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Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. To explore genetic resistance mechanisms, we performed genome-wide CRISPR-Cas9 screens in cells treated with the DNA topoisomerase I inhibitor topotecan. Thus, we here establish that inactivating terminal components of the non-homologous end-joining (NHEJ) machinery or of the BRCA1-A complex specifically confer topotecan resistance to ATM-deficient cells. We show that hypersensitivity of ATM -mutant cells to topotecan or the poly-(ADP-ribose) polymerase (PARP) inhibitor olaparib reflects delayed engagement of homologous recombination at DNA-replication-fork associated single-ended double-strand breaks (DSBs), allowing some to be subject to toxic NHEJ. Preventing DSB ligation by NHEJ, or enhancing homologous recombination by BRCA1-A complex disruption, suppresses this toxicity, highlighting a crucial role for ATM in preventing toxic LIG4-mediated chromosome fusions. Notably, suppressor mutations in ATM -mutant backgrounds are different to those in BRCA1 -mutant scenarios, suggesting new opportunities for patient stratification and additional therapeutic vulnerabilities for clinical exploitation. Mutations in the ATM tumor suppressor gene confer hypersensitivity to DNA-damaging chemotherapeutic agents. Here, the authors provide evidence that these hypersensitivities reflect a crucial role for ATM at damaged replication forks being to prevent toxic DNA end-joining leading to chromosome fusions and cell death.

BackgroundAn increasing number of studies have recently reported that microRNAs packaged in exosomes contribute to multiple biological processes such as cancer progression; however, little is known about their role in the development of radiation-induced bystander effects.MethodsThe exosomes were isolated from the culture medium of BEP2D cells with or without γ-ray irradiation by ultracentrifugation. To monitor DNA damage and repair efficiency, the DNA double-strand break biomarker 53BP1 foci, comet, micronuclei, expression of DNA repair genes and NHEJ repair activity were detected. The miR-1246 targeting sequence of the DNA ligase 4 (LIG4) mRNA 3′UTR was assessed by luciferase reporter vectors.ResultsmiR-1246 was increased in exosomes secreted from 2 Gy-irradiated BEP2D cells and inhibited the proliferation of nonirradiated cells. The miR-1246 mimic, exosomes from irradiated cells, and radiation-conditioned cell culture medium increased the yields of 53BP1 foci, comet tail and micronuclei in nonirradiated cells, and decreased NHEJ efficiency. miR-1246 downregulated LIG4 expression by directly targeting its 3′UTR.ConclusionsOur findings demonstrate that miR-1246 packaged in exosomes could act as a transfer messenger and contribute to DNA damage by directly repressing the LIG4 gene. Exosomal miR-1246 may be a critical predictor of and player in radiation-induced bystander DNA damage.

Radiation resistance is a major cause of lung cancer treatment failure. Armadillo (ARM) superfamily proteins participate in various fundamental cellular processes; however, whether ARM proteins regulate radiation resistance is not fully understood. Here, we used an unbiased CRISPR/Cas9 library screen and identified plakophilin 2 (PKP2), a member of the ARM superfamily of proteins, as a critical driver of radiation resistance in lung cancer. The PKP2 level was significantly higher after radiotherapy than before radiotherapy, and high PKP2 expression after radiotherapy predicted poor overall survival (OS) and postprogression survival (PPS). Mechanistically, mass spectrometry analysis identified that PKP2 was methylated at the arginine site and interacted with protein arginine methyltransferase 1 (PRMT1). Methylation of PKP2 by PRMT1 stabilized β-catenin by recruiting USP7, further inducing LIG4, a key DNA ligase in nonhomologous end-joining (NHEJ) repair. Concomitantly, PKP2-induced radioresistance depended on facilitating LIG4-mediated NHEJ repair in lung cancer. More strikingly, after exposure to irradiation, treatment with the PRMT1 inhibitor C-7280948 abolished PKP2-induced radioresistance, and C-7280948 is a potential radiosensitizer in lung cancer. In summary, our results demonstrate that targeting the PRMT1/PKP2/β-catenin/LIG4 pathway is an effective approach to overcome radiation resistance in lung cancer.

Tumour repressor p53 isoform Δ133p53 is a target gene of p53 and an antagonist of p53-mediated apoptotic activity. We recently demonstrated that Δ133p53 promotes DNA double-strand break (DSB) repair by upregulating transcription of the repair genes RAD51 , LIG4 and RAD52 in a p53-independent manner. However, Δ133p53 lacks the transactivation domain of full-length p53, and the mechanism by which it exerts transcriptional activity independently of full-length p53 remains unclear. In this report, we describe the accumulation of high levels of both Δ133p53 and p73 (a p53 family member) at 24 h post γ-irradiation (hpi). Δ133p53 can form a complex with p73 upon γ-irradiation. The co-expression of Δ133p53 and p73, but not either protein alone, can significantly promote DNA DSB repair mechanisms, including homologous recombination (HR), non-homologous end joining (NHEJ) and single-strand annealing (SSA). p73 and Δ133p53 act synergistically to promote the expression of RAD51 , LIG4 and RAD52 by joining together to bind to region containing a Δ133p53-responsive element (RE) and a p73-RE in the promoters of all three repair genes. In addition to its accumulation at 24 hpi, p73 protein expression also peaks at 4 hpi. The depletion of p73 not only reduces early-stage apoptotic frequency (4–6 hpi), but also significantly increases later-stage DNA DSB accumulation (48 hpi), leading to cell cycle arrest in the G2 phase and, ultimately, cell senescence. In summary, the apoptotic regulator p73 also coordinates with Δ133p53 to promote DNA DSB repair, and the loss of function of p73 in DNA DSB repair may underlie spontaneous and carcinogen-induced tumorigenesis in p73 knockout mice.

The double-strand break (DSB) repair pathway called microhomology-mediated end-joining (MMEJ) is thought to be dependent on DNA polymerase theta (Polθ) and occur independently of nonhomologous end-joining (NHEJ) factors. An unresolved question is whether MMEJ is facilitated by a single Polθ-mediated end-joining pathway or consists of additional undiscovered pathways. We find that human X-family Polλ, which functions in NHEJ, additionally exhibits robust MMEJ activity like Polθ. Polλ promotes MMEJ in mammalian cells independently of essential NHEJ factors LIG4/XRCC4 and Polθ, which reveals a distinct Polλ-dependent MMEJ mechanism. X-ray crystallography employing in situ photo-induced DSB formation captured Polλ in the act of stabilizing a microhomology-mediated DNA synapse with incoming nucleotide at 2.0 Å resolution and reveals how Polλ performs replication across a DNA synapse joined by minimal base-pairing. Last, we find that Polλ is semisynthetic lethal with BRCA1 and BRCA2. Together, these studies indicate Polλ MMEJ as a distinct DSB repair mechanism. Chandramouly et al. discover that human Polλ exhibits robust microhomology-mediated end-joining (MMEJ) activity like PolΦ. Polλ promotes MMEJ in mammalian cells independently of essential nonhomologous end-joining factors LIG4/XRCC4 and Polq, which indicates a distinct Polλ-dependent MMEJ mechanism.

A percentage of colorectal cancer (CRC) patients display low sensitivity to radiotherapy, which affects its therapeutic effect. Cancer cells DNA double-strand breaks (DSBs) repair capacity is crucial for radiosensitivity, but the roles of long noncoding RNAs (lncRNAs) in this process are largely uncharacterized. This study aims to explore whether lnc-RI regulates CRC cell growth and radiosensitivity by regulating the nonhomologous end-joining (NHEJ) repair pathway. CRC cells in which lnc-RI has been silenced showed lower cell growth and higher apoptosis rates due to increased DSBs and cell cycle arrest. We found that miR-4727-5p targets both lnc-RI and LIG4 mRNA and inhibit their expression. CRC cells showed increased radiosensitivity when lnc-RI was silenced. These results reveal novel roles for lnc-RI in both DNA damage repair and radiosensitivity regulation in CRC cells. Our study revealed that lnc-R I regulates LIG4 expression through lnc-RI /miR-4727-5p/ LIG4 axis and regulates NHEJ repair efficiency to participate in DNA damage repair. The level of lnc-RI was negatively correlated with the radiosensitivity of CRC cells, indicates that lnc-RI may be a potential target for CRC therapy. We also present the first report of the function of miR-4727-5p.

The RNA-sensing pathway contributes to type I interferon (IFN) production induced by DNA damaging agents. However, the potential involvement of RNA sensors in DNA repair is unknown. Here, we found that retinoic acid-inducible gene I (RIG-I), a key cytosolic RNA sensor that recognizes RNA virus and initiates the MAVS-IRF3-type I IFN signaling cascade, is recruited to double-stranded breaks (DSBs) and suppresses non-homologous end joining (NHEJ). Mechanistically, RIG-I interacts with XRCC4, and the RIG-I/XRCC4 interaction impedes the formation of XRCC4/LIG4/XLF complex at DSBs. High expression of RIG-I compromises DNA repair and sensitizes cancer cells to irradiation treatment. In contrast, depletion of RIG-I renders cells resistant to irradiation in vitro and in vivo. In addition, this mechanism suggests a protective role of RIG-I in hindering retrovirus integration into the host genome by suppressing the NHEJ pathway. Reciprocally, XRCC4, while suppressed for its DNA repair function, has a critical role in RIG-I immune signaling through RIG-I interaction. XRCC4 promotes RIG-I signaling by enhancing oligomerization and ubiquitination of RIG-I, thereby suppressing RNA virus replication in host cells. In vivo, silencing XRCC4 in mouse lung promotes influenza virus replication in mice and these mice display faster body weight loss, poorer survival, and a greater degree of lung injury caused by influenza virus infection. This reciprocal regulation of RIG-I and XRCC4 reveals a new function of RIG-I in suppressing DNA repair and virus integration into the host genome, and meanwhile endues XRCC4 with a crucial role in potentiating innate immune response, thereby helping host to prevail in the battle against virus. The RNA-sensing pathway has been associated with type I interferon (IFN) production induced by DNA damaging agents. Here the authors reveal that RIG-I, a cytosolic RNA sensor that recognizes RNA virus and initiates IFN signaling, is recruited to double-stranded breaks and suppresses non-homologous end joining.

Global public health facing serious challenges due to the incidence of cancer and the growth of treatment resistance. Mer-tyrosine kinase plays a crucial role in cell biology and correlated with many cancers such as Epithelial ovarian cancer, liver cancer, breast cancer, Metastatic melanoma, and Acute myeloid leukemia (AML). Hence the identification of novel drug for MERTK protein is extreme important. In this research, we used computational techniques, molecular operating environment (MOE 2015) for virtual screening with drug like natural compounds library. We used known compound UNC2025 as positive control and one million compounds was retrieved from different databases (OTAVA, ZINC, ChEMBL) and docked with MERTK protein. Out of million compounds the 4 top hit inhibitors chosen from docking were further screened for ADMET profiling confirming their compliance with drug designing and toxicological principle and subjected to molecular dynamic (MD) simulation and MM-PBSA analysis. The results of these analyses showed that only four compounds that make strong interactions with MERTK protein via highest binding affinity hydrogen bond and hydrophobic contacts (lig1, lig2, lig3, lig4). The computed binding affinity ranges from –22.977 to –18.707 kcal/mol. The increased helix and reduced β–sheet contents in MERTK on the binding of top hit candidates depicted the higher structural stability of MERTK, rather than MERTK alone and MERTK–UNC2025. The study finds critical residues which serve a vital part in binding with the inhibitor and the active site of the MERTK protein, i.e. , Phe598, Gly599, Lys619, Arg629, Glu633, Glu637, Arg722, Asp723, Arg727, Asp741, Gly743, Leu744, Lys746, Arg758, Ala760, and Lys761 through decomposed binding free energy analysis. This study focuses on the pursuit of several MERTK protein targets, which could have consequences for the development of novel therapeutics for various cancers.

DNA double-strand breaks (DSBs) are among the most deleterious types of DNA damage as they can lead to mutations and chromosomal rearrangements, which underlie cancer development. Classical non-homologous end-joining (cNHEJ) is the dominant pathway for DSB repair in human cells, involving the DNA-binding proteins XRCC6 (Ku70) and XRCC5 (Ku80). Other DNA-binding proteins such as Zinc Finger (ZnF) domain-containing proteins have also been implicated in DNA repair, but their role in cNHEJ remained elusive. Here we show that ZNF384, a member of the C2H2 family of ZnF proteins, binds DNA ends in vitro and is recruited to DSBs in vivo. ZNF384 recruitment requires the poly(ADP-ribosyl) polymerase 1 (PARP1)-dependent expansion of damaged chromatin, followed by binding of its C2H2 motifs to the exposed DNA. Moreover, ZNF384 interacts with Ku70/Ku80 via its N-terminus, thereby promoting Ku70/Ku80 assembly and the accrual of downstream cNHEJ factors, including APLF and XRCC4/LIG4, for efficient repair at DSBs. Altogether, our data suggest that ZNF384 acts as a ‘Ku-adaptor’ that binds damaged DNA and Ku70/Ku80 to facilitate the build-up of a cNHEJ repairosome, highlighting a role for ZNF384 in DSB repair and genome maintenance. Classical non-homologous end-joining (cNHEJ) is the dominant pathway used by human cells to repair DNA double-strand breaks (DSBs) and maintain genome stability. Here the authors show that PARP1-driven chromatin expansion allows the recruitment of ZNF384, which in turn recruits Ku70/Ku80 to facilitate cNHEJ.

Classical nonhomologous end joining (C-NHEJ) repairs DNA double-strand breaks (DSBs) throughout interphase but predominates in G1 phase when homologous recombination is unavailable. Complexes containing the Ku70/80 (“Ku”) and XRCC4/ligase IV (Lig4) core C-NHEJ factors are required, respectively, for sensing and joining DSBs. While XRCC4/Lig4 are absolutely required for joining RAG1/2 endonuclease (“RAG”)-initiated DSBs during V(D)J recombination in G1-phase progenitor lymphocytes, cycling cells deficient for XRCC4/Lig4 also can join chromosomal DSBs by alternative end-joining (A-EJ) pathways. Restriction of V(D)J recombination by XRCC4/Lig4-mediated joining has been attributed to RAG shepherding V(D)J DSBs exclusively into the C-NHEJ pathway. Here, we report that A-EJ of DSB ends generated by RAG1/2, Cas9:gRNA, and Zinc finger endonucleases in Lig4-deficient G1-arrested progenitor B cell lines is suppressed by Ku. Thus, while diverse DSBs remain largely as free broken ends in Lig4-deficient G1-arrested progenitor B cells, deletion of Ku70 increases DSB rejoining and translocation levels to those observed in Ku70-deficient counterparts. Correspondingly, while RAG-initiated V(D)J DSB joining is abrogated in Lig4-deficient G1-arrested progenitor B cell lines, joining of RAG-generated DSBs in Ku70-deficient and Ku70/Lig4 double-deficient lines occurs through a translocation-like A-EJ mechanism. Thus, in G1-arrested, Lig4-deficient progenitor B cells are functionally end-joining suppressed due to Ku-dependent blockage of A-EJ, potentially in association with G1-phase down-regulation of Lig1. Finally, we suggest that differential impacts of Ku deficiency versus Lig4 deficiency on V(D)J recombination, neuronal apoptosis, and embryonic development results from Ku-mediated inhibition of A-EJ in the G1 cell cycle phase in Lig4-deficient developing lymphocyte and neuronal cells.