chromosome 1p (del(1p)) are a recurrent genomic aberration associated with poor outcome in Multiple myeloma (MM.) TRIM33, an E3 ligase and transcriptional co-repressor, is located within a commonly deleted region at 1p13.2. TRIM33 is reported to play a role in the regulation of mitosis and PARP-dependent DNA damage response (DDR), both of which are important for maintenance of genome stability. Here, we demonstrate that MM patients with loss of TRIM33 exhibit increased chromosomal instability and poor outcome. Through knockdown studies, we show that TRIM33 loss induces a DDR defect, leading to accumulation of DNA double strand breaks (DSBs) and slower DNA repair kinetics, along with reduced efficiency of non-homologous end joining (NHEJ). Furthermore, TRIM33 loss results in dysregulated ubiquitination of ALC1, an important regulator of response to PARP inhibition. We show that TRIM33 knockdown sensitizes MM cells to the PARP inhibitor Olaparib, and this is synergistic with the standard of care therapy bortezomib, even in co-culture with bone marrow stromal cells (BMSCs). These findings suggest that TRIM33 loss contributes to the pathogenesis of high-risk MM and that this may be therapeutically exploited through the use of PARP inhibitors.


Assuntos
Mieloma Múltiplo , Inibidores de Poli(ADP-Ribose) Polimerases , Humanos , Inibidores de Poli(ADP-Ribose) Polimerases/farmacologia , Reparo do DNA , Mieloma Múltiplo/tratamento farmacológico , Mieloma Múltiplo/genética , Quebras de DNA de Cadeia Dupla , Instabilidade Genômica , Fatores de Transcrição
14.
Cell Cycle ; 23(4): 339-352, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38557443

RESUMO

REV7 is an abundant, multifunctional protein that is a known factor in cell cycle regulation and in several key DNA repair pathways including Trans-Lesion Synthesis (TLS), the Fanconi Anemia (FA) pathway, and DNA Double-Strand Break (DSB) repair pathway choice. Thus far, no direct role has been studied for REV7 in the DNA damage response (DDR) signaling pathway. Here we describe a novel function for REV7 in DSB-induced p53 signaling. We show that REV7 binds directly to p53 to block ATM-dependent p53 Ser15 phosphorylation. We also report that REV7 is involved in the destabilization of p53. These findings affirm REV7's participation in fundamental cell cycle and DNA repair pathways. Furthermore, they highlight REV7 as a critical factor for the integration of multiple processes that determine viability and genome stability.


Assuntos
Proteínas Mutadas de Ataxia Telangiectasia , Dano ao DNA , Transdução de Sinais , Proteína Supressora de Tumor p53 , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Proteína Supressora de Tumor p53/genética , Humanos , Fosforilação , Quebras de DNA de Cadeia Dupla , Ligação Proteica , Reparo do DNA , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação a DNA/genética , Linhagem Celular Tumoral
15.
Pharmacol Res ; 203: 107165, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38561112

RESUMO

The clinical use of the DNA damaging anticancer drug doxorubicin (DOX) is limited by irreversible cardiotoxicity, which depends on the cumulative dose. The RAS-homologous (RHO) small GTPase RAC1 contributes to DOX-induced DNA damage formation and cardiotoxicity. However, the pathophysiological relevance of other RHO GTPases than RAC1 and different cardiac cell types (i.e., cardiomyocytes, non-cardiomyocytes) for DOX-triggered cardiac damage is unclear. Employing diverse in vitro and in vivo models, we comparatively investigated the level of DOX-induced DNA damage in cardiomyocytes versus non-cardiomyocytes (endothelial cells and fibroblasts), in the presence or absence of selected RHO GTPase inhibitors. Non-cardiomyocytes exhibited the highest number of DOX-induced DNA double-strand breaks (DSB), which were efficiently repaired in vitro. By contrast, rather low levels of DSB were formed in cardiomyocytes, which however remained largely unrepaired. Moreover, DOX-induced apoptosis was detected only in non-cardiomyocytes but not in cardiomyocytes. Pharmacological inhibitors of RAC1 and CDC42 most efficiently attenuated DOX-induced DNA damage in all cell types examined in vitro. Consistently, immunohistochemical analyses revealed that the RAC1 inhibitor NSC23766 and the pan-RHO GTPase inhibitor lovastatin reduced the level of DOX-induced residual DNA damage in both cardiomyocytes and non-cardiomyocytes in vivo. Overall, we conclude that endothelial cells, fibroblasts and cardiomyocytes contribute to the pathophysiology of DOX-induced cardiotoxicity, with RAC1- and CDC42-regulated signaling pathways being especially relevant for DOX-stimulated DSB formation and DNA damage response (DDR) activation. Hence, we suggest dual targeting of RAC1/CDC42-dependent mechanisms in multiple cardiac cell types to mitigate DNA damage-dependent cardiac injury evoked by DOX-based anticancer therapy.


Assuntos
Aminoquinolinas , Doxorrubicina , Células Endoteliais , Fibroblastos , Miócitos Cardíacos , Pirimidinas , Proteína cdc42 de Ligação ao GTP , Proteínas rac1 de Ligação ao GTP , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/antagonistas & inibidores , Proteínas rac1 de Ligação ao GTP/genética , Animais , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/patologia , Miócitos Cardíacos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Doxorrubicina/toxicidade , Doxorrubicina/efeitos adversos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/patologia , Células Endoteliais/metabolismo , Cardiotoxicidade , Antibióticos Antineoplásicos/toxicidade , Camundongos , Apoptose/efeitos dos fármacos , Masculino , Humanos , Camundongos Endogâmicos C57BL , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Neuropeptídeos/metabolismo , Dano ao DNA/efeitos dos fármacos , Células Cultivadas
16.
Br J Cancer ; 130(10): 1621-1634, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38575732

RESUMO

BACKGROUND: DNA double-strand break (DSB) induction and repair are important events for determining cell survival and the outcome of cancer radiotherapy. The DNA-dependent protein kinase (DNA-PK) complex functions at the apex of DSBs repair, and its assembly and activity are strictly regulated by post-translation modifications (PTMs)-associated interactions. However, the PTMs of the catalytic subunit DNA-PKcs and how they affect DNA-PKcs's functions are not fully understood. METHODS: Mass spectrometry analyses were performed to identify the crotonylation sites of DNA-PKcs in response to γ-ray irradiation. Co-immunoprecipitation (Co-IP), western blotting, in vitro crotonylation assays, laser microirradiation assays, in vitro DNA binding assays, in vitro DNA-PK assembly assays and IF assays were employed to confirm the crotonylation, identify the crotonylase and decrotonylase, and elucidate how crotonylation regulates the activity and function of DNA-PKcs. Subcutaneous xenografts of human HeLa GCN5 WT or HeLa GCN5 siRNA cells in BALB/c nude mice were generated and utilized to assess tumor proliferation in vivo after radiotherapy. RESULTS: Here, we reveal that K525 is an important site of DNA-PKcs for crotonylation, and whose level is sharply increased by irradiation. The histone acetyltransferase GCN5 functions as the crotonylase for K525-Kcr, while HDAC3 serves as its dedicated decrotonylase. K525 crotonylation enhances DNA binding activity of DNA-PKcs, and facilitates assembly of the DNA-PK complex. Furthermore, GCN5-mediated K525 crotonylation is indispensable for DNA-PKcs autophosphorylation and the repair of double-strand breaks in the NHEJ pathway. GCN5 suppression significantly sensitizes xenograft tumors of mice to radiotherapy. CONCLUSIONS: Our study defines K525 crotonylation of DNA-PKcs is important for the DNA-PK complex assembly and DSBs repair activity via NHEJ pathway. Targeting GCN5-mediated K525 Kcr of DNA-PKcs may be a promising therapeutic strategy for improving the outcome of cancer radiotherapy.


Assuntos
Quebras de DNA de Cadeia Dupla , Reparo do DNA , Proteína Quinase Ativada por DNA , Camundongos Endogâmicos BALB C , Tolerância a Radiação , Fatores de Transcrição de p300-CBP , Humanos , Animais , Proteína Quinase Ativada por DNA/metabolismo , Camundongos , Fatores de Transcrição de p300-CBP/metabolismo , Células HeLa , Camundongos Nus , Feminino , Processamento de Proteína Pós-Traducional , Neoplasias/radioterapia , Neoplasias/metabolismo , Neoplasias/genética , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Microbiol Res ; 284: 127713, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38608339

RESUMO

Deinococcus radiodurans, with its high homologous recombination (HR) efficiency of double-stranded DNA breaks (DSBs), is a model organism for studying genome stability maintenance and an attractive microbe for industrial applications. Here, we developed an efficient CRISPR/Cpf1 genome editing system in D. radiodurans by evaluating and optimizing double-plasmid strategies and four Cas effector proteins from various organisms, which can precisely introduce different types of template-dependent mutagenesis without off-target toxicity. Furthermore, the role of DNA repair genes in determining editing efficiency in D. radiodurans was evaluated by introducing the CRISPR/Cpf1 system into 13 mutant strains lacking various DNA damage response and repair factors. In addition to the crucial role of RecA-dependent HR required for CRISPR/Cpf1 editing, D. radiodurans showed higher editing efficiency when lacking DdrB, the single-stranded DNA annealing (SSA) protein involved in the RecA-independent DSB repair pathway. This suggests a possible competition between HR and SSA pathways in the CRISPR editing of D. radiodurans. Moreover, off-target effects were observed during the genome editing of the pprI knockout strain, a master DNA damage response gene in Deinococcus species, which suggested that precise regulation of DNA damage response is critical for a high-fidelity genome editing system.


Assuntos
Sistemas CRISPR-Cas , Reparo do DNA , Deinococcus , Edição de Genes , Deinococcus/genética , Edição de Genes/métodos , Reparo do DNA/genética , Genoma Bacteriano , Quebras de DNA de Cadeia Dupla , Recombinação Homóloga , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plasmídeos/genética , Mutagênese , Instabilidade Genômica , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Recombinases Rec A/genética , Recombinases Rec A/metabolismo , Dano ao DNA
18.
Cancer Res Commun ; 4(5): 1199-1210, 2024 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-38630886

RESUMO

Homologous recombination (HR)-related gene alterations are present in a significant subset of prostate, breast, ovarian, pancreatic, lung, and colon cancers rendering these tumors as potential responders to specific DNA damaging agents. A small molecule acylfulvene prodrug, LP-184, metabolizes to an active compound by the oxidoreductase activity of enzyme prostaglandin reductase 1 (PTGR1), which is frequently elevated in multiple solid tumor types. Prior work demonstrated that cancer cell lines deficient in a spectrum of DNA damage repair (DDR) pathway genes show increased susceptibility to LP-184. Here, we investigated the potential of LP-184 in targeting multiple tumors with impaired HR function and its mechanism of action as a DNA damaging agent. LP-184 induced elevated DNA double-strand breaks in HR deficient (HRD) cancer cells. Depletion of key HR components BRCA2 or ataxia telangiectasia mutated (ATM) in cancer cells conferred up to 12-fold increased sensitivity to the LP-184. LP-184 showed nanomolar potency in a diverse range of HRD cancer models, including prostate cancer organoids, leiomyosarcoma cell lines, and patient-derived tumor graft models of lung, pancreatic, and prostate cancers. LP-184 demonstrated complete, durable tumor regression in 10 patient-derived xenograft (PDX) models of HRD triple-negative breast cancer (TNBC) including those resistant to PARP inhibitors (PARPi). LP-184 further displayed strong synergy with PARPi in ovarian and prostate cancer cell lines as well as in TNBC PDX models. These preclinical findings illustrate the potential of LP-184 as a pan-HRD cancer therapeutic. Taken together, our results support continued clinical evaluation of LP-184 in a large subset of HRD solid tumors. SIGNIFICANCE: New agents with activity against DDR-deficient solid tumors refractory to standard-of-care therapies are needed. We report multiple findings supporting the potential for LP-184, a novel alkylating agent with three FDA orphan drug designations, to fill this void clinically: strong nanomolar potency; sustained, durable regression of solid tumor xenografts; synthetic lethality with HR defects. LP-184 adult phase IA trial to assess safety in advanced solid tumors is ongoing.


Assuntos
Antineoplásicos , Recombinação Homóloga , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Camundongos , Linhagem Celular Tumoral , Recombinação Homóloga/efeitos dos fármacos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Neoplasias/tratamento farmacológico , Neoplasias/genética , Feminino , Quebras de DNA de Cadeia Dupla/efeitos dos fármacos , Masculino , Reparo do DNA/efeitos dos fármacos
19.
Nature ; 629(8011): 435-442, 2024 May.
Artigo em Inglês | MEDLINE | ID: mdl-38658751

RESUMO

WRN helicase is a promising target for treatment of cancers with microsatellite instability (MSI) due to its essential role in resolving deleterious non-canonical DNA structures that accumulate in cells with faulty mismatch repair mechanisms1-5. Currently there are no approved drugs directly targeting human DNA or RNA helicases, in part owing to the challenging nature of developing potent and selective compounds to this class of proteins. Here we describe the chemoproteomics-enabled discovery of a clinical-stage, covalent allosteric inhibitor of WRN, VVD-133214. This compound selectively engages a cysteine (C727) located in a region of the helicase domain subject to interdomain movement during DNA unwinding. VVD-133214 binds WRN protein cooperatively with nucleotide and stabilizes compact conformations lacking the dynamic flexibility necessary for proper helicase function, resulting in widespread double-stranded DNA breaks, nuclear swelling and cell death in MSI-high (MSI-H), but not in microsatellite-stable, cells. The compound was well tolerated in mice and led to robust tumour regression in multiple MSI-H colorectal cancer cell lines and patient-derived xenograft models. Our work shows an allosteric approach for inhibition of WRN function that circumvents competition from an endogenous ATP cofactor in cancer cells, and designates VVD-133214 as a promising drug candidate for patients with MSI-H cancers.


Assuntos
Helicase da Síndrome de Werner , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Helicase da Síndrome de Werner/metabolismo , Helicase da Síndrome de Werner/química , Animais , Camundongos , Regulação Alostérica/efeitos dos fármacos , Linhagem Celular Tumoral , Feminino , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/enzimologia , Proteômica , Quebras de DNA de Cadeia Dupla , Instabilidade de Microssatélites , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Modelos Moleculares , Masculino , Cisteína/metabolismo , Cisteína/química
20.
Nucleic Acids Res ; 52(8): 4422-4439, 2024 May 08.
Artigo em Inglês | MEDLINE | ID: mdl-38567724

RESUMO

Efficient repair of DNA double-strand breaks in the Ig heavy chain gene locus is crucial for B-cell antibody class switch recombination (CSR). The regulatory dynamics of the repair pathway direct CSR preferentially through nonhomologous end joining (NHEJ) over alternative end joining (AEJ). Here, we demonstrate that the histone acetyl reader BRD2 suppresses AEJ and aberrant recombination as well as random genomic sequence capture at the CSR junctions. BRD2 deficiency impairs switch (S) region synapse, optimal DNA damage response (DDR), and increases DNA break end resection. Unlike BRD4, a similar bromodomain protein involved in NHEJ and CSR, BRD2 loss does not elevate RPA phosphorylation and R-loop formation in the S region. As BRD2 stabilizes the cohesion loader protein NIPBL in the S regions, the loss of BRD2 or NIPBL shows comparable deregulation of S-S synapsis, DDR, and DNA repair pathway choice during CSR. This finding extends beyond CSR, as NIPBL and BRD4 have been linked to Cornelia de Lange syndrome, a developmental disorder exhibiting defective NHEJ and Ig isotype switching. The interplay between these proteins sheds light on the intricate mechanisms governing DNA repair and immune system functionality.


Assuntos
Reparo do DNA por Junção de Extremidades , Switching de Imunoglobulina , Fatores de Transcrição , Switching de Imunoglobulina/genética , Animais , Camundongos , Reparo do DNA por Junção de Extremidades/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Linfócitos B/imunologia , Linfócitos B/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Serina-Treonina Quinases/genética , Humanos , Recombinação Genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Proteínas que Contêm Bromodomínio
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