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1.
Cell Death Dis ; 15(5): 329, 2024 May 13.
Article En | MEDLINE | ID: mdl-38740757

Iron is crucial for cell DNA synthesis and repair, but an excess of free iron can lead to oxidative stress and subsequent cell death. Although several studies suggest that cancer cells display characteristics of 'Iron addiction', an ongoing debate surrounds the question of whether iron can influence the malignant properties of ovarian cancer. In the current study, we initially found iron levels increase during spheroid formation. Furthermore, iron supplementation can promote cancer cell survival, cancer spheroid growth, and migration; vice versa, iron chelators inhibit this process. Notably, iron reduces the sensitivity of ovarian cancer cells to platinum as well. Mechanistically, iron downregulates DNA homologous recombination (HR) inhibitor polymerase theta (POLQ) and relieves its antagonism against the HR repair enzyme RAD51, thereby promoting DNA damage repair to resist chemotherapy-induced damage. Additionally, iron tightly regulated by ferritin (FTH1/FTL) which is indispensable for iron-triggered DNA repair. Finally, we discovered that iron chelators combined with platinum exhibit a synergistic inhibitory effect on ovarian cancer in vitro and in vivo. Our findings affirm the pro-cancer role of iron in ovarian cancer and reveal that iron advances platinum resistance by promoting DNA damage repair through FTH1/FTL/POLQ/RAD51 pathway. Our findings highlight the significance of iron depletion therapy, revealing a promising avenue for advancing ovarian cancer treatment.


DNA Repair , Drug Resistance, Neoplasm , Iron , Ovarian Neoplasms , Rad51 Recombinase , Female , Humans , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Ovarian Neoplasms/genetics , Drug Resistance, Neoplasm/drug effects , DNA Repair/drug effects , Iron/metabolism , Cell Line, Tumor , Rad51 Recombinase/metabolism , Animals , Ferritins/metabolism , Mice , Platinum/pharmacology , Platinum/therapeutic use , Mice, Nude , Oxidoreductases/metabolism
2.
Nat Commun ; 15(1): 4430, 2024 May 24.
Article En | MEDLINE | ID: mdl-38789420

Histone H2AX plays a key role in DNA damage signalling in the surrounding regions of DNA double-strand breaks (DSBs). In response to DNA damage, H2AX becomes phosphorylated on serine residue 139 (known as γH2AX), resulting in the recruitment of the DNA repair effectors 53BP1 and BRCA1. Here, by studying resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient mammary tumours, we identify a function for γH2AX in orchestrating drug-induced replication fork degradation. Mechanistically, γH2AX-driven replication fork degradation is elicited by suppressing CtIP-mediated fork protection. As a result, H2AX loss restores replication fork stability and increases chemoresistance in BRCA1/2-deficient tumour cells without restoring homology-directed DNA repair, as highlighted by the lack of DNA damage-induced RAD51 foci. Furthermore, in the attempt to discover acquired genetic vulnerabilities, we find that ATM but not ATR inhibition overcomes PARP inhibitor (PARPi) resistance in H2AX-deficient tumours by interfering with CtIP-mediated fork protection. In summary, our results demonstrate a role for H2AX in replication fork biology in BRCA-deficient tumours and establish a function of H2AX separable from its classical role in DNA damage signalling and DSB repair.


BRCA1 Protein , BRCA2 Protein , DNA Replication , Drug Resistance, Neoplasm , Histones , Poly(ADP-ribose) Polymerase Inhibitors , Humans , BRCA1 Protein/metabolism , BRCA1 Protein/deficiency , BRCA1 Protein/genetics , Histones/metabolism , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , DNA Replication/drug effects , BRCA2 Protein/metabolism , BRCA2 Protein/genetics , BRCA2 Protein/deficiency , Cell Line, Tumor , Female , Drug Resistance, Neoplasm/genetics , Animals , Ataxia Telangiectasia Mutated Proteins/metabolism , Ataxia Telangiectasia Mutated Proteins/genetics , DNA Breaks, Double-Stranded , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast Neoplasms/drug therapy , Mice , Tumor Suppressor p53-Binding Protein 1/metabolism , Tumor Suppressor p53-Binding Protein 1/genetics , DNA Repair , Carrier Proteins/metabolism , Carrier Proteins/genetics , DNA Damage , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics
3.
Life Sci Alliance ; 7(8)2024 Aug.
Article En | MEDLINE | ID: mdl-38803223

Homologous recombination is a major pathway for the repair of DNA double strand breaks, essential both to maintain genomic integrity and to generate genetic diversity. Mechanistically, homologous recombination involves the use of a homologous DNA molecule as a template to repair the break. In eukaryotes, the search for and invasion of the homologous DNA molecule is carried out by two recombinases, RAD51 in somatic cells and RAD51 and DMC1 in meiotic cells. During recombination, the recombinases bind overhanging single-stranded DNA ends to form a nucleoprotein filament, which is the active species in promoting DNA invasion and strand exchange. RAD51 and DMC1 carry two major DNA-binding sites-essential for nucleofilament formation and DNA strand exchange, respectively. Here, we show that the function of RAD51 DNA-binding site II is conserved in the plant, Arabidopsis. Mutation of three key amino acids in site II does not affect RAD51 nucleofilament formation but inhibits its recombinogenic activity, analogous to results from studies of the yeast and human proteins. We further confirm that recombinogenic function of RAD51 DNA-binding site II is not required for meiotic double-strand break repair when DMC1 is present. The Arabidopsis AtRAD51-II3A separation of function mutant shows a dominant negative phenotype, pointing to distinct biochemical properties of eukaryotic RAD51 proteins.


Arabidopsis Proteins , Arabidopsis , Homologous Recombination , Rad51 Recombinase , Arabidopsis/metabolism , Arabidopsis/genetics , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Binding Sites , Mutation , DNA Breaks, Double-Stranded , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Meiosis/genetics , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , DNA Repair
4.
Cell Death Dis ; 15(5): 321, 2024 May 08.
Article En | MEDLINE | ID: mdl-38719812

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.


Acetylglucosamine , DNA-Binding Proteins , Proliferating Cell Nuclear Antigen , Rad51 Recombinase , Recombinational DNA Repair , Ubiquitin-Protein Ligases , Humans , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Ubiquitin-Protein Ligases/metabolism , Acetylglucosamine/metabolism , Rad51 Recombinase/metabolism , Proliferating Cell Nuclear Antigen/metabolism , Phosphorylation , DNA Replication , Ubiquitination , DNA Breaks, Double-Stranded , DNA-Directed DNA Polymerase/metabolism , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/genetics , DNA Damage , DNA/metabolism , HEK293 Cells , Ultraviolet Rays , Protein Binding , Glycosylation , Translesion DNA Synthesis
5.
Cell Rep ; 43(5): 114205, 2024 May 28.
Article En | MEDLINE | ID: mdl-38753485

The advent of PARP inhibitors (PARPis) has profoundly changed the treatment landscape of BRCA1/BRCA2-mutated cancers. Despite this, the development of resistance to these compounds has become a major challenge. Hence, a detailed understanding of the mechanisms underlying PARPi sensitivity is crucially needed. Here, we show that loss of the POLE3-POLE4 subunits of DNA polymerase epsilon (Polε) strongly sensitizes cancer cells to PARPis in a Polε level-independent manner. Loss of POLE3-POLE4 is not associated with defective RAD51 foci formation, excluding a major defect in homologous recombination. On the contrary, treatment with PARPis triggers replicative gap accumulation in POLE3-POLE4 knockout (KO) cells in a PRIMPOL-dependent manner. In addition to this, the loss of POLE3-POLE4 further sensitizes BRCA1-silenced cells to PARPis. Importantly, the knockdown of 53BP1 does not rescue PARPi sensitivity in POLE3-POLE4 KO cells, bypassing a common PARPi resistance mechanism and outlining a potential strategy to sensitize cancer cells to PARPis.


Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Humans , DNA Replication/drug effects , Cell Line, Tumor , BRCA1 Protein/metabolism , BRCA1 Protein/genetics , DNA Polymerase II/metabolism , Tumor Suppressor p53-Binding Protein 1/metabolism , Rad51 Recombinase/metabolism
6.
Cancer Immunol Immunother ; 73(5): 95, 2024 Apr 12.
Article En | MEDLINE | ID: mdl-38607586

BACKGROUND: Homologous recombination deficiency (HRD), though largely uncharacterized in clear cell renal cell carcinoma (ccRCC), was found associated with RAD51 loss of expression. PBRM1 is the second most common mutated genes in ccRCC. Here, we introduce a HRD function-based PBRM1-RAD51 ccRCC classification endowed with diverse immune checkpoint blockade (ICB) responses. METHODS: Totally 1542 patients from four independent cohorts were enrolled, including our localized Zhongshan hospital (ZSHS) cohort and Zhongshan hospital metastatic RCC (ZSHS-mRCC) cohort, The Cancer Genome Atlas (TCGA) cohort and CheckMate cohort. The genomic profile and immune microenvironment were depicted by genomic, transcriptome data and immunohistochemistry. RESULTS: We observed that PBRM1-loss ccRCC harbored enriched HRD-associated mutational signature 3 and loss of RAD51. Dual-loss of PBRM1 and RAD51 identified patients hyper-sensitive to immunotherapy. This dual-loss subtype was featured by M1 macrophage infiltration. Dual-loss was, albeit homologous recombination defective, with high chromosomal stability. CONCLUSIONS: PBRM1 and RAD51 dual-loss ccRCC indicates superior responses to immunotherapy. Dual-loss ccRCC harbors an immune-desert microenvironment but enriched with M1 macrophages. Dual-loss ccRCC is susceptible to defective homologous recombination but possesses high chromosomal stability.


Carcinoma, Renal Cell , Carcinoma , Kidney Neoplasms , Humans , Carcinoma, Renal Cell/genetics , Carcinoma, Renal Cell/therapy , Immunotherapy , Kidney Neoplasms/genetics , Kidney Neoplasms/therapy , Chromosomal Instability , Tumor Microenvironment , Rad51 Recombinase , DNA-Binding Proteins/genetics , Transcription Factors/genetics
7.
Chirality ; 36(4): e23664, 2024 Apr.
Article En | MEDLINE | ID: mdl-38561319

Linear dichroism spectroscopy is used to investigate the structure of RecA family recombinase filaments (RecA and Rad51 proteins) with DNA for clarifying the molecular mechanism of DNA strand exchange promoted by these proteins and its activation. The measurements show that the recombinases promote the perpendicular base orientation of single-stranded DNA only in the presence of activators, indicating the importance of base orientation in the reaction. We summarize the results and discuss the role of DNA base orientation.


DNA , Rad51 Recombinase , Rad51 Recombinase/chemistry , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism , Stereoisomerism , DNA/chemistry , DNA, Single-Stranded
8.
Health Phys ; 126(6): 397-404, 2024 Jun 01.
Article En | MEDLINE | ID: mdl-38568172

ABSTRACT: Experiments that examine the impacts of subnatural background radiation exposure provide a unique approach to studying the biological effects of low-dose radiation. These experiments often need to be conducted in deep underground laboratories in order to filter surface-level cosmic radiation. This presents some logistical challenges in experimental design and necessitates a model organism with minimal maintenance. As such, desiccated yeast ( Saccharomyces cerevisiae ) is an ideal model system for these investigations. This study aimed to determine the impact of prolonged sub-background radiation exposure in anhydrobiotic (desiccated) yeast at SNOLAB in Sudbury, Ontario, Canada. Two yeast strains were used: a normal wild type and an isogenic recombinational repair-deficient rad51 knockout strain ( rad51 Δ). Desiccated yeast samples were stored in the normal background surface control laboratory (68.0 nGy h -1 ) and in the sub-background environment within SNOLAB (10.1 nGy h -1 ) for up to 48 wk. Post-rehydration survival, growth rate, and metabolic activity were assessed at multiple time points. Survival in the sub-background environment was significantly reduced by a factor of 1.39 and 2.67 in the wild type and rad51 ∆ strains, respectively. Post-rehydration metabolic activity measured via alamarBlue reduction remained unchanged in the wild type strain but was 26% lower in the sub-background rad51 ∆ strain. These results demonstrate that removing natural background radiation negatively impacts the survival and metabolism of desiccated yeast, highlighting the potential importance of natural radiation exposure in maintaining homeostasis of living organisms.


Desiccation , Saccharomyces cerevisiae , Saccharomyces cerevisiae/radiation effects , Rad51 Recombinase/metabolism , Radiation Exposure/adverse effects , Radiation Exposure/analysis , Radiation Dosage
9.
Molecules ; 29(8)2024 Apr 10.
Article En | MEDLINE | ID: mdl-38675528

Glioblastoma (GBM), the most frequent and lethal brain cancer in adults, is characterized by short survival times and high mortality rates. Due to the resistance of GBM cells to conventional therapeutic treatments, scientific interest is focusing on the search for alternative and efficient adjuvant treatments. S-Adenosylmethionine (AdoMet), the well-studied physiological methyl donor, has emerged as a promising anticancer compound and a modulator of multiple cancer-related signaling pathways. We report here for the first time that AdoMet selectively inhibited the viability and proliferation of U87MG, U343MG, and U251MG GBM cells. In these cell lines, AdoMet induced S and G2/M cell cycle arrest and apoptosis and downregulated the expression and activation of proteins involved in homologous recombination DNA repair, including RAD51, BRCA1, and Chk1. Furthermore, AdoMet was able to maintain DNA in a damaged state, as indicated by the increased γH2AX/H2AX ratio. AdoMet promoted mitotic catastrophe through inhibiting Aurora B kinase expression, phosphorylation, and localization causing GBM cells to undergo mitotic catastrophe-induced death. Finally, AdoMet inhibited DNA repair and induced cell cycle arrest, apoptosis, and mitotic catastrophe in patient-derived GBM cells. In light of these results, AdoMet could be considered a potential adjuvant in GBM therapy.


Antineoplastic Agents , Apoptosis , Cell Proliferation , Glioblastoma , S-Adenosylmethionine , Humans , Glioblastoma/drug therapy , Glioblastoma/metabolism , Glioblastoma/pathology , S-Adenosylmethionine/pharmacology , Cell Line, Tumor , Apoptosis/drug effects , Cell Proliferation/drug effects , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistry , Cell Survival/drug effects , DNA Repair/drug effects , Aurora Kinase B/metabolism , Aurora Kinase B/antagonists & inhibitors , Brain Neoplasms/drug therapy , Brain Neoplasms/metabolism , Brain Neoplasms/pathology , Rad51 Recombinase/metabolism , Cell Cycle Checkpoints/drug effects , Mitosis/drug effects
10.
Mol Med ; 30(1): 54, 2024 Apr 22.
Article En | MEDLINE | ID: mdl-38649802

BACKGROUND: Bleomycin, a potent antitumor agent, is limited in clinical use due to the potential for fatal pulmonary toxicity. The accelerated DNA damage and senescence in alveolar epithelial cells (AECs) is considered a key factor in the development of lung pathology. Understanding the mechanisms for bleomycin-induced lung injury is crucial for mitigating its adverse effects. METHODS: Human lung epithelial (A549) cells were exposed to bleomycin and subsequently assessed for cellular senescence, DNA damage, and double-strand break (DSB) repair. The impact of Rad51 overexpression on DSB repair and senescence in AECs was evaluated in vitro. Additionally, bleomycin was intratracheally administered in C57BL/6 mice to establish a pulmonary fibrosis model. RESULTS: Bleomycin exposure induced dose- and time-dependent accumulation of senescence hallmarks and DNA lesions in AECs. These effects are probably due to the inhibition of Rad51 expression, consequently suppressing homologous recombination (HR) repair. Mechanistic studies revealed that bleomycin-mediated transcriptional inhibition of Rad51 might primarily result from E2F1 depletion. Furthermore, the genetic supplement of Rad51 substantially mitigated bleomycin-mediated effects on DSB repair and senescence in AECs. Notably, decreased Rad51 expression was also observed in the bleomycin-induced mouse pulmonary fibrosis model. CONCLUSIONS: Our works suggest that the inhibition of Rad51 plays a pivotal role in bleomycin-induced AECs senescence and lung injury, offering potential strategies to alleviate the pulmonary toxicity of bleomycin.


Bleomycin , Cellular Senescence , DNA Repair , Rad51 Recombinase , Bleomycin/adverse effects , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics , Animals , Cellular Senescence/drug effects , Cellular Senescence/genetics , Humans , Mice , DNA Repair/drug effects , Mice, Inbred C57BL , Pulmonary Fibrosis/chemically induced , Pulmonary Fibrosis/genetics , Pulmonary Fibrosis/metabolism , Pulmonary Fibrosis/pathology , Disease Models, Animal , Down-Regulation/drug effects , A549 Cells , DNA Damage/drug effects , DNA Breaks, Double-Stranded/drug effects , E2F1 Transcription Factor/metabolism , E2F1 Transcription Factor/genetics , Alveolar Epithelial Cells/metabolism , Alveolar Epithelial Cells/drug effects
11.
J Exp Clin Cancer Res ; 43(1): 122, 2024 Apr 23.
Article En | MEDLINE | ID: mdl-38654320

BACKGROUND: Radiation therapy stands to be one of the primary approaches in the clinical treatment of malignant tumors. Nasopharyngeal Carcinoma, a malignancy predominantly treated with radiation therapy, provides an invaluable model for investigating the mechanisms underlying radiation therapy resistance in cancer. While some reports have suggested the involvement of circRNAs in modulating resistance to radiation therapy, the underpinning mechanisms remain unclear. METHODS: RT-qPCR and in situ hybridization were used to detect the expression level of circCDYL2 in nasopharyngeal carcinoma tissue samples. The effect of circCDYL2 on radiotherapy resistance in nasopharyngeal carcinoma was demonstrated by in vitro and in vivo functional experiments. The HR-GFP reporter assay determined that circCDYL2 affected homologous recombination repair. RNA pull down, RIP, western blotting, IF, and polysome profiling assays were used to verify that circCDYL2 promoted the translation of RAD51 by binding to EIF3D protein. RESULTS: We have identified circCDYL2 as highly expressed in nasopharyngeal carcinoma tissues, and it was closely associated with poor prognosis. In vitro and in vivo experiments demonstrate that circCDYL2 plays a pivotal role in promoting radiotherapy resistance in nasopharyngeal carcinoma. Our investigation unveils a specific mechanism by which circCDYL2, acting as a scaffold molecule, recruits eukaryotic translation initiation factor 3 subunit D protein (EIF3D) to the 5'-UTR of RAD51 mRNA, a crucial component of the DNA damage repair pathway to facilitate the initiation of RAD51 translation and enhance homologous recombination repair capability, and ultimately leads to radiotherapy resistance in nasopharyngeal carcinoma. CONCLUSIONS: These findings establish a novel role of the circCDYL2/EIF3D/RAD51 axis in nasopharyngeal carcinoma radiotherapy resistance. Our work not only sheds light on the underlying molecular mechanism but also highlights the potential of circCDYL2 as a therapeutic sensitization target and a promising prognostic molecular marker for nasopharyngeal carcinoma.


Nasopharyngeal Carcinoma , Rad51 Recombinase , Radiation Tolerance , Recombinational DNA Repair , Humans , Nasopharyngeal Carcinoma/radiotherapy , Nasopharyngeal Carcinoma/genetics , Nasopharyngeal Carcinoma/metabolism , Nasopharyngeal Carcinoma/pathology , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics , Mice , Animals , Radiation Tolerance/genetics , RNA, Circular/genetics , Nasopharyngeal Neoplasms/radiotherapy , Nasopharyngeal Neoplasms/genetics , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/pathology , Cell Line, Tumor , Female , Male , Prognosis , Mice, Nude
12.
J Cancer Res Clin Oncol ; 150(4): 179, 2024 Apr 07.
Article En | MEDLINE | ID: mdl-38584230

PURPOSE: The present study aims to determine the molecular mechanism mediated by RAD51 antisense RNA 1 (RAD51-AS1) in ovarian cancer (OvCA). METHODS: The data associated with RAD51-AS1 in OvCA were obtained from the Cancer Genome Atlas (TCGA) and the Gene Expression Omnibus (GEO) database. Relative expression of RAD51-AS1 was detected. Determination of cell proliferation, metastasis, and invasion was performed by cell counting, colony formation, would-healing, and transwell invasion assays. Protein levels were detected by western blotting. The molecular mechanism mediated by RAD51-AS1 was predicted by bioinformatics analysis and verified by dual-luciferase reporter assays. Subcutaneous tumorigenesis models were used to confirm the function of RAD51-AS1 in vivo. RESULTS: Data from TCGA and GEO showed that RAD51-AS1 was associated with poor prognosis in OvCA patients and DNA repair, cell cycle, focal adhesion, and apoptosis in SKOV3.ip cells. High levels of RAD51-AS1 were detected in OvCA cells. Overexpressing RAD51-AS1 enhanced the proliferative, invading, and migratory capabilities of OvCA cells in vitro while silencing RAD51-AS1 exhibited the opposite effects. Mechanically, RAD51-AS1 elevated eukaryotic initiation factor 5A2 (EIF5A2) expression as a sponge for microRNA (miR)-140-3p. Finally, the role of RAD51-AS1 was verified by subcutaneous tumorigenesis models. CONCLUSION: RAD51-AS1 promoted OvCA progression by the regulation of the miR-140-3p/EIF5A2 axis, which illustrated the potential therapeutic target for OvCA.


MicroRNAs , Ovarian Neoplasms , RNA, Long Noncoding , Female , Humans , Carcinogenesis/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation/genetics , Cell Transformation, Neoplastic/genetics , Gene Expression Regulation, Neoplastic , MicroRNAs/genetics , MicroRNAs/metabolism , Ovarian Neoplasms/genetics , Rad51 Recombinase/genetics , RNA, Long Noncoding/genetics
13.
Int J Mol Sci ; 25(7)2024 Mar 24.
Article En | MEDLINE | ID: mdl-38612444

Human Rad51 protein (HsRad51)-promoted DNA strand exchange, a crucial step in homologous recombination, is regulated by proteins and calcium ions. Both the activator protein Swi5/Sfr1 and Ca2+ ions stimulate different reaction steps and induce perpendicular DNA base alignment in the presynaptic complex. To investigate the role of base orientation in the strand exchange reaction, we examined the Ca2+ concentration dependence of strand exchange activities and structural changes in the presynaptic complex. Our results show that optimal D-loop formation (strand exchange with closed circular DNA) required Ca2+ concentrations greater than 5 mM, whereas 1 mM Ca2+ was sufficient for strand exchange between two oligonucleotides. Structural changes indicated by increased fluorescence intensity of poly(dεA) (a poly(dA) analog) reached a plateau at 1 mM Ca2+. Ca2+ > 2 mM was required for saturation of linear dichroism signal intensity at 260 nm, associated with rigid perpendicular DNA base orientation, suggesting a correlation with the stimulation of D-loop formation. Therefore, Ca2+ exerts two different effects. Thermal stability measurements suggest that HsRad51 binds two Ca2+ ions with KD values of 0.2 and 2.5 mM, implying that one step is stimulated by one Ca2+ bond and the other by two Ca2+ bonds. Our results indicate parallels between the Mg2+ activation of RecA and the Ca2+ activation of HsRad51.


Oligonucleotides , Rad51 Recombinase , Humans , Calcium , Ions , DNA
14.
Cell Cycle ; 23(4): 369-384, 2024 Feb.
Article En | MEDLINE | ID: mdl-38571319

Acetaldehyde, a chemical that can cause DNA damage and contribute to cancer, is prevalently present in our environment, e.g. in alcohol, tobacco, and food. Although aldehyde potentially promotes crosslinking reactions among biological substances including DNA, RNA, and protein, it remains unclear what types of DNA damage are caused by acetaldehyde and how they are repaired. In this study, we explored mechanisms involved in the repair of acetaldehyde-induced DNA damage by examining the cellular sensitivity to acetaldehyde in the collection of human TK6 mutant deficient in each genome maintenance system. Among the mutants, mismatch repair mutants did not show hypersensitivity to acetaldehyde, while mutants deficient in base and nucleotide excision repair pathways or homologous recombination (HR) exhibited higher sensitivity to acetaldehyde than did wild-type cells. We found that acetaldehyde-induced RAD51 foci representing HR intermediates were prolonged in HR-deficient cells. These results indicate a pivotal role of HR in the repair of acetaldehyde-induced DNA damage. These results suggest that acetaldehyde causes complex DNA damages that require various types of repair pathways. Mutants deficient in the removal of protein adducts from DNA ends such as TDP1-/- and TDP2-/- cells exhibited hypersensitivity to acetaldehyde. Strikingly, the double mutant deficient in both TDP1 and RAD54 showed similar sensitivity to each single mutant. This epistatic relationship between TDP1-/- and RAD54-/- suggests that the protein-DNA adducts generated by acetaldehyde need to be removed for efficient repair by HR. Our study would help understand the molecular mechanism of the genotoxic and mutagenic effects of acetaldehyde.


Acetaldehyde , DNA Damage , DNA Repair , Homologous Recombination , Acetaldehyde/toxicity , Humans , Homologous Recombination/drug effects , Homologous Recombination/genetics , DNA Repair/drug effects , Rad51 Recombinase/metabolism , Rad51 Recombinase/genetics , Mutation/genetics , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Cell Line
15.
JAMA Netw Open ; 7(4): e247811, 2024 Apr 01.
Article En | MEDLINE | ID: mdl-38648056

Importance: RAD51C and RAD51D are involved in DNA repair by homologous recombination. Germline pathogenic variants (PVs) in these genes are associated with an increased risk of ovarian and breast cancer. Understanding the homologous recombination deficiency (HRD) status of tumors from patients with germline PVs in RAD51C/D could guide therapeutic decision-making and improve survival. Objective: To characterize the clinical and tumor characteristics of germline RAD51C/D PV carriers, including the evaluation of HRD status. Design, Setting, and Participants: This retrospective cohort study included 91 index patients plus 90 relatives carrying germline RAD51C/D PV (n = 181) in Spanish hospitals from January 1, 2014, to December 31, 2021. Genomic and functional HRD biomarkers were assessed in untreated breast and ovarian tumor samples (n = 45) from June 2022 to February 2023. Main Outcomes and Measures: Clinical and pathologic characteristics were assessed using descriptive statistics. Genomic HRD by genomic instability scores, functional HRD by RAD51, and gene-specific loss of heterozygosity were analyzed. Associations between HRD status and tumor subtype, age at diagnosis, and gene-specific loss of heterozygosity in RAD51C/D were investigated using logistic regression or the t test. Results: A total of 9507 index patients were reviewed, and 91 patients (1.0%) were found to carry a PV in RAD51C/D; 90 family members with a germline PV in RAD51C/D were also included. A total of 157 of carriers (86.7%) were women and 181 (55.8%) had received a diagnosis of cancer, mainly breast cancer or ovarian cancer. The most prevalent PVs were c.1026+5_1026+7del (11 of 56 [19.6%]) and c.709C>T (9 of 56 [16.1%]) in RAD51C and c.694C>T (20 of 35 [57.1%]) in RAD51D. In untreated breast cancer and ovarian cancer, the prevalence of functional and genomic HRD was 55.2% (16 of 29) and 61.1% (11 of 18) for RAD51C, respectively, and 66.7% (6 of 9) and 90.0% (9 of 10) for RAD51D. The concordance between HRD biomarkers was 91%. Tumors with the same PV displayed contrasting HRD status, and age at diagnosis did not correlate with the occurrence of HRD. All breast cancers retaining the wild-type allele were estrogen receptor positive and lacked HRD. Conclusions and Relevance: In this cohort study of germline RAD51C/D breast cancer and ovarian cancer, less than 70% of tumors displayed functional HRD, and half of those that did not display HRD were explained by retention of the wild-type allele, which was more frequent among estrogen receptor-positive breast cancers. Understanding which tumors are associated with RAD51C/D and HRD is key to identify patients who can benefit from targeted therapies, such as PARP (poly [adenosine diphosphate-ribose] polymerase) inhibitors.


Breast Neoplasms , Germ-Line Mutation , Homologous Recombination , Ovarian Neoplasms , Rad51 Recombinase , Adult , Female , Humans , Breast Neoplasms/genetics , Breast Neoplasms/epidemiology , DNA-Binding Proteins/genetics , Genetic Predisposition to Disease , Homologous Recombination/genetics , Ovarian Neoplasms/genetics , Ovarian Neoplasms/epidemiology , Prevalence , Retrospective Studies , Spain/epidemiology , Rad51 Recombinase/genetics
16.
Sci Rep ; 14(1): 9906, 2024 04 30.
Article En | MEDLINE | ID: mdl-38689033

CUL4B, a crucial scaffolding protein in the largest E3 ubiquitin ligase complex CRL4B, is involved in a broad range of physiological and pathological processes. While previous research has shown that CUL4B participates in maintaining intestinal homeostasis and function, its involvement in facilitating intestinal recovery following ionizing radiation (IR) damage has not been fully elucidated. Here, we utilized in vivo and in vitro models to decipher the role of CUL4B in intestinal repair after IR-injury. Our findings demonstrated that prior to radiation exposure, CUL4B inhibited the ubiquitination modification of PSME3, which led to the accumulation of PSME3 and subsequent negative regulation of p53-mediated apoptosis. In contrast, after radiation, CUL4B dissociated from PSME3 and translocated into the nucleus at phosphorylated histones H2A (γH2AX) foci, thereby impeding DNA damage repair and augmenting p53-mediated apoptosis through inhibition of BRCA1 phosphorylation and RAD51. Our study elucidated the dynamic role of CUL4B in the repair of radiation-induced intestinal damage and uncovered novel molecular mechanisms underlying the repair process, suggesting a potential therapeutic strategy of intestinal damage after radiation therapy for cancers.


Apoptosis , Cullin Proteins , Intestines , Regeneration , Tumor Suppressor Protein p53 , Animals , Humans , Mice , Apoptosis/radiation effects , BRCA1 Protein/metabolism , BRCA1 Protein/genetics , Cullin Proteins/metabolism , Cullin Proteins/genetics , DNA Damage , DNA Repair , Histones/metabolism , Intestines/radiation effects , Intestines/pathology , Mice, Inbred C57BL , Phosphorylation/radiation effects , Rad51 Recombinase/metabolism , Radiation, Ionizing , Regeneration/radiation effects , Tumor Suppressor Protein p53/metabolism , Ubiquitination
17.
JCO Precis Oncol ; 8: e2300483, 2024 Feb.
Article En | MEDLINE | ID: mdl-38427930

PURPOSE: To meet the urgent need for accessible homologous recombination-deficient (HRD) test options, we validated a laboratory-developed test (LDT) and a functional RAD51 assay to assess patients with ovarian cancer and predict the clinical benefit of poly(ADP-ribose) polymerase inhibitor therapy. METHODS: Optimization of the LDT cutoff and validation on the basis of samples from 91 patients enrolled in the ENGOT-ov24/NSGO-AVANOVA1&2 trial (ClinicalTrials.gov identifier: NCT02354131), previously subjected to commercial CDx HRD testing (CDx). RAD51 foci analysis was performed and tumors with ≥five foci/nucleus were classified as RAD51-positive (homologous recombination-proficient). RESULTS: The optimal LDT cutoff is 54. Comparing CDx genome instability score and LDT HRD scores show a Spearman's correlation of rho = 0.764 (P < .0001). Cross-tabulation analysis shows that the sensitivity of the LDT HRD score is 86% and of the LDT HRD status is 91.8% (Fisher's exact test P < .001). Survival analysis on progression-free survival (PFS) of LDT-assessed patients show a Cox regression P < .05. RAD51 assays show a correlation between low RAD51 foci detection (<20% RAD51+ cells) and significantly prolonged PFS (P < .001). CONCLUSION: The robust concordance between the open standard LDT and the CDx, especially the correlation with PFS, warrants future validation and implementation of the open standard LDT for HRD testing in diagnostic settings.


Antineoplastic Agents , Ovarian Neoplasms , Humans , Female , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors/therapeutic use , Homologous Recombination/genetics , Ovarian Neoplasms/diagnosis , Ovarian Neoplasms/drug therapy , Ovarian Neoplasms/genetics , Progression-Free Survival , Rad51 Recombinase/genetics
19.
Nature ; 628(8006): 212-220, 2024 Apr.
Article En | MEDLINE | ID: mdl-38509361

RAD51 is the central eukaryotic recombinase required for meiotic recombination and mitotic repair of double-strand DNA breaks (DSBs)1,2. However, the mechanism by which RAD51 functions at DSB sites in chromatin has remained elusive. Here we report the cryo-electron microscopy structures of human RAD51-nucleosome complexes, in which RAD51 forms ring and filament conformations. In the ring forms, the N-terminal lobe domains (NLDs) of RAD51 protomers are aligned on the outside of the RAD51 ring, and directly bind to the nucleosomal DNA. The nucleosomal linker DNA that contains the DSB site is recognized by the L1 and L2 loops-active centres that face the central hole of the RAD51 ring. In the filament form, the nucleosomal DNA is peeled by the RAD51 filament extension, and the NLDs of RAD51 protomers proximal to the nucleosome bind to the remaining nucleosomal DNA and histones. Mutations that affect nucleosome-binding residues of the RAD51 NLD decrease nucleosome binding, but barely affect DNA binding in vitro. Consistently, yeast Rad51 mutants with the corresponding mutations are substantially defective in DNA repair in vivo. These results reveal an unexpected function of the RAD51 NLD, and explain the mechanism by which RAD51 associates with nucleosomes, recognizes DSBs and forms the active filament in chromatin.


Cryoelectron Microscopy , DNA Breaks, Double-Stranded , Nucleosomes , Rad51 Recombinase , Saccharomyces cerevisiae Proteins , Humans , DNA/chemistry , DNA/metabolism , DNA/ultrastructure , DNA Repair/genetics , Nucleosomes/chemistry , Nucleosomes/metabolism , Nucleosomes/ultrastructure , Protein Subunits/chemistry , Protein Subunits/metabolism , Rad51 Recombinase/chemistry , Rad51 Recombinase/metabolism , Rad51 Recombinase/ultrastructure , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Saccharomyces cerevisiae Proteins/genetics , Saccharomyces cerevisiae Proteins/metabolism , Mutation , Protein Domains , Histones/chemistry , Histones/metabolism , Histones/ultrastructure , Protein Binding
20.
Nat Commun ; 15(1): 2132, 2024 Mar 08.
Article En | MEDLINE | ID: mdl-38459011

Growth factor receptor-bound protein 2 (GRB2) is a cytoplasmic adapter for tyrosine kinase signaling and a nuclear adapter for homology-directed-DNA repair. Here we find nuclear GRB2 protects DNA at stalled replication forks from MRE11-mediated degradation in the BRCA2 replication fork protection axis. Mechanistically, GRB2 binds and inhibits RAD51 ATPase activity to stabilize RAD51 on stalled replication forks. In GRB2-depleted cells, PARP inhibitor (PARPi) treatment releases DNA fragments from stalled forks into the cytoplasm that activate the cGAS-STING pathway to trigger pro-inflammatory cytokine production. Moreover in a syngeneic mouse metastatic ovarian cancer model, GRB2 depletion in the context of PARPi treatment reduced tumor burden and enabled high survival consistent with immune suppression of cancer growth. Collective findings unveil GRB2 function and mechanism for fork protection in the BRCA2-RAD51-MRE11 axis and suggest GRB2 as a potential therapeutic target and an enabling predictive biomarker for patient selection for PARPi and immunotherapy combination.


DNA Replication , Neoplasms , Animals , Humans , Mice , DNA , Genomic Instability , GRB2 Adaptor Protein/genetics , GRB2 Adaptor Protein/metabolism , Immunity, Innate , MRE11 Homologue Protein/metabolism , Neoplasms/genetics , Rad51 Recombinase/genetics , Rad51 Recombinase/metabolism
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