Saturation genome editing-based clinical classification of BRCA2 variants

Nature, Год журнала: 2025, Номер unknown

Опубликована: Янв. 8, 2025

Язык: Английский

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Redox regulation: mechanisms, biology and therapeutic targets in diseases

Signal Transduction and Targeted Therapy, Год журнала: 2025, Номер 10(1)

Опубликована: Март 7, 2025

Redox signaling acts as a critical mediator in the dynamic interactions between organisms and their external environment, profoundly influencing both onset progression of various diseases. Under physiological conditions, oxidative free radicals generated by mitochondrial respiratory chain, endoplasmic reticulum, NADPH oxidases can be effectively neutralized NRF2-mediated antioxidant responses. These responses elevate synthesis superoxide dismutase (SOD), catalase, well key molecules like nicotinamide adenine dinucleotide phosphate (NADPH) glutathione (GSH), thereby maintaining cellular redox homeostasis. Disruption this finely tuned equilibrium is closely linked to pathogenesis wide range Recent advances have broadened our understanding molecular mechanisms underpinning dysregulation, highlighting pivotal roles genomic instability, epigenetic modifications, protein degradation, metabolic reprogramming. findings provide foundation for exploring regulation mechanistic basis improving therapeutic strategies. While antioxidant-based therapies shown early promise conditions where stress plays primary pathological role, efficacy diseases characterized complex, multifactorial etiologies remains controversial. A deeper, context-specific signaling, particularly redox-sensitive proteins, designing targeted aimed at re-establishing balance. Emerging small molecule inhibitors that target specific cysteine residues proteins demonstrated promising preclinical outcomes, setting stage forthcoming clinical trials. In review, we summarize current intricate relationship disease also discuss how these insights leveraged optimize strategies practice.

Язык: Английский

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Positive and negative regulators of RAD51/DMC1 in homologous recombination and DNA replication

DNA repair, Год журнала: 2023, Номер 134, С. 103613 - 103613

Опубликована: Дек. 13, 2023

Язык: Английский

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Mismatch repair protein MLH1 suppresses replicative stress in BRCA2-deficient breast tumors

Journal of Clinical Investigation, Год журнала: 2024, Номер 134(7)

Опубликована: Янв. 25, 2024

Loss of BRCA2 (breast cancer 2) is lethal for normal cells. Yet it remains poorly understood how, in mutation carriers, cells undergoing loss heterozygosity overcome the lethality and undergo tissue-specific neoplastic transformation. Here, we identified mismatch repair gene mutL homolog 1 (MLH1) as a genetic interactor whose overexpression supports viability Brca2-null Mechanistically, showed that MLH1 interacts with Flap endonuclease (FEN1) competes to process RNA flaps Okazaki fragments. Together, they restrained DNA2 nuclease activity on reversed forks lagging strands, leading replication fork (RF) stability BRCA2-deficient In these cells, also attenuated R-loops, allowing progression stable RFs, which suppressed genomic instability supported cell viability. We demonstrated significance their interaction by Brca2-mutant mice inhibition Brca2-deficient tumor growth Mlh1 loss. Furthermore, described estrogen inducing expression through receptor α (ERα), might explain why majority carriers develop ER-positive breast cancer. Taken together, our findings reveal role relieving replicative stress show how may contribute establishment tumors.

Язык: Английский

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Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor

Nature Communications, Год журнала: 2024, Номер 15(1)

Опубликована: Авг. 9, 2024

Despite the potential of small molecules and recombinant proteins to enhance efficiency homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed chemically modified, remain suboptimal for precise gene editing. Here, we screen biased ssDNA binding sequences repair-related engineer RAD51-preferred into HDR-boosting modules donors. Donors with these exhibit an augmented affinity RAD51, thereby enhancing HDR across various genomic loci cell types when cooperated Cas9, nCas9, Cas12a. By combining inhibitor non-homologous end joining (NHEJ) or HDRobust strategy, modular donors achieve up 90.03% (median 74.81%) efficiency. The targeting endogenous protein enable a chemical modification-free strategy improve efficacy Single-stranded using current design parameters inefficient authors types.

Язык: Английский

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Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants

PLoS Genetics, Год журнала: 2023, Номер 19(9), С. e1010940 - e1010940

Опубликована: Сен. 15, 2023

The unknown pathogenicity of a significant number variants found in cancer-related genes is attributed to limited epidemiological data, resulting their classification as variant uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest VUSs, which necessitated development several robust functional assays determine significance. Here we report use humanized-mouse embryonic stem cell (mESC) line expressing single copy human BRCA2 for CRISPR-Cas9-based high-throughput assay. As proof-of-principle, have saturated 11 codons encoded by exons 3, 18, 19 and all possible single-nucleotide exon 13 multiplexed these categorization. Specifically, used pool 180-mer single-stranded donor DNA generate combination variants. Using high throughput sequencing-based approach, show drop frequency non-functional variants, whereas are enriched cells. We further demonstrate response DNA-damaging agents, cisplatin olaparib, allowing us cellular survival drug parameters classification. this categorized 599 including 93-single nucleotide (SNVs) across codons, 28 reported ClinVar. also functionally 252 SNVs from into 188 60 demonstrating that saturation genome editing (SGE) coupled with sensitivity can enhance annotation VUS.

Язык: Английский

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Heavy water inhibits DNA double-strand break repairs and disturbs cellular transcription, presumably via quantum-level mechanisms of kinetic isotope effects on hydrolytic enzyme reactions

PLoS ONE, Год журнала: 2024, Номер 19(10), С. e0309689 - e0309689

Опубликована: Окт. 3, 2024

Heavy water, containing the heavy hydrogen isotope, is toxic to cells, although underlying mechanism remains incompletely understood. In addition, certain enzymatic proton transfer reactions exhibit kinetic isotope effects attributed isotopes and their temperature dependencies, indicative of quantum tunneling phenomena. However, correlation between biological water mediated by elusive. this study, we elucidated arising from dependencies in vitro, focusing on deacetylation, DNA cleavage, protein which are crucial hydrolysis. Intriguingly, intracellular related vitro effects, significantly impeded multiple double-strand break repair mechanisms for cell survival. Additionally, exposure enhanced histone acetylation associated transcriptional activation consistent with observed deacetylation reactions. Moreover, as cytotoxic effect proliferation induced exhibited temperature-dependency. These findings reveal substantial impact water-induced cellular functions governed hydrolytic reactions, potentially quantum-level effects.

Язык: Английский

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Genetically engineered mouse models for hereditary cancer syndromes

Cancer Science, Год журнала: 2023, Номер 114(5), С. 1800 - 1815

Опубликована: Янв. 30, 2023

Advances in molecular diagnostics have led to improved diagnosis and understanding of hereditary cancers the clinic. Improving management, treatment, potential prevention carriers predisposing mutations requires preclinical experimental models that reflect key pathogenic features specific syndrome associated with mutations. Numerous genetically engineered mouse (GEM) cancer been developed. In this review, we describe Lynch breast ovarian syndrome, two most common predisposition syndromes. We focus on as illustrative for using devise approaches a high-risk population. GEM are an invaluable tool models. Here, review some their use studies.

Язык: Английский

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IGF‐I protects porcine granulosa cells from hypoxia‐induced apoptosis by promoting homologous recombination repair through the PI3K/AKT/E2F8/RAD51 pathway

The FASEB Journal, Год журнала: 2023, Номер 38(1)

Опубликована: Дек. 14, 2023

Abstract Severe hypoxia induced by vascular compromise (ovarian torsion, surgery), obliteration of vessels (aging, chemotherapy, particularly platinum drugs) can cause massive follicle atresia. On the other hand, increases occurrence DNA double‐strand breaks (DSBs) and triggers cellular damage repair mechanisms; however, if is not promptly repaired, it also induce apoptosis program. Insulin‐like growth factor‐I (IGF‐I) a polypeptide hormone that plays essential roles in stimulating mammalian follicular development. Here, we report novel role for IGF‐I protecting hypoxic GCs from promoting through homologous recombination (HR) process. Indeed, environment within follicles significantly inhibited efficiency HR‐directed repair. The presence IGF‐I‐induced HR pathway to alleviate hypoxia‐induced primarily upregulating expression RAD51 recombinase. Importantly, identified new transcriptional regulator , namely E2F8, which mediates protective effects on facilitating activation . Furthermore, demonstrated PI3K/AKT crucial E2F8 expression, resulting increased enhanced activity, mitigates thereby protects against apoptosis. Together, these findings define mechanism IGF‐I‐mediated protection activating PI3K/AKT/E2F8/RAD51 under hypoxia.

Язык: Английский

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A Multifunctional and Highly Adaptable Reporter System for CRISPR/Cas Editing

International Journal of Molecular Sciences, Год журнала: 2023, Номер 24(9), С. 8271 - 8271

Опубликована: Май 5, 2023

CRISPR/Cas systems are some of the most promising tools for therapeutic genome editing. The use these is contingent on optimal designs guides and homology-directed repair (HDR) templates. While this design can be achieved in silico, validation further optimization usually performed with help reporter systems. Here, we describe a novel system, termed BETLE, that allows fast, sensitive, cell-specific detection editing template-specific HDR by encoding multiple proteins different open-reading frames. Out-of-frame non-homologous end joining (NHEJ) leads to expression either secretable NanoLuc luciferase, enabling highly sensitive low-cost analysis editing, or fluorescent mTagBFP2, allowing enumeration tissue-specific localization genome-edited cells. BETLE includes site validate sequence-of-interest, making it broadly adaptable. We evaluated using defective moxGFP 39-base-pair deletion showed spCas9, saCas9, asCas12a as well sequence-specific cell lines single integrants. Taken together, data show rapid vitro represents state-of art tool future applications vivo.

Язык: Английский

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