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Cell Jan 2024Lactylation is a lactate-induced post-translational modification best known for its roles in epigenetic regulation. Herein, we demonstrate that MRE11, a crucial...
Lactylation is a lactate-induced post-translational modification best known for its roles in epigenetic regulation. Herein, we demonstrate that MRE11, a crucial homologous recombination (HR) protein, is lactylated at K673 by the CBP acetyltransferase in response to DNA damage and dependent on ATM phosphorylation of the latter. MRE11 lactylation promotes its binding to DNA, facilitating DNA end resection and HR. Inhibition of CBP or LDH downregulated MRE11 lactylation, impaired HR, and enhanced chemosensitivity of tumor cells in patient-derived xenograft and organoid models. A cell-penetrating peptide that specifically blocks MRE11 lactylation inhibited HR and sensitized cancer cells to cisplatin and PARPi. These findings unveil lactylation as a key regulator of HR, providing fresh insights into the ways in which cellular metabolism is linked to DSB repair. They also imply that the Warburg effect can confer chemoresistance through enhancing HR and suggest a potential therapeutic strategy of targeting MRE11 lactylation to mitigate the effects.
Topics: Humans; DNA; DNA Breaks, Double-Stranded; DNA Repair; DNA-Binding Proteins; Epigenesis, Genetic; Homologous Recombination; MRE11 Homologue Protein; Recombinational DNA Repair; Lactic Acid
PubMed: 38128537
DOI: 10.1016/j.cell.2023.11.022 -
Nature Reviews. Molecular Cell Biology Jul 2023All organisms possess molecular mechanisms that govern DNA repair and associated DNA damage response (DDR) processes. Owing to their relevance to human disease, most... (Review)
Review
All organisms possess molecular mechanisms that govern DNA repair and associated DNA damage response (DDR) processes. Owing to their relevance to human disease, most notably cancer, these mechanisms have been studied extensively, yet new DNA repair and/or DDR factors and functional interactions between them are still being uncovered. The emergence of CRISPR technologies and CRISPR-based genetic screens has enabled genome-scale analyses of gene-gene and gene-drug interactions, thereby providing new insights into cellular processes in distinct DDR-deficiency genetic backgrounds and conditions. In this Review, we discuss the mechanistic basis of CRISPR-Cas genetic screening approaches and describe how they have contributed to our understanding of DNA repair and DDR pathways. We discuss how DNA repair pathways are regulated, and identify and characterize crosstalk between them. We also highlight the impacts of CRISPR-based studies in identifying novel strategies for cancer therapy, and in understanding, overcoming and even exploiting cancer-drug resistance, for example in the contexts of PARP inhibition, homologous recombination deficiencies and/or replication stress. Lastly, we present the DDR CRISPR screen (DDRcs) portal , in which we have collected and reanalysed data from CRISPR screen studies and provide a tool for systematically exploring them.
Topics: Humans; CRISPR-Cas Systems; DNA Repair; Neoplasms; Genome; DNA Damage
PubMed: 36781955
DOI: 10.1038/s41580-022-00571-x -
Cell Sep 2023Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer's disease (AD), with the...
Persistent DNA double-strand breaks (DSBs) in neurons are an early pathological hallmark of neurodegenerative diseases including Alzheimer's disease (AD), with the potential to disrupt genome integrity. We used single-nucleus RNA-seq in human postmortem prefrontal cortex samples and found that excitatory neurons in AD were enriched for somatic mosaic gene fusions. Gene fusions were particularly enriched in excitatory neurons with DNA damage repair and senescence gene signatures. In addition, somatic genome structural variations and gene fusions were enriched in neurons burdened with DSBs in the CK-p25 mouse model of neurodegeneration. Neurons enriched for DSBs also had elevated levels of cohesin along with progressive multiscale disruption of the 3D genome organization aligned with transcriptional changes in synaptic, neuronal development, and histone genes. Overall, this study demonstrates the disruption of genome stability and the 3D genome organization by DSBs in neurons as pathological steps in the progression of neurodegenerative diseases.
Topics: Animals; Humans; Mice; Alzheimer Disease; DNA; DNA Breaks, Double-Stranded; DNA Repair; Neurodegenerative Diseases; Neurons; Single-Cell Analysis; Sequence Analysis, RNA; Genomic Instability
PubMed: 37774679
DOI: 10.1016/j.cell.2023.08.038 -
Nature Communications Aug 2023Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low...
Genome editing, specifically CRISPR/Cas9 technology, has revolutionized biomedical research and offers potential cures for genetic diseases. Despite rapid progress, low efficiency of targeted DNA integration and generation of unintended mutations represent major limitations for genome editing applications caused by the interplay with DNA double-strand break repair pathways. To address this, we conduct a large-scale compound library screen to identify targets for enhancing targeted genome insertions. Our study reveals DNA-dependent protein kinase (DNA-PK) as the most effective target to improve CRISPR/Cas9-mediated insertions, confirming previous findings. We extensively characterize AZD7648, a selective DNA-PK inhibitor, and find it to significantly enhance precise gene editing. We further improve integration efficiency and precision by inhibiting DNA polymerase theta (Polϴ). The combined treatment, named 2iHDR, boosts templated insertions to 80% efficiency with minimal unintended insertions and deletions. Notably, 2iHDR also reduces off-target effects of Cas9, greatly enhancing the fidelity and performance of CRISPR/Cas9 gene editing.
Topics: Gene Editing; CRISPR-Cas Systems; Protein Kinases; DNA Repair; DNA
PubMed: 37580318
DOI: 10.1038/s41467-023-40344-4 -
Cell Stem Cell Nov 2023Somatic mutations accumulate in all cells with age and can confer a selective advantage, leading to clonal expansion over time. In hematopoietic cells, mutations in a...
Somatic mutations accumulate in all cells with age and can confer a selective advantage, leading to clonal expansion over time. In hematopoietic cells, mutations in a subset of genes regulating DNA repair or epigenetics frequently lead to clonal hematopoiesis (CH). Here, we describe the context and mechanisms that lead to enrichment of hematopoietic stem cells (HSCs) with mutations in SRCAP, which encodes a chromatin remodeler that also influences DNA repair. We show that SRCAP mutations confer a selective advantage in human cells and in mice upon treatment with the anthracycline-class chemotherapeutic doxorubicin and bone marrow transplantation. Furthermore, Srcap mutations lead to a lymphoid-biased expansion, driven by loss of SRCAP-regulated H2A.Z deposition and increased DNA repair. Altogether, we demonstrate that SRCAP operates at the intersection of multiple pathways in stem and progenitor cells, offering a new perspective on the functional impact of genetic variants that promote stem cell competition in the hematopoietic system.
Topics: Animals; Humans; Mice; Adenosine Triphosphatases; Clonal Hematopoiesis; DNA Repair; Epigenesis, Genetic; Hematopoiesis; Mutation
PubMed: 37863054
DOI: 10.1016/j.stem.2023.09.011 -
Molecular Cell Jul 2023Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We...
Insertions and deletions (indels) are common sources of structural variation, and insertions originating from spontaneous DNA lesions are frequent in cancer. We developed a highly sensitive assay called insertion and deletion sequencing (Indel-seq) to monitor rearrangements in human cells at the TRIM37 acceptor locus that reports indels stemming from experimentally induced and spontaneous genome instability. Templated insertions, which derive from sequences genome wide, require contact between donor and acceptor loci, require homologous recombination, and are stimulated by DNA end-processing. Insertions are facilitated by transcription and involve a DNA/RNA hybrid intermediate. Indel-seq reveals that insertions are generated via multiple pathways. The broken acceptor site anneals with a resected DNA break or invades the displaced strand of a transcription bubble or R-loop, followed by DNA synthesis, displacement, and then ligation by non-homologous end joining. Our studies identify transcription-coupled insertions as a critical source of spontaneous genome instability that is distinct from cut-and-paste events.
Topics: Humans; DNA Repair; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA; Genomic Instability; Tripartite Motif Proteins; Ubiquitin-Protein Ligases
PubMed: 37402370
DOI: 10.1016/j.molcel.2023.06.016 -
Nature Methods Sep 2023Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA...
Homology-directed repair (HDR), a method for repair of DNA double-stranded breaks can be leveraged for the precise introduction of mutations supplied by synthetic DNA donors, but remains limited by low efficiency and off-target effects. In this study, we report HDRobust, a high-precision method that, via the combined transient inhibition of nonhomologous end joining and microhomology-mediated end joining, resulted in the induction of point mutations by HDR in up to 93% (median 60%, s.e.m. 3) of chromosomes in populations of cells. We found that, using this method, insertions, deletions and rearrangements at the target site, as well as unintended changes at other genomic sites, were largely abolished. We validated this approach for 58 different target sites and showed that it allows efficient correction of pathogenic mutations in cells derived from patients suffering from anemia, sickle cell disease and thrombophilia.
Topics: Humans; Gene Editing; CRISPR-Cas Systems; Recombinational DNA Repair; DNA Breaks, Double-Stranded; DNA End-Joining Repair; DNA
PubMed: 37474806
DOI: 10.1038/s41592-023-01949-1 -
International Journal of Molecular... Nov 2023The intricate interplay between DNA damage response (DDR) and metabolism unveils a profound insight into the fundamental mechanisms governing the maintenance of genomic...
The intricate interplay between DNA damage response (DDR) and metabolism unveils a profound insight into the fundamental mechanisms governing the maintenance of genomic integrity [...].
Topics: Humans; DNA Repair; DNA Damage; Neoplasms
PubMed: 38003620
DOI: 10.3390/ijms242216430 -
Nature Cell Biology Jul 2023Proper repair of DNA damage lesions is essential to maintaining genome integrity and preventing the development of human diseases, including cancer. Increasing evidence...
Proper repair of DNA damage lesions is essential to maintaining genome integrity and preventing the development of human diseases, including cancer. Increasing evidence suggests the importance of the nuclear envelope in the spatial regulation of DNA repair, although the mechanisms of such regulatory processes remain poorly defined. Through a genome-wide synthetic viability screen for PARP-inhibitor resistance using an inducible CRISPR-Cas9 platform and BRCA1-deficient breast cancer cells, we identified a transmembrane nuclease (renamed NUMEN) that could facilitate compartmentalized and non-homologous end joining-dependent repair of double-stranded DNA breaks at the nuclear periphery. Collectively, our data demonstrate that NUMEN generates short 5' overhangs through its endonuclease and 3'→5' exonuclease activities, promotes the repair of DNA lesions-including heterochromatic lamina-associated domain breaks as well as deprotected telomeres-and functions as a downstream effector of DNA-dependent protein kinase catalytic subunit. These findings underline the role of NUMEN as a key player in DNA repair pathway choice and genome-stability maintenance, and have implications for ongoing research into the development and treatment of genome instability disorders.
Topics: Humans; DNA Repair; DNA Breaks, Double-Stranded; DNA-Binding Proteins; DNA End-Joining Repair; Endonucleases
PubMed: 37322289
DOI: 10.1038/s41556-023-01165-1 -
Molecular Cell Aug 2023To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human...
To maintain genome integrity, cells must accurately duplicate their genome and repair DNA lesions when they occur. To uncover genes that suppress DNA damage in human cells, we undertook flow-cytometry-based CRISPR-Cas9 screens that monitored DNA damage. We identified 160 genes whose mutation caused spontaneous DNA damage, a list enriched in essential genes, highlighting the importance of genomic integrity for cellular fitness. We also identified 227 genes whose mutation caused DNA damage in replication-perturbed cells. Among the genes characterized, we discovered that deoxyribose-phosphate aldolase DERA suppresses DNA damage caused by cytarabine (Ara-C) and that GNB1L, a gene implicated in 22q11.2 syndrome, promotes biogenesis of ATR and related phosphatidylinositol 3-kinase-related kinases (PIKKs). These results implicate defective PIKK biogenesis as a cause of some phenotypes associated with 22q11.2 syndrome. The phenotypic mapping of genes that suppress DNA damage therefore provides a rich resource to probe the cellular pathways that influence genome maintenance.
Topics: Humans; CRISPR-Cas Systems; DNA Damage; Mutation; DNA Repair; Phenotype
PubMed: 37478847
DOI: 10.1016/j.molcel.2023.06.025