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1.
Nat Commun ; 15(1): 8648, 2024 Oct 05.
Artículo en Inglés | MEDLINE | ID: mdl-39368985

RESUMEN

Break-induced replication (BIR) is mutagenic, and thus its use requires tight regulation, yet the underlying mechanisms remain elusive. Here we uncover an important role of 53BP1 in suppressing BIR after end resection at double strand breaks (DSBs), distinct from its end protection activity, providing insight into the mechanisms governing BIR regulation and DSB repair pathway selection. We demonstrate that loss of 53BP1 induces BIR-like hyperrecombination, in a manner dependent on Polα-primase-mediated end fill-in DNA synthesis on single-stranded DNA (ssDNA) overhangs at DSBs, leading to PCNA ubiquitination and PIF1 recruitment to activate BIR. On broken replication forks, where BIR is required for repairing single-ended DSBs (seDSBs), SMARCAD1 displaces 53BP1 to facilitate the localization of ubiquitinated PCNA and PIF1 to DSBs for BIR activation. Hyper BIR associated with 53BP1 deficiency manifests template switching and large deletions, underscoring another aspect of 53BP1 in suppressing genome instability. The synthetic lethal interaction between the 53BP1 and BIR pathways provides opportunities for targeted cancer treatment.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Replicación del ADN , Antígeno Nuclear de Célula en Proliferación , Proteína 1 de Unión al Supresor Tumoral P53 , Ubiquitinación , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Humanos , Animales , Ratones , ADN Helicasas/metabolismo , ADN Helicasas/genética , ADN Helicasas/deficiencia , ADN de Cadena Simple/metabolismo , ADN de Cadena Simple/genética , Inestabilidad Genómica
2.
Methods Enzymol ; 705: 271-309, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39389667

RESUMEN

In humans, DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, proliferating cell nuclear antigen (PCNA), carry out DNA synthesis during lagging strand replication, the initiation of leading strand DNA replication as well as most of the major DNA damage repair pathways. In each of these contexts, pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process that involves the PCNA clamp loader, replication factor C and, depending on the DNA synthesis pathway, the major single strand DNA-binding protein complex, replication protein A (RPA). In a recent report from our laboratory, we designed and utilized direct, ensemble Förster Resonance Energy Transfer approaches to monitor the transient state kinetics of pol δ holoenzyme assembly and initiation of DNA synthesis on P/T junctions engaged by RPA. In this chapter, we detail the original approaches and discuss adaptations that can be utilized to monitor fast kinetic reactions in the millisecond (ms) timescale. All approaches described in this chapter utilize a commercially-available fluorescence spectrophotometer, can be readily evolved for alternative DNA polymerases and P/T DNA substrates, and permit incorporation of protein posttranslational modifications, accessory factors, DNA covalent modifications, accessory factors, enzymes, etc. Hence, these approaches are widely accessible and broadly applicable for characterizing DNA polymerase holoenzyme assembly and initiation of DNA synthesis during any PCNA-dependent DNA synthesis pathway.


Asunto(s)
ADN Polimerasa III , Replicación del ADN , ADN , Transferencia Resonante de Energía de Fluorescencia , Antígeno Nuclear de Célula en Proliferación , Transferencia Resonante de Energía de Fluorescencia/métodos , ADN Polimerasa III/metabolismo , ADN Polimerasa III/química , Humanos , Cinética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Holoenzimas/metabolismo , Holoenzimas/química , ADN/metabolismo , ADN/química , Proteína de Replicación A/metabolismo , Proteína de Replicación A/química , Proteína de Replicación C/metabolismo , Proteína de Replicación C/genética , Proteína de Replicación C/química
3.
Front Cell Infect Microbiol ; 14: 1458913, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39346898

RESUMEN

Introduction: Hepatocellular carcinoma (HCC) is a pressing global issue, with Hepatitis B virus (HBV) infection remaining the primary. Emodin, an anthraquinone compound extracted from the natural plant's. This study investigates the molecular targets and possible mechanisms of emodin in treating HBV-related HCC based on network pharmacology and molecular docking and validates the screened molecular targets through in vitro experiments. Methods: Potential targets related to emodin were obtained through PubChem, CTD, PharmMapper, SuperPred, and TargetNet databases. Potential disease targets for HBV and HCC were identified using the DisGeNET, GeneCards, OMIM, and TTD databases. A Venn diagram was used to determine overlapping genes between the drug and the diseases. Enrichment analysis of these genes was performed using GO and KEGG via bioinformatics websites. The overlapping genes were imported into STRING to construct a protein-protein interaction network. Cytoscape 3.9.1 software was used for visualizing and analyzing the core targets. Molecular docking analysis of the drug and core targets was performed using Schrodinger. The regulatory effects of emodin on these core targets were validate through in vitro experiments. Results: A total of 43 overlapping genes were identified. GO analysis recognized 926 entries, and KEGG analysis identified 135 entries. The main pathways involved in the KEGG analysis included cancer, human cytomegalovirus infection and prostate cancer. The binding energies of emodin with HSP90AA1, PTGS2, GSTP1, SOD2, MAPK3, and PCNA were all less than -5 kcal/mol. Compared to normal liver tissue, the mRNA levels of XRCC1, MAPK3, and PCNA were significantly elevated in liver cancer tissue. The expression levels of XRCC1, HIF1A, MAPK3, and PCNA genes were closely related to HCC progression. High expressions of HSP90AA1, TGFB1, HIF1A, MAPK3, and PCNA were all closely associated with poor prognosis in HCC. In vitro experiments demonstrated that emodin significantly downregulated the expression of HSP90AA1, MAPK3, XRCC1, PCNA, and SOD2, while significantly upregulating the expression of PTGS2 and GSTP1. Conclusion: This study, based on network pharmacology and molecular docking validation, suggests that emodin may exert therapeutic effects on HBV-related HCC by downregulating the expression of XRCC1, MAPK3, PCNA, HSP90AA1, and SOD2, and upregulating the expression of PTGS2 and GSTP1.


Asunto(s)
Carcinoma Hepatocelular , Emodina , Virus de la Hepatitis B , Neoplasias Hepáticas , Simulación del Acoplamiento Molecular , Farmacología en Red , Mapas de Interacción de Proteínas , Emodina/farmacología , Emodina/uso terapéutico , Humanos , Carcinoma Hepatocelular/virología , Carcinoma Hepatocelular/tratamiento farmacológico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/virología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Mapas de Interacción de Proteínas/efectos de los fármacos , Biología Computacional , Antivirales/farmacología , Antivirales/uso terapéutico , Hepatitis B/tratamiento farmacológico , Hepatitis B/complicaciones , Hepatitis B/virología , Células Hep G2 , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral
4.
Am J Hum Genet ; 111(9): 1970-1993, 2024 Sep 05.
Artículo en Inglés | MEDLINE | ID: mdl-39106866

RESUMEN

The precise regulation of DNA replication is vital for cellular division and genomic integrity. Central to this process is the replication factor C (RFC) complex, encompassing five subunits, which loads proliferating cell nuclear antigen onto DNA to facilitate the recruitment of replication and repair proteins and enhance DNA polymerase processivity. While RFC1's role in cerebellar ataxia, neuropathy, and vestibular areflexia syndrome (CANVAS) is known, the contributions of RFC2-5 subunits on human Mendelian disorders is largely unexplored. Our research links bi-allelic variants in RFC4, encoding a core RFC complex subunit, to an undiagnosed disorder characterized by incoordination and muscle weakness, hearing impairment, and decreased body weight. We discovered across nine affected individuals rare, conserved, predicted pathogenic variants in RFC4, all likely to disrupt the C-terminal domain indispensable for RFC complex formation. Analysis of a previously determined cryo-EM structure of RFC bound to proliferating cell nuclear antigen suggested that the variants disrupt interactions within RFC4 and/or destabilize the RFC complex. Cellular studies using RFC4-deficient HeLa cells and primary fibroblasts demonstrated decreased RFC4 protein, compromised stability of the other RFC complex subunits, and perturbed RFC complex formation. Additionally, functional studies of the RFC4 variants affirmed diminished RFC complex formation, and cell cycle studies suggested perturbation of DNA replication and cell cycle progression. Our integrated approach of combining in silico, structural, cellular, and functional analyses establishes compelling evidence that bi-allelic loss-of-function RFC4 variants contribute to the pathogenesis of this multisystemic disorder. These insights broaden our understanding of the RFC complex and its role in human health and disease.


Asunto(s)
Proteína de Replicación C , Humanos , Proteína de Replicación C/genética , Proteína de Replicación C/metabolismo , Masculino , Células HeLa , Femenino , Fenotipo , Replicación del ADN/genética , Adulto , Mutación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Alelos
5.
Protein Sci ; 33(9): e5093, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39180489

RESUMEN

RTEL1 is an essential DNA helicase which plays an important role in various aspects of genome stability, from telomere metabolism to DNA replication, repair and recombination. RTEL1 has been implicated in a number of genetic diseases and cancer development, including glioma, breast, lung and gastrointestinal tumors. RTEL1 is a FeS helicase but, in addition to the helicase core, it comprises a long C-terminal region which includes a number of folded domains connected by intrinsically disordered loops and mediates RTEL1 interaction with factors involved in pivotal cellular pathways. However, information on the architecture and the function of this region is still limited. We expressed and purified a variety of fragments encompassing the folded domains and the unstructured regions. We determined the crystal structure of the second repeat, confirming that it has a fold similar to the harmonin homology domains. SAXS data provide low-resolution information on all the fragments and suggest that the presence of the RING domain affects the overall architecture of the C-terminal region, making the structure significantly more compact. NMR data provide experimental information on the interaction between PCNA and the RTEL1 C-terminal region, revealing a putative low-affinity additional site of interaction. A biochemical analysis shows that the C-terminal region, in addition to a preference for telomeric RNA and DNA G-quadruplexes, has a high affinity for R-loops and D-loops, consistent with the role played by the RTEL1 helicase in homologous recombination, telomere maintenance and preventing replication-transcription conflicts. We further dissected the contribution of each domain in binding different substrates.


Asunto(s)
ADN Helicasas , Humanos , ADN Helicasas/química , ADN Helicasas/metabolismo , ADN Helicasas/genética , Cristalografía por Rayos X , Modelos Moleculares , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Dominios Proteicos , Dispersión del Ángulo Pequeño
6.
Proc Natl Acad Sci U S A ; 121(34): e2315759121, 2024 Aug 20.
Artículo en Inglés | MEDLINE | ID: mdl-39145935

RESUMEN

Ubiquitination status of proliferating cell nuclear antigen (PCNA) is crucial for regulating DNA lesion bypass. After the resolution of fork stalling, PCNA is subsequently deubiquitinated, but the underlying mechanism remains undefined. We found that the N-terminal domain of ATAD5 (ATAD5-N), the largest subunit of the PCNA-unloading complex, functions as a scaffold for Ub-PCNA deubiquitination. ATAD5 recognizes DNA-loaded Ub-PCNA through distinct DNA-binding and PCNA-binding motifs. Furthermore, ATAD5 forms a heterotrimeric complex with UAF1-USP1 deubiquitinase, facilitating the deubiquitination of DNA-loaded Ub-PCNA. ATAD5 also enhances the Ub-PCNA deubiquitination by USP7 and USP11 through specific interactions. ATAD5 promotes the distinct deubiquitination process of UAF1-USP1, USP7, and USP11 for poly-Ub-PCNA. Additionally, ATAD5 mutants deficient in UAF1-binding had increased sensitivity to DNA-damaging agents. Our results ultimately reveal that ATAD5 and USPs cooperate to efficiently deubiquitinate Ub-PCNA prior to its release from the DNA in order to safely deactivate the DNA repair process.


Asunto(s)
ATPasas Asociadas con Actividades Celulares Diversas , Proteínas de Unión al ADN , Antígeno Nuclear de Célula en Proliferación , Ubiquitina Tiolesterasa , Peptidasa Específica de Ubiquitina 7 , Ubiquitinación , ATPasas Asociadas con Actividades Celulares Diversas/metabolismo , ATPasas Asociadas con Actividades Celulares Diversas/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Humanos , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Ubiquitina Tiolesterasa/metabolismo , Ubiquitina Tiolesterasa/genética , Peptidasa Específica de Ubiquitina 7/metabolismo , Peptidasa Específica de Ubiquitina 7/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Tioléster Hidrolasas/metabolismo , Tioléster Hidrolasas/genética , Ubiquitina/metabolismo , Daño del ADN , Unión Proteica , Proteasas Ubiquitina-Específicas
7.
Int J Mol Sci ; 25(16)2024 Aug 08.
Artículo en Inglés | MEDLINE | ID: mdl-39201337

RESUMEN

The fidelity of replication, especially in the presence of DNA damage, is essential for the proper function of cells. Mutations that inactivate genes involved in DNA damage repair or bypass are enriched in several types of cancer cells. Thus, it is important to further our understanding of the mechanisms governing replication fidelity. PCNA is a ring-shaped complex that encircles DNA at the front of the replication fork, at the double-stranded/single-stranded DNA junction. It serves as a processivity factor for the different DNA replication polymerases, allowing them to replicate longer stretches of DNA by physically tethering them to the DNA and preventing their detachment. In addition, PCNA also regulates and coordinates different DNA damage bypass pathways meant to allow DNA replication in the presence of DNA damage. Due to its essentiality and the numerous functions it has in the cell, much is still unclear about PCNA. Here, we utilize PCNA mutants that lower the stability of the PCNA complex on the chromatin, and thus tend to disassociate and fall from the DNA. Using these mutants, we show that PCNA's physical presence on the DNA can prevent DNA misalignment at repetitive sequences, leading to increased mutation formation. We also show that PCNA-interacting proteins play an important role in strengthening the ring's stability on the chromatin. Such repetitive sequence-induced mutations are common in several human diseases and it is important to study their formation and the mechanisms guarding against them.


Asunto(s)
Replicación del ADN , Mutación , Antígeno Nuclear de Célula en Proliferación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Humanos , Daño del ADN , Estabilidad Proteica , Cromatina/metabolismo , Cromatina/genética , ADN/metabolismo , ADN/genética
8.
J Mol Biol ; 436(16): 168695, 2024 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-38969056

RESUMEN

Proliferating cell nuclear antigen (PCNA), the homotrimeric eukaryotic sliding clamp protein, recruits and coordinates the activities of a multitude of proteins that function on DNA at the replication fork. Chromatin assembly factor 1 (CAF-1), one such protein, is a histone chaperone that deposits histone proteins onto DNA immediately following replication. The interaction between CAF-1 and PCNA is essential for proper nucleosome assembly at silenced genomic regions. Most proteins that bind PCNA contain a PCNA-interacting peptide (PIP) motif, a conserved motif containing only eight amino acids. Precisely how PCNA is able to discriminate between binding partners at the replication fork using only these small motifs remains unclear. Yeast CAF-1 contains a PIP motif on its largest subunit, Cac1. We solved the crystal structure of the PIP motif of CAF-1 bound to PCNA using a new strategy to produce stoichiometric quantities of one PIP motif bound to each monomer of PCNA. The PIP motif of CAF-1 binds to the hydrophobic pocket on the front face of PCNA in a similar manner to most known PIP-PCNA interactions. However, several amino acids immediately flanking either side of the PIP motif bind the IDCL or C-terminus of PCNA, as observed for only a couple other known PIP-PCNA interactions. Furthermore, mutational analysis suggests positively charged amino acids in these flanking regions are responsible for the low micromolar affinity of CAF-1 for PCNA, whereas the presence of a negative charge upstream of the PIP prevents a more robust interaction with PCNA. These results provide additional evidence that positive charges within PIP-flanking regions of PCNA-interacting proteins are crucial for specificity and affinity of their recruitment to PCNA at the replication fork.


Asunto(s)
Factor 1 de Ensamblaje de la Cromatina , Modelos Moleculares , Antígeno Nuclear de Célula en Proliferación , Unión Proteica , Saccharomyces cerevisiae , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sitios de Unión , Factor 1 de Ensamblaje de la Cromatina/metabolismo , Factor 1 de Ensamblaje de la Cromatina/química , Factor 1 de Ensamblaje de la Cromatina/genética , Cristalografía por Rayos X , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/química , Antígeno Nuclear de Célula en Proliferación/genética , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética
9.
Zhongguo Dang Dai Er Ke Za Zhi ; 26(7): 757-764, 2024 Jul 15.
Artículo en Chino | MEDLINE | ID: mdl-39014954

RESUMEN

OBJECTIVES: To investigate the protective effects of 2-methoxyestradiol (2ME) against hypoxic pulmonary hypertension (HPH) in neonatal rats. METHODS: Ninety-six Wistar neonatal rats were randomly divided into a normoxia group, a hypoxia group, and a hypoxia + 2ME group, with each group further subdivided into 3-day, 7-day, 14-day, and 21-day subgroups, containing eight rats each. The hypoxia and hypoxia + 2ME groups received daily subcutaneous injections of saline and 2ME (240 µg/kg), respectively, while the normoxia group was raised in a normoxic environment with daily saline injections. Right ventricular systolic pressure (RVSP) was measured using the direct pressure method. Pulmonary vascular morphology was assessed using hematoxylin and eosin staining, with metrics including the percentage of medial thickness of small pulmonary arteries relative to the external diameter (MT%) and the cross-sectional area of the media of small pulmonary arteries relative to the total cross-sectional area (MA%). Immunohistochemistry was used to detect the expression levels of hypoxia-inducible factor-1α (HIF-1α) and proliferating cell nuclear antigen (PCNA) proteins, while real-time quantitative PCR was used to to assess HIF-1α and PCNA mRNA levels. RESULTS: Compared to the normoxia group, the hypoxia and hypoxia + 2ME groups showed increased RVSP and upregulated HIF-1α and PCNA protein and mRNA expression levels at 3, 7, 14, and 21 days after hypoxia (P<0.05). Furthermore, at 7, 14, and 21 days after hypoxia, the hypoxia group showed increased MT% and MA% (P<0.05). In comparison to the hypoxia group, the hypoxia + 2ME group exhibited reduced RVSP and downregulated HIF-1α and PCNA protein and mRNA expression levels, along with decreased MT% and MA% at 7, 14, and 21 days after hypoxia (P<0.05). CONCLUSIONS: 2ME may protect against HPH in neonatal rats by inhibiting the expression of HIF-1α and PCNA and reducing pulmonary vascular remodeling. Citation:Chinese Journal of Contemporary Pediatrics, 2024, 26(7): 757-764.


Asunto(s)
2-Metoxiestradiol , Animales Recién Nacidos , Hipertensión Pulmonar , Subunidad alfa del Factor 1 Inducible por Hipoxia , Hipoxia , Antígeno Nuclear de Célula en Proliferación , Arteria Pulmonar , Ratas Wistar , Animales , 2-Metoxiestradiol/farmacología , Ratas , Hipertensión Pulmonar/prevención & control , Hipertensión Pulmonar/tratamiento farmacológico , Antígeno Nuclear de Célula en Proliferación/análisis , Antígeno Nuclear de Célula en Proliferación/genética , Hipoxia/complicaciones , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Arteria Pulmonar/efectos de los fármacos , Masculino , Femenino , Estradiol/farmacología , Estradiol/análogos & derivados , ARN Mensajero/análisis
10.
Genes (Basel) ; 15(7)2024 Jun 24.
Artículo en Inglés | MEDLINE | ID: mdl-39062611

RESUMEN

Translesion synthesis (TLS) is a mechanism of DNA damage tolerance utilized by eukaryotic cells to replicate DNA across lesions that impede the high-fidelity replication machinery. In TLS, a series of specialized DNA polymerases are employed, which recognize specific DNA lesions, insert nucleotides across the damage, and extend the distorted primer-template. This allows cells to preserve genetic integrity at the cost of mutations. In humans, TLS enzymes include the Y-family, inserter polymerases, Polη, Polι, Polκ, Rev1, and the B-family extender polymerase Polζ, while in S. cerevisiae only Polη, Rev1, and Polζ are present. To bypass DNA lesions, TLS polymerases cooperate, assembling into a complex on the eukaryotic sliding clamp, PCNA, termed the TLS mutasome. The mutasome assembly is contingent on protein-protein interactions (PPIs) between the modular domains and subunits of TLS enzymes, and their interactions with PCNA and DNA. While the structural mechanisms of DNA lesion bypass by the TLS polymerases and PPIs of their individual modules are well understood, the mechanisms by which they cooperate in the context of TLS complexes have remained elusive. This review focuses on structural studies of TLS polymerases and describes the case of TLS holoenzyme assemblies in action emerging from recent high-resolution Cryo-EM studies.


Asunto(s)
Daño del ADN , Reparación del ADN , Replicación del ADN , ADN Polimerasa Dirigida por ADN , Antígeno Nuclear de Célula en Proliferación , ADN Polimerasa Dirigida por ADN/metabolismo , ADN Polimerasa Dirigida por ADN/genética , Humanos , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Replicación del ADN/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , ADN/genética , ADN/metabolismo , Síntesis Translesional de ADN
11.
Food Chem Toxicol ; 191: 114889, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39059691

RESUMEN

Bisphenol A (BPA) has been a substantial additive in plastics until the reports on its adverse effects have led to its restrictions and replacement. Monitoring studies document the increasing occurrence of bisphenol analogs, however, data on their effects and risks is still insufficient. Based on the indications that BPA might contribute to ovarian cancer pathogenesis, we examined effects of the analogs AF (BPAF), S (BPS) and F (BPF) (10-9-10-4 M) on the Caov-3 epithelial cancer cells, including the impact on cell viability, proliferation, oxidative stress, and production and expression of several factors and genes related to ovarian cancer. At environmentally relevant doses, bisphenols did not exert significant effects. At the highest concentration, BPAF caused varied alterations, including decreased cell viability and proliferation, caspase activation, down-regulation of PCNA and BIRC5, elevation of IL8, VEGFA, MYC, PTGS2 and ABCB1 expressions. Only BPA (10-4 M) increased IL-6, IL-8 and VEGFA output by the Caov-3 cells. Each bisphenol induced generation of reactive oxygen species and decreased superoxide dismutase activity at the highest concentration. Although the effects were observed only in the supraphysiological doses, the results indicate that certain bisphenol analogs might affect several ovarian cancer cell characteristics and merit further investigation.


Asunto(s)
Compuestos de Bencidrilo , Proliferación Celular , Supervivencia Celular , Neoplasias Ováricas , Fenoles , Humanos , Fenoles/toxicidad , Compuestos de Bencidrilo/toxicidad , Proliferación Celular/efectos de los fármacos , Femenino , Supervivencia Celular/efectos de los fármacos , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/patología , Línea Celular Tumoral , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismo , Sulfonas/toxicidad , Estrés Oxidativo/efectos de los fármacos , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Survivin/genética , Survivin/metabolismo , Subfamilia B de Transportador de Casetes de Unión a ATP/genética , Subfamilia B de Transportador de Casetes de Unión a ATP/metabolismo , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Interleucina-8/metabolismo , Interleucina-8/genética , Ciclooxigenasa 2/genética , Ciclooxigenasa 2/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Proteínas Inhibidoras de la Apoptosis/genética , Proteínas Inhibidoras de la Apoptosis/metabolismo , Fluorocarburos
12.
Zhonghua Zhong Liu Za Zhi ; 46(7): 676-685, 2024 Jul 23.
Artículo en Chino | MEDLINE | ID: mdl-39034803

RESUMEN

Objective: To explore the effect and molecular mechanism of circ_0000263 on HeLa cell activity, apoptosis, telomerase activity, and radiosensitivity. Methods: The Hela cells were divided into si-NC, si-circ, vector, circ_0000263, anti-NC, anti-miR-338-3p, miR-NC, miR-338-3p, si-circ+anti-NC, si-circ+ anti-miR-338-3p, si-circ+vector, si-circ+TERT, sh-NC, sh-circ groups. Reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) was used to detect the expressions of circ_0000263 and miR-338-3p. Cell clone formation array was used to detect cell survival; cell counting kit-8 (CCK-8) to detect cell proliferation; flow cytometry to detect apoptosis; western blot method to detect the expressions of proliferating cell nuclear antigen (PCNA), Cleaved-casp3, telomerase reverse transcriptase (TERT) proteins; double luciferase assay to detect the targeting relationships of circ_0000263 and miR-338-3p, miR-338-3p and TERT; telomere repeat amplification enzyme linked immunosorbent assay (TRAR-ELISA) to detect telomerase activity. Results: Circ_0000263 was highly expressed in Hela cells, miR-338-3p was low expressed, and TERT was highly expressed; circ_0000263 was also highly expressed in Hela cells treated with radiation (P<0.05). Knockdown of circ_0000263 inhibited the clone formation and cell proliferation ability of HeLa cells, and enhanced the radiosensitivity and apoptosis of HeLa cells. In contrast, knockdown of circ_0000263 decreased PCNA protein expression level and enhanced Cleaved-casp3 protein expression level in HeLa cells (P<0.05). The apoptosis rate in the si-circ group was (13.19±1.12)%, which was higher than (6.80±0.62)% of si-NC group (P<0.05). The apoptosis rate in the si-circ+4 Gy group was (24.82±1.57)%, which was higher than (17.00±0.96)% of si-NC+4 Gy group (P<0.05). Circ_0000263 targeted regulated miR-338-3p, and miR-338-3p targeted regulated TERT. MiR-338-3p was lowly expressed in HeLa cells, and knockdown of circ_0000263 elevated miR-338-3p expression level in HeLa cells. Circ_0000263 regulated TERT expression and inhibited telomerase activity through miR-338-3p. MiR-338-3p/TERT can restore the effect of circ_0000263 on the radiosensitivity of Hela cells. The apoptosis rate in the si-circ+anti-NC group was (27.37±0.89)%, which was higher than (18.22±1.18)% of the si-circ+anti-miR-338-3p group (P<0.05). The apoptosis rate in the si-circ+vector group was (27.55±0.48)%, which was higher than (20.10±0.68)% of si-circ+TERT group (P<0.05). After 72 hours of radiation by 4 Gy, the cell survival fraction of si-circ+anti-NC group was 0.41±0.02, which was lower than 0.66±0.03 of the si-circ+anti-miR-338-3p group (P<0.05); the cell survival fraction of si-circ+vector group was 0.42±0.05, which was lower than 0.70±0.03 of si-circ+TERT group (P<0.05). Conclusion: Inhibiting the expression of circ_0000263 supresses the proliferation of Hela cells by regulating miR-338-3p/TERT, promotes apoptosis, inhibits telomerase activity, increases the radiosensitivity of cancer cells, and provides a theoretical basis for improving the radiosensitivity of Hela cells.


Asunto(s)
Apoptosis , Proliferación Celular , MicroARNs , Tolerancia a Radiación , Telomerasa , Humanos , Células HeLa , MicroARNs/metabolismo , MicroARNs/genética , Telomerasa/genética , Telomerasa/metabolismo , Apoptosis/efectos de la radiación , ARN Circular/genética , ARN Circular/metabolismo , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética
13.
Cell Biochem Biophys ; 82(3): 2237-2248, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38849695

RESUMEN

LncRNAs involvement in heart disease, however, the effect of lncRNA prostate cancer-associated transcript 19 (PCAT19) in coronary artery disease (CAD) remains unclear. In the current study, we aimed to verify the role of PCAT19 in CAD. We first investigated the differentially expressed lncRNAs in different Genes Expression Omnibus (GEO) database. We then detected lncRNAs expression in healthy volunteers and acute myocardial infarction (AMI) patients by qRT­PCR. The correlation of PCAT19 and Glucosaminyl (N-Acetyl) Transferase 2 (GCNT2) was analyzed. Human coronary artery endothelial cells (HCAECs) was used to conduct cell hypoxia-reoxygenation (H/R) injury model to imitate AMI injury. CCK8, BrdU, tube formation assay were used to detect cell viability, proliferation, and angiogenesis. Immunofluorescence, western blotting were used to detect ki67, VEGFA, PCNA, CD31, and GCNT2 expression, respectively. We obtained six different lncRNAs from GEO database and identified PCAT19 high expression in AMI patients. PCAT19 was positive correlation to GCNT2. Further experiments presented that PCAT19 knockdown promoted cell viability, proliferation and angiogenesis, GCNT2 knockdown also promoted cell viability, proliferation, and angiogenesis. These results confirmed by the inhibition of Ki67 and VEGFA. Importantly, PCAT19 overexpression suppressed cell proliferation and angiogenesis, these results also confirmed by the inhibition of PCNA and CD31. However, the inhibitory effect of PCAT19 overexpression was reversed by GCNT2 knockdown. Our study indicated that PCAT19 plays an important role in the CAD disease, its effects was related to GCNT2. Our research provides a novel sight for the effect of PCAT19 on CAD.


Asunto(s)
Proliferación Celular , Enfermedad de la Arteria Coronaria , ARN Largo no Codificante , Humanos , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/metabolismo , Enfermedad de la Arteria Coronaria/patología , Células Endoteliales/metabolismo , Células Endoteliales/citología , Infarto del Miocardio/metabolismo , Infarto del Miocardio/genética , Infarto del Miocardio/patología , Supervivencia Celular , Factor A de Crecimiento Endotelial Vascular/metabolismo , Factor A de Crecimiento Endotelial Vascular/genética , Masculino , Células Cultivadas , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Interferencia de ARN , Neovascularización Patológica/genética , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/metabolismo , Molécula-1 de Adhesión Celular Endotelial de Plaqueta/genética , Antígeno Ki-67/metabolismo , Antígeno Ki-67/genética , Angiogénesis
14.
Nat Commun ; 15(1): 5392, 2024 Jun 25.
Artículo en Inglés | MEDLINE | ID: mdl-38918391

RESUMEN

DNA double-strand breaks (DSBs), such as those produced by radiation and radiomimetics, are amongst the most toxic forms of cellular damage, in part because they involve extensive oxidative modifications at the break termini. Prior to completion of DSB repair, the chemically modified termini must be removed. Various DNA processing enzymes have been implicated in the processing of these dirty ends, but molecular knowledge of this process is limited. Here, we demonstrate a role for the metallo-ß-lactamase fold 5'-3' exonuclease SNM1A in this vital process. Cells disrupted for SNM1A manifest increased sensitivity to radiation and radiomimetic agents and show defects in DSB damage repair. SNM1A is recruited and is retained at the sites of DSB damage via the concerted action of its three highly conserved PBZ, PIP box and UBZ interaction domains, which mediate interactions with poly-ADP-ribose chains, PCNA and the ubiquitinated form of PCNA, respectively. SNM1A can resect DNA containing oxidative lesions induced by radiation damage at break termini. The combined results reveal a crucial role for SNM1A to digest chemically modified DNA during the repair of DSBs and imply that the catalytic domain of SNM1A is an attractive target for potentiation of radiotherapy.


Asunto(s)
Roturas del ADN de Doble Cadena , Enzimas Reparadoras del ADN , Reparación del ADN , Exodesoxirribonucleasas , Humanos , Roturas del ADN de Doble Cadena/efectos de la radiación , Exodesoxirribonucleasas/metabolismo , Exodesoxirribonucleasas/genética , Enzimas Reparadoras del ADN/metabolismo , Enzimas Reparadoras del ADN/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , ADN/metabolismo , ADN/genética , Ubiquitinación , Proteínas de Ciclo Celular
15.
Sci Adv ; 10(23): eadn5175, 2024 Jun 07.
Artículo en Inglés | MEDLINE | ID: mdl-38838138

RESUMEN

Inheritance of epigenetic information is critical for maintaining cell identity. The transfer of parental histone H3-H4 tetramers, the primary carrier of epigenetic modifications on histone proteins, represents a crucial yet poorly understood step in the inheritance of epigenetic information. Here, we show the lagging strand DNA polymerase, Pol δ, interacts directly with H3-H4 and that the interaction between Pol δ and the sliding clamp PCNA regulates parental histone transfer to lagging strands, most likely independent of their roles in DNA synthesis. When combined, mutations at Pol δ and Mcm2 that compromise parental histone transfer result in a greater reduction in nucleosome occupancy at nascent chromatin than mutations in either alone. Last, PCNA contributes to nucleosome positioning on nascent chromatin. On the basis of these results, we suggest that the PCNA-Pol δ complex couples lagging strand DNA synthesis to parental H3-H4 transfer, facilitating epigenetic inheritance.


Asunto(s)
ADN Polimerasa III , Replicación del ADN , Epigénesis Genética , Histonas , Antígeno Nuclear de Célula en Proliferación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Histonas/metabolismo , ADN Polimerasa III/metabolismo , ADN Polimerasa III/genética , Nucleosomas/metabolismo , Nucleosomas/genética , ADN/metabolismo , Humanos , Unión Proteica , Mutación , Cromatina/metabolismo , Cromatina/genética
16.
Nucleic Acids Res ; 52(15): 8861-8879, 2024 Aug 27.
Artículo en Inglés | MEDLINE | ID: mdl-38943334

RESUMEN

BRCA1/2 proteins function in genome stability by promoting repair of double-stranded DNA breaks through homologous recombination and by protecting stalled replication forks from nucleolytic degradation. In BRCA1/2-deficient cancer cells, extensively degraded replication forks can be rescued through distinct fork recovery mechanisms that also promote cell survival. Here, we identified a novel pathway mediated by the E3 ubiquitin ligase RAD18, the E2-conjugating enzyme UBC13, the recombination factor PALB2, the E3 ubiquitin ligase RNF168 and PCNA ubiquitination that promotes fork recovery in BRCA1- but not BRCA2-deficient cells. We show that this pathway does not promote fork recovery by preventing replication fork reversal and degradation in BRCA1-deficient cells. We propose a mechanism whereby the RAD18-UBC13-PALB2-RNF168 axis facilitates resumption of DNA synthesis by promoting re-annealing of the complementary single-stranded template strands of the extensively degraded forks, thereby allowing re-establishment of a functional replication fork. We also provide preliminary evidence for the potential clinical relevance of this novel fork recovery pathway in BRCA1-mutated cancers, as RAD18 is over-expressed in BRCA1-deficient cancers, and RAD18 loss compromises cell viability in BRCA1-deficient cancer cells.


Asunto(s)
Proteína BRCA1 , Replicación del ADN , Proteínas de Unión al ADN , Proteína del Grupo de Complementación N de la Anemia de Fanconi , Enzimas Ubiquitina-Conjugadoras , Ubiquitina-Proteína Ligasas , Humanos , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Proteína BRCA1/metabolismo , Proteína BRCA1/genética , Proteína BRCA1/deficiencia , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/genética , Enzimas Ubiquitina-Conjugadoras/metabolismo , Enzimas Ubiquitina-Conjugadoras/genética , Línea Celular Tumoral , Proteína del Grupo de Complementación N de la Anemia de Fanconi/genética , Proteína del Grupo de Complementación N de la Anemia de Fanconi/metabolismo , Ubiquitinación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Reparación del ADN
17.
Nucleic Acids Res ; 52(13): 7650-7664, 2024 Jul 22.
Artículo en Inglés | MEDLINE | ID: mdl-38842913

RESUMEN

DNA polymerase δ (pol δ) holoenzymes, comprised of pol δ and the processivity sliding clamp, PCNA, carry out DNA synthesis during lagging strand replication, initiation of leading strand replication, and the major DNA damage repair and tolerance pathways. Pol δ holoenzymes are assembled at primer/template (P/T) junctions and initiate DNA synthesis in a stepwise process involving the major single strand DNA (ssDNA)-binding protein complex, RPA, the processivity sliding clamp loader, RFC, PCNA and pol δ. During this process, the interactions of RPA, RFC and pol δ with a P/T junction all significantly overlap. A burning issue that has yet to be resolved is how these overlapping interactions are accommodated during this process. To address this, we design and utilize novel, ensemble FRET assays that continuously monitor the interactions of RPA, RFC, PCNA and pol δ with DNA as pol δ holoenzymes are assembled and initiate DNA synthesis. Results from the present study reveal that RPA remains engaged with P/T junctions throughout this process and the RPA•DNA complexes dynamically re-organize to allow successive binding of RFC and pol δ. These results have broad implications as they highlight and distinguish the functional consequences of dynamic RPA•DNA interactions in RPA-dependent DNA metabolic processes.


Asunto(s)
ADN Polimerasa III , Replicación del ADN , ADN , Antígeno Nuclear de Célula en Proliferación , Proteína de Replicación A , Proteína de Replicación C , Moldes Genéticos , Proteína de Replicación A/metabolismo , ADN Polimerasa III/metabolismo , ADN Polimerasa III/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Holoenzimas/metabolismo , ADN/metabolismo , ADN/biosíntesis , Proteína de Replicación C/metabolismo , Proteína de Replicación C/genética , Cartilla de ADN/genética , Transferencia Resonante de Energía de Fluorescencia , Humanos
18.
FASEB J ; 38(9): e23630, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38713100

RESUMEN

Heat shock proteins (HSPs) are a group of highly conserved proteins found in a wide range of organisms. In recent years, members of the HSP family were overexpressed in various tumors and widely involved in oncogenesis, tumor development, and therapeutic resistance. In our previous study, DNAJC24, a member of the DNAJ/HSP40 family of HSPs, was found to be closely associated with the malignant phenotype of hepatocellular carcinoma. However, its relationship with other malignancies needs to be further explored. Herein, we demonstrated that DNAJC24 exhibited upregulated expression in LUAD tissue samples and predicted poor survival in LUAD patients. The upregulation of DNAJC24 expression promoted proliferation and invasion of LUAD cells in A549 and NCI-H1299 cell lines. Further studies revealed that DNAJC24 could regulate the PI3K/AKT signaling pathway by affecting AKT phosphorylation. In addition, a series of experiments such as Co-IP and mass spectrometry confirmed that DNAJC24 could directly interact with PCNA and promoted the malignant phenotypic transformation of LUAD. In conclusion, our results suggested that DNAJC24 played an important role in the progression of LUAD and may serve as a specific prognostic biomarker for LUAD patients. The DNAJC24/PCNA/AKT axis may be a potential target for future individualized and precise treatment of LUAD patients.


Asunto(s)
Proliferación Celular , Proteínas del Choque Térmico HSP40 , Antígeno Nuclear de Célula en Proliferación , Proteínas Proto-Oncogénicas c-akt , Animales , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Línea Celular Tumoral , Progresión de la Enfermedad , Regulación Neoplásica de la Expresión Génica , Proteínas del Choque Térmico HSP40/metabolismo , Proteínas del Choque Térmico HSP40/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Neoplasias Pulmonares/genética , Ratones Endogámicos BALB C , Ratones Desnudos , Fosforilación , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal
19.
Zhongguo Zhong Yao Za Zhi ; 49(9): 2355-2363, 2024 May.
Artículo en Chino | MEDLINE | ID: mdl-38812136

RESUMEN

This study explored the effects of 4-hydroxy-2(3H)-benzoxazolone(HBOA) on the proliferation and apoptosis of pancreatic cancer cells and its molecular mechanism. The L3.6 cells cultured in vitro were treated with HBOA of 0-1.0 mmol·L~(-1). The cell viability was detected by the cell counting kit-8(CCK-8) method, and the half inhibitory concentration(IC_(50)) was analyzed to determine the drug concentration and time. The cell morphology was observed under an inverted microscope and by acridine orange(AO) staining. The ability of proliferation and self-renewal were evaluated through live cell counting and colony formation experiments. The cell cycle progression and cell apoptosis rate were detected by flow cytometry. The morphology of cell apoptosis was observed by scanning electron microscopy. The mRNA expression of proliferating cell nuclear antigen(PCNA), cyclinA1, cyclinA2, cyclin dependent kinase 2(CDK2), and cyclin dependent kinase inhibitor 1A(P21) were determined by qPCR. The level of reactive oxygen species(ROS), lipid peroxide, and mitochondrial membrane potential were measured by flow cytometry. The activity of protein kinase B(Akt)/mammalian target of rapamycin(mTOR) signaling pathway was detected by Western blot. Compared with the control group, the cells treated with HBOA exhibited a significant decrease in viability. Then the optimal concentration and intervention time of HBOA were determined to be 0.4 mmol·L~(-1), 0.6 mmol·L~(-1), and 48 h. Compared with the control group, groups with HBOA of 0.4 mmol·L~(-1 )and 0.6 mmol·L~(-1) showed a significant suppression in cell proliferation and colony formation ability, down-regulated mRNA of PCNA, cyclinA1, cyclinA2, and CDK2, up-regulated P21 mRNA, S-phase cell cycle arrest, and increased cell apoptosis rate. There was an appearance of apoptotic bodies, increased ROS and lipid peroxide, decreased mitochondrial membrane potential(with a significant decrease in 0.6 mmol·L~(-1) group), and down-regulated p-Akt and p-mTOR proteins. The results show that HBOA inhibits the proliferation of pancreatic cancer L3.6 cells and induces cell apoptosis, which may be related to the increase in reactive oxygen species and the inhibition of the Akt/mTOR pathway.


Asunto(s)
Apoptosis , Proliferación Celular , Neoplasias Pancreáticas , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/metabolismo , Proliferación Celular/efectos de los fármacos , Apoptosis/efectos de los fármacos , Humanos , Línea Celular Tumoral , Benzoxazoles/farmacología , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Ciclo Celular/efectos de los fármacos , Quinasa 2 Dependiente de la Ciclina/genética , Quinasa 2 Dependiente de la Ciclina/metabolismo , Supervivencia Celular/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo
20.
Microb Biotechnol ; 17(4): e14471, 2024 04.
Artículo en Inglés | MEDLINE | ID: mdl-38646975

RESUMEN

Proliferating cell nuclear antigen (PCNA) is an essential factor for DNA metabolism. The influence of PCNA on DNA replication and repair, combined with the high expression rate of PCNA in various tumours renders PCNA a promising target for cancer therapy. In this context, an autodisplay-based screening method was developed to identify peptidic PCNA interaction inhibitors. A 12-mer randomized peptide library consisting of 2.54 × 106 colony-forming units was constructed and displayed at the surface of Escherichia coli BL21 (DE3) cells by autodisplay. Cells exhibiting an enhanced binding to fluorescent mScarlet-I-PCNA were enriched in four sorting rounds by flow cytometry. This led to the discovery of five peptide variants with affinity to mScarlet-I-PCNA. Among these, P3 (TCPLRWITHDHP) exhibited the highest binding signal. Subsequent flow cytometric analysis revealed a dissociation constant of 0.62 µM for PCNA-P3 interaction. Furthermore, the inhibition of PCNA interactions was investigated using p15, a PIP-box containing protein involved in DNA replication and repair. P3 inhibited the PCNA-p1551-70 interaction with a half maximal inhibitory activity of 16.2 µM, characterizing P3 as a potent inhibitor of the PCNA-p15 interaction.


Asunto(s)
Escherichia coli , Biblioteca de Péptidos , Antígeno Nuclear de Célula en Proliferación , Unión Proteica , Antígeno Nuclear de Célula en Proliferación/metabolismo , Antígeno Nuclear de Célula en Proliferación/genética , Antígeno Nuclear de Célula en Proliferación/química , Escherichia coli/genética , Escherichia coli/metabolismo , Citometría de Flujo , Evaluación Preclínica de Medicamentos/métodos , Técnicas de Visualización de Superficie Celular/métodos , Humanos , Péptidos/metabolismo , Péptidos/genética , Péptidos/química , Péptidos/farmacología
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