Large-scale phase III clinical trials of Olaparib have revealed benefits for ovarian cancer patients with BRCA gene mutations or homologous recombination deficiency (HRD). However, fewer than 50% of ovarian cancer patients have both BRCA mutations and HRD. Therefore, improving the effect of Olaparib in HR-proficient patients is of great clinical value. Here, a combination strategy comprising Olaparib and CDK12-IN-3 effectively inhibited the growth of HR-proficient ovarian cancer in cell line, patient-derived organoid (PDO), and mouse xenograft models. Furthermore, the combination strategy induced severe DNA double-strand break (DSB) formation, increased NHEJ activity in the G2 phase, and reduced HR activity in cancer cells. Mechanistically, the combination treatment impaired Ku80 poly(ADP-ribosyl)ation (PARylation) and phosphorylation, resulting in PARP1-Ku80 complex dissociation. After dissociation, Ku80 occupancy at DSBs and the resulting Ku80-primed NHEJ activity were increased. Owing to Ku80-mediated DNA end protection, MRE11 and Rad51 foci formation was inhibited after the combination treatment, suggesting that this treatment suppressed HR activity. Intriguingly, the combination strategy expedited cGAS nuclear relocalization, further suppressing HR and, conversely, increasing genomic instability. Moreover, the inhibitory effect on cell survival persisted after drug withdrawal. These findings provide a rationale for the clinical application of CDK12-IN-3 in combination with Olaparib.

DNA double-strand breaks (DSBs) are considered one of the most harmful forms of DNA damage. These DSBs are repaired through non-homologous end joining (NHEJ) and homologous recombination (HR) pathways and defects in these processes can lead to genomic instability and promote tumorigenesis. Phosphatase and Tensin homolog (PTEN) are crucial in HR repair. However, its involvement in the NHEJ repair pathway has remained elusive. In this study, we investigate the function of epigenetic regulation of PTEN in the NHEJ repair pathway. Our findings indicate that both the phosphorylation and phosphatase activity of PTEN are required for efficient NHEJ-mediated DSB repair. During the DNA damage response, we observed a reduced expression and chromatin attachment of the key NHEJ proteins, including Ku70/80, DNA-PKcs, XRCC4, and XLF, in PTEN-null cells. This reduction was attributed to the instability of these NHEJ proteins, as confirmed by our protein half-life assay. We have demonstrated that the DNA-PKcs inhibitor, NU7026, suppresses the DNA damage-induced phosphorylation of the C-terminal of PTEN. Thus, our study indicates that PTEN could be a target of DNA-PKcs. Protein-protein docking analysis also shows that PTEN interacts with the C-terminal region of DNA-PKcs. PTEN null cells exhibit compromised DNA-PKcs foci after DNA damage as it is in a hyper-phosphorylated state. Phospho-PTEN assists in recruiting DNA-PKcs on the DNA damage site by maintaining its hypo-phosphorylated state which also depends on its phosphatase activity. Therefore, after DNA damage, crosstalk between PTEN and DNA-PKcs modulates the NHEJ pathway. Thus, during DNA damage, PTEN gets phosphorylated directly or indirectly by DNA-PKcs and attaches to chromatin, resulting in the dephosphorylation of DNA-PKcs and subsequently recruitment of other NHEJ factors on chromatin occurs for efficient execution of the NHEJ pathway. Thus, our research provides a molecular understanding of the epigenetic regulation of PTEN and its significant role in controlling the NHEJ pathway.

Thyroid cancer incidence increases worldwide annually, primarily due to factors such as ionizing radiation (IR), iodine intake, and genetics. Papillary carcinoma of the thyroid (PTC) accounts for about 80% of thyroid cancer cases. RET/PTC1 (coiled-coil domain containing 6 [CCDC6]-rearranged during transfection) rearrangement is a distinctive feature in over 70% of thyroid cancers who exposed to low doses of IR in Chernobyl and Hiroshima‒Nagasaki atomic bombings. This study aims to elucidate mechanism between RET/PTC1 rearrangement and IR in PTC. N-thy-ori-3-1 cells were subjected to varying doses of IR (2/1/0.5/0.2/0.1/0.05 Gy) of IR at different days, and result showed low-dose IR-induced RET/PTC1 rearrangement in a dose-dependent manner. RET/PTC1 has been observed to promote PTC both in vivo and in vitro. To delineate the role of different DNA repair pathways, SCR7, RI-1, and Olaparib were employed to inhibit non-homologous end joining (NHEJ), homologous recombination (HR), and microhomology-mediated end joining (MMEJ), respectively. Notably, inhibiting NHEJ enhanced HR repair efficiency and reduced IR-induced RET/PTC1 rearrangement. Conversely, inhibiting HR increased NHEJ repair efficiency and subsequent RET/PTC1 rearrangement. The MMEJ did not show a markable role in this progress. Additionally, inhibiting DNA-dependent protein kinase catalytic subunit (DNA-PKcs) decreased the efficiency of NHEJ and thus reduced IR-induced RET/PTC1 rearrangement. To conclude, the data suggest that NHEJ, rather than HR or MMEJ, is the critical cause of IR-induced RET/PTC1 rearrangement. Targeting DNA-PKcs to inhibit the NHEJ has emerged as a promising therapeutic strategy for addressing IR-induced RET/PTC1 rearrangement in PTC.

Non-homologous end-joining (NHEJ) stands as a pivotal DNA repair pathway crucial for the survival and persistence of Mycobacterium tuberculosis (Mtb) during its dormant, non-replicating phase, a key aspect of its long-term resilience. Mycobacterial NHEJ is a remarkably simple two-component system comprising the rate-limiting DNA binding protein Ku (mKu) and Ligase D. To elucidate mKu\'s role in NHEJ, we conducted a series of in silico and in vitro experiments. Molecular dynamics simulations and in vitro assays revealed that mKu\'s DNA binding stabilizes both the protein and DNA, while also shielding DNA ends from exonuclease degradation. Surface plasmon resonance (SPR) and electrophoretic mobility shift assays (EMSA) demonstrated mKu\'s robust affinity for linear double-stranded DNA (dsDNA), showing positive cooperativity for DNA substrates of 40 base pairs or longer, and its ability to slide along DNA strands. Moreover, analytical ultracentrifugation, size exclusion chromatography, and negative stain electron microscopy (EM) unveiled mKu\'s unique propensity to form higher-order oligomers exclusively with DNA, suggesting a potential role in mycobacterial NHEJ synapsis. This comprehensive characterization sheds new light on mKu\'s function within the Mtb NHEJ repair pathway. Targeting this pathway may thus impede the pathogen\'s ability to persist in its latent state within the host for prolonged periods.

Non-pathogenic mycobacteria, including Mycolicibacterium neoaurum, can directly utilize phytosterols for large-scale industrial production of steroid medicine intermediates due to their natural steroid metabolism pathway. The targeted genetic modification of M. neoaurum is conducive to the selection of high-yield engineering bacteria with high-value-added product, such as Pregnadien-20-carboxylic acid (PDC), which is an important precursor for synthesizing some corticosteroids. Based on heterologous type II CRISPR/sth1Cas9 system, a simple strategy was developed to genetic engineer M. neoaurum genome. Here, a customizable plasmid tool pMSC9 was constructed from pMV261 with integration of sth1Cas9 protein and corresponding sgRNA scaffold. Subsequently, the pMSC9 was inserted with spacer sequences corresponding to different targeted genes, generating editing plasmids, and then transformed into M. neoaurum. As a result, the targeted genes were introduced with DNA double stand breaks (DSBs) by CRISPR/sth1Cas9 system and then repaired by innate non-homologous end-joining (NHEJ) mechanism. Finally, editing plasmids were cured from correctly edited M. neoaurum mutants by means of no resistance cultivation, and the resulting mutant deleting the one target gene was used as the host to which another target gene could be deleted via the same process. This study demonstrated that the CRISPR/sth1Cas9 tool allowed M. neoaurum strains to be rapidly edited. And the editing mode of CRISPR/sth1Cas9 system indicated that this tool was an important supplement to the gene editing toolbox of M. neoaurum.

CRISPR-Cas9-mediated gene editing has vast applications in basic and clinical research and is a promising tool for several disorders. Our lab previously developed a non-integrating RNA virus, measles virus (MeV), as a single-cycle reprogramming vector by replacing the viral attachment protein with the reprogramming factors for induced pluripotent stem cell generation. Encouraged by the MeV reprogramming vector efficiency, in this study, we develop a single-cycle MeV vector to deliver the gRNA(s) and Cas9 nuclease to human cells for efficient gene editing. We show that the MeV vector achieved on-target gene editing of the reporter (mCherry) and endogenous genes (HBB and FANCD1) in human cells. Additionally, the MeV vector achieved precise knock-in via homology-directed repair using a single-stranded oligonucleotide donor. The MeV vector is a new and flexible platform for gene knock-out and knock-in modifications in human cells, capable of incorporating new technologies as they are developed.

Repair of DNA double-strand breaks by the non-homologous end-joining pathway is initiated by the binding of Ku to DNA ends. Multiple Ku proteins load onto linear DNAs in vitro. However, in cells, Ku loading is limited to ∼1-2 molecules per DNA end. The mechanisms enforcing this limit are currently unclear. Here, we show that the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), but not its protein kinase activity, is required to prevent excessive Ku entry into chromatin. Ku accumulation is further restricted by two mechanisms: a neddylation/FBXL12-dependent process that actively removes loaded Ku molecules throughout the cell cycle and a CtIP/ATM-dependent mechanism that operates in S phase. Finally, we demonstrate that the misregulation of Ku loading leads to impaired transcription in the vicinity of DNA ends. Together, our data shed light on the multiple mechanisms operating to prevent Ku from invading chromatin and interfering with other DNA transactions.

[This corrects the article DOI: 10.3389/fimmu.2024.1405022.].

Given its central role in life, DNA is remarkably easy to damage. Double strand breaks (DSBs) are the most toxic form of DNA damage, and DSBs pose the greatest danger to genomic integrity. In higher vertebrates, the non-homologous end joining pathway (NHEJ) is the predominate pathway that repairs DSBs. NHEJ has three steps: 1) DNA end recognition by the DNA dependent protein kinase [DNA-PK], 2) DNA end-processing by numerous NHEJ accessory factors, and 3) DNA end ligation by the DNA ligase IV complex (LX4). Although this would appear to be a relatively simple mechanism, it has become increasingly apparent that it is not. Recently, much insight has been derived regarding the mechanism of non-homologous end joining through a proliferation of cryo-EM studies, structure-function mutational experiments informed by these new structural data, and novel single-molecule imaging approaches. An emerging consensus in the field is that NHEJ progresses from initial DSB end recognition by DNA-PK to synapsis of the two DNA ends in a long-range synaptic complex where ends are held too far apart (115 Å) for ligation, and then progress to a short-range synaptic complex where ends are positioned close enough for ligation. What was surprising from these structural studies was the observation of two distinct types of DNA-PK dimers that represent NHEJ long-range complexes. In this review, we summarize current knowledge about the function of the distinct NHEJ synaptic complexes and align this new information with emerging cellular single-molecule microscopy studies as well as with previous studies of DNA-PK\'s function in repair.

The KU heterodimer (KU70/80) is rapidly recruited to DNA double-strand breaks (DSBs) to regulate their processing and repair. Previous work has revealed that the amino-terminal von Willebrand-like (vWA-like) domain in KU80 harbours a conserved hydrophobic pocket that interacts with a short peptide motif known as the Ku-binding motif (KBM). The KBM is present in a variety of DNA repair proteins such as APLF, CYREN, and Werner protein (WRN). Here, to investigate the importance of KBM-mediated protein-protein interactions for KU80 function, we employed KU80-deficient Chinese Hamster Ovary (Xrs-6) cells transfected with RFP-tagged wild-type human KU80 or KU80 harbouring a mutant vWA-like domain (KU80L68R). Surprisingly, while mutant RFP-KU80L68R largely or entirely restored NHEJ efficiency and radiation resistance in KU80-deficient Xrs-6 cells, it failed to restore cellular resistance to DNA replication stress induced by camptothecin (CPT) or hydroxyurea (HU). Moreover, KU80-deficient Xrs-6 cells expressing RFP-KU80L68R accumulated pan-nuclear γH2AX in an S/G2-phase-dependent manner following treatment with CPT or HU, suggesting that the binding of KU80 to one or more KBM-containing proteins is required for the processing and/or repair of DNA ends that arise during DNA replication stress. Consistent with this idea, depletion of WRN helicase/exonuclease recapitulated the CPT-induced γH2AX phenotype, and did so epistatically with mutation of the KU80 vWA-like domain. These data identify a role for the KBM-binding by KU80 in the response and resistance of CHO cells to arrested and/or collapsed DNA replication forks, and implicate the KBM-mediated interaction of KU80 with WRN as a critical effector of this role.