Non-homologous End Joining Research Articles

A Boolean network model of the double-strand break repair pathway choice

Double strand break (DSB) repair is critical to maintaining the integrity of the genome. DSB repair deficiency underlies multiple pathologies, including cancer, chromosome instability syndromes, and, potentially, neurodevelopmental defects. DSB repair is mainly handled by two pathways: highly accurate homologous recombination (HR), which requires a sister chromatid for template-based repair, limited to S/G2 phases of the cell cycle, and canonical non-homologous end joining (c-NHEJ), available throughout the cell cycle in which minimum homology is sufficient for highly efficient yet error-prone repair. Some circumstances, such as cancer, require alternative highly mutagenic DSB repair pathways like microhomology-mediated end-joining (MMEJ) and single-strand annealing (SSA), which are triggered to attend to DNA damage. These non-canonical repair alternatives are emerging as prominent drivers of resistance in drug-based tumor therapies. Multiple DSB repair options require tight inter-pathway regulation to prevent unscheduled activities. In addition to this complexity, epigenetic modifications of the histones surrounding the DSB region are emerging as critical regulators of the DSB repair pathway choice. Modeling approaches to understanding DSBs repair pathway choice are advantageous to perform simulations and generate predictions on previously uncharacterized aspects of DSBs response.In this work, we present a Boolean network model of the DSB repair pathway choice that incorporates the knowledge, into a dynamic system, of the inter-pathways regulation involved in DSB repair, i.e., HR, c-NHEJ, SSA, and MMEJ. Our model recapitulates the well-characterized HR activity observed in wild-type cells in response to DSBs. It also recovers clinically relevant behaviors of BRCA1/FANCS mutants, and their corresponding drug resistance mechanisms ascribed to DNA repair gain-of-function pathogenic variants. Since epigenetic modifiers are dynamic and possible druggable targets, we incorporated them into our model to better characterize their involvement in DSB repair. Our model predicted that loss of the TIP60 complex and its corresponding histone acetylation activity leads to activation of SSA in response to DSBs. Our experimental validation showed that TIP60 effectively prevents activation of RAD52, a key SSA executor, and confirms the suitable use of Boolean network modeling for understanding DNA DSB repair.

Holding it together: DNA end synapsis during non-homologous end joining

DNA double strand breaks (DSBs) are common lesions whose misrepair are drivers of oncogenic transformations. The non-homologous end joining (NHEJ) pathway repairs the majority of these breaks in vertebrates by directly ligating DNA ends back together. Upon formation of a DSB, a multiprotein complex is assembled on DNA ends which tethers them together within a synaptic complex. Synapsis is a critical step of the NHEJ pathway as loss of synapsis can result in mispairing of DNA ends and chromosome translocations. As DNA ends are commonly incompatible for ligation, the NHEJ machinery must also process ends to enable rejoining. This review describes how recent progress in single-molecule approaches and cryo-EM have advanced our molecular understanding of DNA end synapsis during NHEJ and how synapsis is coordinated with end processing to determine the fidelity of repair.

Arsenic affects homologous recombination and single-strand annealing but not end-joining pathways during DNA double-strand break repair.

Arsenic is a carcinogen that can cause skin, lung, and bladder cancer. While DNA double-strand breaks (DSBs) have been implicated in arsenic-induced carcinogenesis, the exact mechanism remains unclear. In this study, we performed genetic analysis to examine the impact of arsenic trioxide (As2 O3 ) on four different DSB repair pathways using the human pre-B cell line Nalm-6. Random integration analysis showed that As2 O3 does not negatively affect non-homologous end joining or polymerase theta-mediated end joining. In contrast, chromosomal DSB repair analysis revealed that As2 O3 decreases the efficiency of homologous recombination (HR) and, less prominently, single-strand annealing. Consistent with this finding, As2 O3 decreased gene-targeting efficiency, owing to a significant reduction in the frequency of HR-mediated targeted integration. To further verify the inhibitory effect of arsenic on HR, we examined cellular sensitivity to olaparib and camptothecin, which induce one-ended DSBs requiring HR for precise repair. Intriguingly, we found that As2 O3 significantly enhances sensitivity to those anticancer agents in HR-proficient cells. Our results suggest that arsenic-induced genomic instability is attributed to HR suppression, providing valuable insights into arsenic-associated carcinogenesis and therapeutic options.

CSB Regulates Pathway Choice in Response to DNA Replication Stress Induced by Camptothecin.

Topoisomerase inhibitor camptothecin (CPT) induces fork stalling and is highly toxic to proliferating cells. However, how cells respond to CPT-induced fork stalling has not been fully characterized. Here, we report that Cockayne syndrome group B (CSB) protein inhibits PRIMPOL-dependent fork repriming in response to a low dose of CPT. At a high concentration of CPT, CSB is required to promote the restart of DNA replication through MUS81-RAD52-POLD3-dependent break-induced replication (BIR). In the absence of CSB, resumption of DNA synthesis at a high concentration of CPT can occur through POLQ-LIG3-, LIG4-, or PRIMPOL-dependent pathways, which are inhibited, respectively, by RAD51, BRCA1, and BRCA2 proteins. POLQ and LIG3 are core components of alternative end joining (Alt-EJ), whereas LIG4 is a core component of nonhomologous end joining (NHEJ). These results suggest that CSB regulates fork restart pathway choice following high-dosage CPT-induced fork stalling, promoting BIR but inhibiting Alt-EJ, NHEJ, and fork repriming. We find that loss of CSB and BRCA2 is a toxic combination to genomic stability and cell survival at a high concentration of CPT, which is likely due to accumulation of ssDNA gaps, underscoring an important role of CSB in regulating the therapy response in cancers lacking functional BRCA2.

High-efficiency transgene integration by homology-directed repair in human primary cells using DNA-PKcs inhibition.

Therapeutic applications of nuclease-based genome editing would benefit from improved methods for transgene integration via homology-directed repair (HDR). To improve HDR efficiency, we screened six small-molecule inhibitors of DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a key protein in the alternative repair pathway of non-homologous end joining (NHEJ), which generates genomic insertions/deletions (INDELs). From this screen, we identified AZD7648 as the most potent compound. The use of AZD7648 significantly increased HDR (up to 50-fold) and concomitantly decreased INDELs across different genomic loci in various therapeutically relevant primary human cell types. In all cases, the ratio of HDR to INDELs markedly increased, and, in certain situations, INDEL-free high-frequency (>50%) targeted integration was achieved. This approach has the potential to improve the therapeutic efficacy of cell-based therapies and broaden the use of targeted integration as a research tool.

DNA-PK inhibition extends the therapeutic effects of Top2 poisoning to non-proliferating cells, increasing activity at a cost

Type II topoisomerase (Top2) poisoning therapy is used to treat a broad range of cancers via induction of double strand breaks (DSBs) in cells undergoing replication and transcription. Preventing the repair of DSBs via inhibition of DNA-PK, an inhibitor of non-homologous end-joining (NHEJ), increases cell kill with Top2 poisons and has led to the initiation of several clinical trials. To elucidate the cellular mechanisms leading to synergistic activity of dual DNA-PK/Top2 inhibition we looked at their effects in cycling versus non-cycling cells, in 3D spheroids and in xenograft models. Combined DNA-PK/Top2 inhibition was found to not only increase the cell kill in proliferating cells, the cell population that is typically most vulnerable to Top2 poisoning, but also in non-proliferative but transcriptionally active cells. This effect was observed in both cancer and normal tissue models, killing more cells than high concentrations of etoposide alone. The combination treatment delayed tumor growth in mice compared to Top2 poisoning alone, but also led to increased toxicity. These findings demonstrate sensitization of Top2β-expressing, non-cycling cells to Top2 poisoning by DNA-PK inhibition. Expansion of the target cell population of Top2 poison treatment to include non-proliferating cells via combination with DNA damage repair inhibitors has implications for efficacy and toxicity of these combinations, including for inhibitors of DNA-PK currently in clinical trial.

Functional inhibition of RECQL5 helicase elicits non-homologous end joining response and sensitivity of breast cancers to PARP inhibitor.

Poly (ADPRibose) Polymerase inhibitor (PARPi) are clinically approved for the treatment of BRCA-mutated hereditary breast and ovarian cancers with homologous recombination (HR) deficiency, based on synthetic lethality concept. However, ∼90% of breast cancers are BRCA-wild type; they repair PARPi mediated damage through HR, leading to intrinsic de novo resistance. Hence, there is an unmet need of exploring novel targets in HR-proficient aggressive breast cancers for PARPi treatment. RECQL5 physically interacts and disrupts RAD51 from pre-synaptic filaments, aiding HR resolution, replication fork protection and preventing illegitimate recombination. In the current investigation, we show that targeted inhibition of HR by stabilization of RAD51-RECQL5 complex by a pharmacological inhibitor of RECQL5 (4a; 1,3,4-oxadiazole derivative) in the presence of PARPi [talazoparib (BMN673)] leads to abolition of functional HR with uncontrolled activation of NHEJ repair. This was assessed by GFP based NHEJ reporter assay, KU80 recruitment and in vitro NHEJ based plasmid ligation assay. Concomitant treatment with talazoparib and 4a generates copious amounts of replication stress, prolonged cell cycle arrest, extensive double strand breaks (DSBs) and mitotic catastrophe, leading to sensitization of HR-proficient breast cancers. Suppression of NHEJ activity abolishes 4a-mediated sensitization of breast cancers to PARPi treatment. Imperatively, 4a was ineffective against normal mammary epithelial cells, which expresses low RECQL5 vis-à-vis breast cancer cells. Moreover, functional inhibition of RECQL5 suppresses metastatic potential of breast cancer cells in response to PARPi. Together, we identified RECQL5 as a novel pharmacological target for expanding PARPi based treatment horizon for HR-proficient cancers.

Structure and mechanism in non-homologous end joining

DNA double-stranded breaks (DSBs) are a particularly challenging form of DNA damage to repair because the damaged DNA must not only undergo the chemical reactions responsible for returning it to its original state, but, additionally, the two free ends can become physically separated in the nucleus and must be bridged prior to repair. In nonhomologous end joining (NHEJ), one of the major pathways of DSB repair, repair is carried out by a number of repair factors capable of binding to and directly joining DNA ends. It has been unclear how these processes are carried out at a molecular level, owing in part to the lack of structural evidence describing the coordination of the NHEJ factors with each other and a DNA substrate. Advances in cryo-Electron Microscopy (cryo-EM), allowing for the structural characterization of large protein complexes that would be intractable using other techniques, have led to the visualization several key steps of the NHEJ process, which support a model of sequential assembly of repair factors at the DSB, followed by end-bridging mediated by protein-protein complexes and transition to full synapsis. Here we examine the structural evidence for these models, devoting particular attention to recent work identifying a new NHEJ intermediate state and incorporating new NHEJ factors into the general mechanism. We also discuss the evolving understanding of end-bridging mechanisms in NHEJ and DNA-PKcs’s role in mediating DSB repair.

CRISPR/Cas9-induced DNA breaks trigger crossover, chromosomal loss, and chromothripsis-like rearrangements.

DNA double-stranded breaks (DSBs) generated by the Cas9 nuclease are commonly repaired via non-homologous end-joining (NHEJ) or homologous recombination (HR). However, little is known about unrepaired DSBs and the type of damage they trigger in plants. We designed an assay that detects loss of heterozygosity (LOH) in somatic cells, enabling the study of a broad range of DSB-induced genomic events. The system relies on a mapped phenotypic marker which produces a light purple color (betalain pigment) in all plant tissues. Plants with sectors lacking the Betalain marker upon DSB induction between the marker and the centromere were tested for LOH events. Using this assay, we detected a tomato (Solanum lycopersicum) flower with a twin yellow and dark purple sector, corresponding to a germinally transmitted somatic crossover event. We also identified instances of small deletions of genomic regions spanning the T-DNA and whole chromosome loss. In addition, we show that major chromosomal rearrangements including loss of large fragments, inversions, and translocations were clearly associated with the CRISPR-induced DSB. Detailed characterization of complex rearrangements by whole genome sequencing and molecular and cytological analyses supports a model in which a breakage-fusion-bridge cycle followed by chromothripsis-like rearrangements had been induced. Our LOH assay provides a tool for precise breeding via targeted crossover detection. It also uncovers CRISPR-mediated chromothripsis-like events in plants.

Non-homologous end-joining-deficient filamentous fungal strains mitigate the impact of off-target mutations during the application of CRISPR/Cas9.

CRISPR/Cas9 genome editing technology has been implemented in almost all living organisms. Its editing precision appears to be very high and therefore could represent a big change from conventional genetic engineering approaches. However, guide RNA binding to nucleotides similar to the target site could result in undesired off-target mutations. Despite this, evaluating whether mutations occur is rarely performed in genome editing studies. In this study, we generated CRISPR/Cas9-derived filamentous fungal strains and analyzed them for the occurrence of mutations, and to which extent genome stability affects their occurrence. As a test case, we deleted the (hemi-)cellulolytic regulator-encoding gene xlnR in two Aspergillus niger strains: a wild type (WT) and a non-homologous end-joining (NHEJ)-deficient strain ΔkusA. Initial phenotypic analysis suggested a much higher prevalence of mutations in the WT compared to NHEJ-deficient strains, which was confirmed and quantified by whole-genome sequencing analysis. Our results clearly demonstrate that CRISPR/Cas9 applied to an NHEJ-deficient strain is an efficient strategy to avoid unwanted mutations. IMPORTANCE Filamentous fungi are commonly used biofactories for the production of industrially relevant proteins and metabolites. Often, fungal biofactories undergo genetic development (genetic engineering, genome editing, etc.) aimed at improving production yields. In this context, CRISPR/Cas9 has gained much attention as a genome editing strategy due to its simplicity, versatility, and precision. However, despite the high level of accuracy reported for CRISPR/Cas9, in some cases unintentional cleavages in non-targeted loci-known as off-target mutations-could arise. While biosafety should be a central feature of emerging biotechnologies to minimize unintended consequences, few studies quantitatively evaluate the risk of off-target mutations. This study demonstrates that the use of non-homologous end-joining-deficient fungal strains drastically reduces the number of unintended genomic mutations, ensuring that CRISPR/Cas9 can be safely applied for strain development.

Efficient high-precision homology-directed repair-dependent genome editing by HDRobust.

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.

Long-read sequence analysis of MMEJ-mediated CRISPR genome editing reveals complex on-target vector insertions that may escape standard PCR-based quality control

CRISPR genome editing is a powerful tool for elucidating biological functions. To modify the genome as intended, it is essential to understand the various modes of recombination that can occur. In this study, we report complex vector insertions that were identified during the generation of conditional alleles by CRISPR editing using microhomology-mediated end joining (MMEJ). The targeting vector contained two loxP sequences and flanking 40-bp microhomologies. The genomic regions corresponding to the loxP sequences were cleaved with Cas9 in mouse embryonic stem cells. PCR screening for targeted recombination revealed a high frequency of bands of a larger size than expected. Nanopore sequencing of these bands revealed complex vector insertions mediated not only by MMEJ but also by non-homologous end joining and homologous recombination in at least 17% of the clones. A new band appeared upon improving the PCR conditions, suggesting the presence of unintentionally modified alleles that escape standard PCR screening. This prompted us to characterize the recombination of each allele of the genome-edited clones using heterozygous single nucleotide polymorphisms, leading to confirmation of the presence of homozygous alleles. Our study indicates that careful quality control of genome-edited clones is needed to exclude complex, unintended, on-target vector insertion.

MiR-4796 enhances the sensitivity of breast cancer cells to ionising radiation by impairing the DNA repair pathway.

MicroRNAs (miRNAs) are important regulators of DNA damage response (DDR) through post-transcriptional regulation on their target genes, which are implicated in DDR and DNA repair (DR). In this study, we investigated the functional roles and target genes of miR-4796 and miR-1287 in breast cancer cells in response to radiation. The molecular mechanism of miR-4796 in regulating the radiosensitivity of breast cancer cells was also elucidated. Real-time polymerase chain reaction detected miR-4796 and miR-1287 expression; colony formation assay and irradiation therapy tumour xenograft in vivo examined radiosensitising effect; comet assay assessed DNA damage; immunofluorescence imaging determined the formation of γ-H2AX foci; targetscan and RegRNA predicted target mRNAs; luciferase reporter and mutation assays validated target genes; western blotting detected the expression of genes at the protein level; and flow cytometry quantified the activities of nonhomologous end-joining (NHEJ) and homologous recombination (HR). The expressions of miR-4796 and miR-1287 were acutely fluctuated in response to ionising radiation. In the absence of radiation, overexpression of miR-1287 dramatically promoted growth of breast cancer cells in vitro and in vivo, whereas overexpression of miR-4796 did not affect cell growth. When under the treatment with radiation, overexpression of miR-4796 suppressed DR and sensitised cancer cells to radiation both in vitro and in vivo. However, such effect was only observed in cell assays in the overexpressed miR-1287 group, and not confirmed in vivo. We therefore further explored the molecular mechanism of action of miR-4796, and found that miR-4796 targeted multiple components of DDR and DR, including ATM, BRCA1, PARP and RAD51. Moreover, overexpression of miR-4796 inhibited the expression of these DDR components at the protein level. In addition, miR-4796 inhibited HR and NHEJ repair pathways and aggravated radiation-induced DNA damage. The findings here suggest that miR-4796 can enhance radiation-induced cell death by directly targeting multiple DDR components, and repress NHEJ and HR DNA repair pathways. miR-4796 can act as an effective radiation sensitising agent.

RHINO directs MMEJ to repair DNA breaks in mitosis.

Nonhomologous end-joining (NHEJ) and homologous recombination (HR) are the primary pathways for repairing DNA double-strand breaks (DSBs) during interphase, whereas microhomology-mediated end-joining (MMEJ) has been regarded as a backup mechanism. Through CRISPR-Cas9-based synthetic lethal screens in cancer cells, we identified subunits of the 9-1-1 complex (RAD9A-RAD1-HUS1) and its interacting partner, RHINO, as crucial MMEJ factors. We uncovered an unexpected function for RHINO in restricting MMEJ to mitosis. RHINO accumulates in M phase, undergoes Polo-like kinase 1 (PLK1) phosphorylation, and interacts with polymerase θ (Polθ), enabling its recruitment to DSBs for subsequent repair. Additionally, we provide evidence that MMEJ activity in mitosis repairs persistent DSBs that originate in S phase. Our findings offer insights into the synthetic lethal relationship between the genes POLQ and BRCA1 and BRAC2 and the synergistic effect of Polθ and poly(ADP-ribose) polymerase (PARP) inhibitors.

PC4-mediated Ku complex PARylation facilitates NHEJ-dependent DNA damage repair

Radiotherapy is one of the mainstay treatments for hepatocellular carcinoma (HCC). However, a substantial number of patients with HCC develop radioresistance and eventually suffer from tumour progression or relapse, which is a major impediment to the use of radiotherapy. Therefore, elucidating the mechanisms underlying radioresistance and identifying novel therapeutic targets to improve patient prognosis are important in HCC management. In this study, using in vitro and in vivo models, laser microirradiation and live cell imaging methods and coimmunoprecipitation assays, we report that a DNA repair enhancer, human positive cofactor 4 (PC4), promotes nonhomologous end joining (NHEJ)-based DNA repair and renders HCC cells resistant to radiation. Mechanistically, PC4 interacts with poly (ADP-ribose) polymerase 1 (PARP1) and directs Ku complex PARylation, resulting in the successful recruitment of the Ku complex to damaged chromatin and increasing the efficiency of NHEJ repair. Clinically, PC4 is highly expressed in tumour tissues and is correlated with poor prognosis in patients with HCC. Taken together, our data suggest that PC4 is a DNA repair driver that can be targeted to radiosensitise HCC cells.

Base editing enables duplex point mutagenesis in Clostridium autoethanogenum at the price of numerous off-target mutations.

Base editors are recent multiplex gene editing tools derived from the Cas9 nuclease of Streptomyces pyogenes. They can target and modify a single nucleotide in the genome without inducing double-strand breaks (DSB) of the DNA helix. As such, they hold great potential for the engineering of microbes that lack effective DSB repair pathways such as homologous recombination (HR) or non-homologous end-joining (NHEJ). However, few applications of base editors have been reported in prokaryotes to date, and their advantages and drawbacks have not been systematically reported. Here, we used the base editors Target-AID and Target-AID-NG to introduce nonsense mutations into four different coding sequences of the industrially relevant Gram-positive bacterium Clostridium autoethanogenum. While up to two loci could be edited simultaneously using a variety of multiplexing strategies, most colonies exhibited mixed genotypes and most available protospacers led to undesired mutations within the targeted editing window. Additionally, fifteen off-target mutations were detected by sequencing the genome of the resulting strain, among them seven single-nucleotide polymorphisms (SNP) in or near loci bearing some similarity with the targeted protospacers, one 15 nt duplication, and one 12kb deletion which removed uracil DNA glycosylase (UDG), a key DNA repair enzyme thought to be an obstacle to base editing mutagenesis. A strategy to process prokaryotic single-guide RNA arrays by exploiting tRNA maturation mechanisms is also illustrated.

Genetic control of the error-prone repair of a chromosomal double-strand break with 5' overhangs in yeast.

A targeted double-strand break introduced into the genome of Saccharomyces cerevisiae is repaired by the relatively error-prone nonhomologous end joining (NHEJ) pathway when homologous recombination is not an option. A zinc finger nuclease cleavage site was inserted out-of-frame into the LYS2 locus of a haploid yeast strain to study the genetic control of NHEJ when the ends contain 5' overhangs. Repair events that destroyed the cleavage site were identified either as Lys+ colonies on selective medium or as surviving colonies on rich medium. Junction sequences in Lys+ events solely reflected NHEJ and were influenced by the nuclease activity of Mre11 as well as by the presence/absence of the NHEJ-specific polymerase Pol4 and the translesion-synthesis DNA polymerases Pol ζ and Pol η. Although most NHEJ events were dependent on Pol4, a 29-bp deletion with endpoints in 3-bp repeats was an exception. The Pol4-independent deletion required translesion synthesis polymerases as well as the exonuclease activity of the replicative Pol δ DNA polymerase. Survivors were equally split between NHEJ events and 1.2 or 11.7 kb deletions that reflected microhomology-mediated end joining (MMEJ). MMEJ events required the processive resection activity of Exo1/Sgs1, but there unexpectedly was no dependence on the Rad1-Rad10 endonuclease for the removal of presumptive 3' tails. Finally, NHEJ was more efficient in nongrowing than in growing cells and was most efficient in G0 cells. These studies provide novel insights into the flexibility and complexity of error-prone DSB repair in yeast.

Xrcc5/Ku80 is required for the repair of DNA damage in fully grown meiotically arrested mammalian oocytes

Mammalian oocytes spend most of their life in a unique state of cell cycle arrest at meiotic prophase I, during which time they are exposed to countless DNA-damaging events. Recent studies have shown that DNA double-strand break repair occurs predominantly via the homologous recombination (HR) pathway in small non-growing meiotically arrested oocytes (primordial follicle stage). However, the DNA repair mechanisms employed by fully grown meiotically arrested oocytes (GV-stage) have not been studied in detail. Here we established a conditional knockout mouse model to explore the role of Ku80, a critical component of the nonhomologous end joining (NHEJ) pathway, in the repair of DNA damage in GV oocytes. GV oocytes lacking Ku80 failed to repair etoposide-induced DNA damage, even when only low levels of damage were sustained. This indicates Ku80 is needed to resolve DSBs and that HR cannot compensate for a compromised NHEJ pathway in fully-grown oocytes. When higher levels of DNA damage were induced, a severe delay in M-phase entry was observed in oocytes lacking XRCC5 compared to wild-type oocytes, suggesting that Ku80-dependent repair of DNA damage is important for the timely release of oocytes from prophase I and resumption of meiosis. Ku80 was also found to be critical for chromosome integrity during meiotic maturation following etoposide exposure. These data demonstrate that Ku80, and NHEJ, are vital for quality control in mammalian GV stage oocytes and reveal that DNA repair pathway choice differs in meiotically arrested oocytes according to growth status.

Application of CRISPR in Filamentous Fungi and Macrofungi: From Component Function to Development Potentiality.

Fungi, particularly filamentous fungi and macrofungi, have a very powerful ability to produce secondary metabolites and can serve as excellent chassis cells for the production of enzymes or natural products of great value in synthetic biology. Thus, it is imperative to establish simple, reliable, and efficient techniques for their genetic modification. However, the heterokaryosis of some fungi and the dominance of nonhomologous end-joining (NHEJ) repair mechanisms in vivo have been greatly affecting the efficiency of fungal gene editing. In recent years, the CRISPR/Cas9 system has been applied as a widely used gene editing technology in life science research and has also played an important role in the genetic modification of filamentous and macrofungi. The various functional components (cas9, sgRNA, promoter, and screening marker) of the CRISPR/Cas9 system and its development, as well as the difficulties and potential of the CRISPR/Cas9 system in filamentous fungus and macrofungi, are the main topics of this paper.

DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.