Homo sapiens
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PROVIDER: PRJNA731981 | ENA |
REPOSITORIES: ENA
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Project description:Analysis of NHEJ-based DNA repair after CRISPR-mediated DNA cleavage
| PRJNA731544 | ENA
Project description:Non-homologous end-joining (NHEJ) plays an important role in double-strand break (DSB) repair of DNA. Recent studies have shown that the error patterns of NHEJ are strongly biased by sequence context, but these studies were based on relatively few templates. To investigate this more thoroughly, we systematically profiled ~1.16 million independent mutational events resulting from CRISPR/Cas9-mediated cleavage and NHEJ-mediated DSB repair of 6,872 synthetic target sequences, introduced into a human cell line via lentiviral infection. We find that: 1) insertions are dominated by 1 bp events templated by sequence immediately upstream of the cleavage site, 2) deletions are predominantly associated with microhomology, and 3) targets exhibit variable but reproducible diversity with respect to the number and relative frequency of the mutational outcomes to which they give rise. From these data, we trained a model (Lindel) that uses local sequence context to predict the distribution of mutational outcomes. Exploiting the bias of NHEJ outcomes towards microhomology mediated events, we demonstrate the programming of deletion patterns by introducing microhomology to specific locations in the vicinity of the DSB site. We anticipate that our results will inform investigations of DSB repair mechanisms as well as the design of CRISPR/Cas9 experiments for diverse applications including genome-wide screens, gene therapy, lineage tracing and molecular recording.
2019-06-07 | GSE131421 | GEO
Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.
2022-09-11 | E-MTAB-12037 | biostudies-arrayexpress
Project description:DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.
2022-08-25 | E-MTAB-12061 | biostudies-arrayexpress
Project description:To determine whether a predisposition to DNA damage exists in SCA7 and how extensive the predilection to DNA damage might be in SCA7, we used LAM-HTGTS, a powerful high throughput next generation sequencing technique developed for monitoring of DNA double-strand break formation. We modified the LAM-HTGTS protocol by utilizing CRISPR-Cas9 to create the double-strand DNA break at a specific site and also added a step with a 5’ methyl cytosine modified primer to promote LpnPI endonuclease cleavage of sealed breaks to enrich for translocation events. Our unbiased native chromosome DNA repair experimentation revealed that expression of polyglutamine-expanded ataxin-7 yielded greatly reduced translocations in comparison to normal ataxin-7, which is consistent with retained canonical NHEJ repair, decreased HDR activity, and decreased SSA repair in SCA7 cells, as the classical NHEJ pathway is known to prevent translocation by ligating broken double-strand breaks.
2021-02-04 | GSE166119 | GEO
Project description:DNA double-strand breaks (DSBs) contribute to genome instability, a key feature of cancer. DSBs are mainly repaired by homologous recombination (HR) and non-homologous end-joining (NHEJ). We investigated the role of an isoform of the multifunctional cyclin-dependent kinase 9, CDK9-55, in DNA repair, by generating CDK9-55-knockout HeLa clones (through CRISPR-36 Cas9), which showed potential HR dysfunction. A phosphoproteomic screening in these clones treated with camptothecin revealed that CDC23 (cell division cycle 23), a component of the E3 ubiquitin ligase APC/C (anaphase-promoting complex/cyclosome), is a new substrate of CDK9-55, with S588 being its putative phosphorylation site. Mutated non-phosphorylatable CDC23(S588A) affected the repair pathway choice by impairing HR and favouring error prone NHEJ. Moreover, CDC23(S588A) promoted the ubiquitination of UFL1, a recently identified HR player. Overall, CDK9-55 could guide APC/C in choosing the correct DNA repair pathway, possibly by regulating UFL1 stability. This CDK9 role should be considered when designing CDK-inhibitor-based cancer therapies.
2021-07-20 | MSV000087856 | MassIVE
Project description:Mutations in the ATM tumor suppressor gene confer cellular hypersensitivity to various DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms towards such drugs, we performed genome-wide CRISPR-Cas9 loss-of-function screens in cells treated with the DNA topoisomerase I poison topotecan. Our ensuing characterizations of hits established that loss of terminal components of the non-homologous end joining (NHEJ) machinery or components of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. Our findings indicate that hypersensitivity of ATM-mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib is due to delayed engagement of homologous recombination repair (HRR) at a subset of DNA-replication-fork associated single ended double-strand breaks (seDSBs), which allows non-homologous end joining (NHEJ) mediated repair, resulting in toxic chromosome fusions. Thus, restoration of legitimate repair in ATM-deficient cells – either by preventing the DNA ligation step of NHEJ or by enhancing HRR engagement by deregulating the BRCA1-A complex – markedly suppresses this toxicity. We conclude that the crucial role for ATM at seDSBs is to prevent toxic LIG4-mediated NHEJ at damaged replication forks. Furthermore, our observation that suppressor mutations in ATM-mutant backgrounds are fundamentally different to those that operate in BRCA1-mutant scenarios suggests new opportunities for patient stratification in the clinic, as well as additional therapeutic vulnerabilities that might be exploited in drug-resistant cancers.
2019-11-12 | PXD011108 | Pride
Project description:Gene disruption by CRISPR/Cas9 is highly efficient and relies on the error-prone non-homologous end-joining (NHEJ) pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than NHEJ in mammalian cells. Here, by testing whether manipulation of DNA repair factors would improve HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative 53BP1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem (iPS) cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of NHEJ-mediated DSB repair in the presence of these two factors is not suppressed, and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, co-expression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing.
2017-11-23 | GSE103241 | GEO
Project description:Maintenance of genome integrity requires tight control of DNA damage signalling and repair activities at DNA lesions, as well as their restriction at telomeres. Key components of the mechanisms underlying appropriate responses to DNA damage include phosphorylation events by DNA damage response (DDR) kinases, as well as regulatory and proteolytic ubiquitination events by the ubiquitin machinery. How these events are coordinated and controlled in a concerted manner to achieve productive DNA repair is not well understood. Here we identify the ubiquitin-conjugating enzyme UBE2D3 (or UBCH5C) as a critical multi-level regulator of ATM kinase-induced DDR signalling that controls the activity of the ubiquitin-ligase RNF168 to promote non-homologous end-joining (NHEJ) mediated DNA repair at telomeres. We find that UBE2D3 contributes to DDR-induced chromatin ubiquitination and recruitment of the NHEJ-promoting factor 53BP1, both of which are mediated by RNF168 upon DNA damage-induced ATM activation. In addition, we find that UBE2D3 promotes NHEJ by limiting RNF168 accumulation and facilitating ATM-mediated KAP1-S824 phosphorylation, important for heterochromatic DNA repair. Mechanistically, we show that defective KAP1-S824 phosphorylation upon UBE2D3-deficiency is linked to RNF168 hyperaccumulation and caused by aberrant PP2A phosphatase activity, the counteraction of which restores both KAP1-S824 phosphorylation and telomeric NHEJ in UBE2D3-deficient cells. Our results identify UBE2D3 as a novel multi-level regulator of NHEJ that orchestrates the activities of RNF168 in DNA repair. Moreover, we reveal the existence of a negative regulatory circuit in the DDR that is constrained by UBE2D3 and consists of RNF168- and PP2A-mediated restriction of ATM-dependent KAP1 phosphorylation.
2024-04-30 | PXD044410 | Pride
Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
| S-EPMC2531291 | biostudies-literature