Nonhomologous DNA End Joining Research Articles

Robust DNA repair in PAXX-deficient mammalian cells.

To ensure genome stability, mammalian cells employ several DNA repair pathways. Nonhomologous DNA end joining (NHEJ) is the DNA repair process that fixes double‐strand breaks throughout the cell cycle. NHEJ is involved in the development of B and T lymphocytes through its function in V(D)J recombination and class switch recombination (CSR). NHEJ consists of several core and accessory factors, including Ku70, Ku80, XRCC4, DNA ligase 4, DNA‐PKcs, Artemis, and XLF. Paralog of XRCC4 and XLF (PAXX) is the recently described accessory NHEJ factor that structurally resembles XRCC4 and XLF and interacts with Ku70/Ku80. To determine the physiological role of PAXX in mammalian cells, we purchased and characterized a set of custom‐generated and commercially available NHEJ‐deficient human haploid HAP1 cells, PAXX Δ , XRCC4 Δ, and XLF Δ. In our studies, HAP1 PAXX Δ cells demonstrated modest sensitivity to DNA damage, which was comparable to wild‐type controls. By contrast, XRCC4 Δ and XLF Δ HAP1 cells possessed significant DNA repair defects measured as sensitivity to double‐strand break inducing agents and chromosomal breaks. To investigate the role of PAXX in CSR, we generated and characterized Paxx −/− and Aid −/− murine lymphoid CH12F3 cells. CSR to IgA was nearly at wild‐type levels in the Paxx −/− cells and completely ablated in the absence of activation‐induced cytidine deaminase (AID). In addition, Paxx −/− CH12F3 cells were hypersensitive to zeocin when compared to wild‐type controls. We concluded that Paxx‐deficient mammalian cells maintain robust NHEJ and CSR.

Normal development of mice lacking PAXX, the paralogue of XRCC4 and XLF.

DNA repair consists of several cellular pathways which recognize and repair damaged DNA. The classical nonhomologous DNA end‐joining (NHEJ) pathway repairs double‐strand breaks in DNA. It is required for maturation of both B and T lymphocytes by supporting V(D)J recombination as well as B‐cell differentiation during class switch recombination (CSR). Inactivation of NHEJ factors Ku70, Ku80, XRCC4, DNA ligase 4, DNA‐PKcs, and Artemis impairs V(D)J recombination and blocks lymphocyte development. Paralogue of XRCC4 and XLF (PAXX) is an accessory NHEJ factor that has a significant impact on the repair of DNA lesions induced by ionizing radiation in human, murine, and chicken cells. However, the role of PAXX during development is poorly understood. To determine the physiological role of PAXX, we deleted part of the Paxx promoter and the first two exons in mice. Further, we compared Paxx‐knockout mice with wild‐type (WT) and NHEJ‐deficient controls including Ku80‐ and Dna‐pkcs‐null and severe combined immunodeficiency mice. Surprisingly, Paxx‐deficient mice were not distinguishable from the WT littermates; they were the same weight and size, fertility status, had normal spleen, thymus, and bone marrow. Paxx‐deficient mice had the same number of chromosomal and chromatid breaks as WT mice. Moreover, Paxx‐deficient primary B lymphocytes had the same level of CSR as lymphocytes isolated from WT mice. We concluded that PAXX is dispensable for normal mouse development.

Erratum: Increasing the efficiency of homology-directed repair for CRISPR-Cas9-induced precise gene editing in mammalian cells.

The insertion of precise genetic modifications by genome editing tools such as CRISPR-Cas9 is limited by the relatively low efficiency of homology-directed repair (HDR) compared with the higher efficiency of the nonhomologous end-joining (NHEJ) pathway. To enhance HDR, enabling the insertion of precise genetic modifications, we suppressed the NHEJ key molecules KU70, KU80 or DNA ligase IV by gene silencing, the ligase IV inhibitor SCR7 or the coexpression of adenovirus 4 E1B55K and E4orf6 proteins in a 'traffic light' and other reporter systems. Suppression of KU70 and DNA ligase IV promotes the efficiency of HDR 4-5-fold. When co-expressed with the Cas9 system, E1B55K and E4orf6 improved the efficiency of HDR up to eightfold and essentially abolished NHEJ activity in both human and mouse cell lines. Our findings provide useful tools to improve the frequency of precise gene modifications in mammalian cells.

Abstract LB-A11: Targeting DNA-PK and the DNA damage response via small molecule Ku inhibitors

Abstract The DNA dependent protein kinase (DNA-PK) is a validated target for cancer therapeutics that drives the DNA damage response (DDR) and plays a critical role as a primary sensor in the non-homologous end joining (NHEJ) DNA double strand break (DSB) repair pathway. Various anti-cancer therapeutic strategies mediate their cytotoxic effects by inducing DSBs, including ionizing radiation (IR), and clinical outcomes are directly related to the repair of DNA damage. Modulating the pathway responsible for repairing DSBs will have a profound impact on the efficacy of DNA damaging agents in the clinic. To date, development of inhibitors for DNA-PK has focused on targeting the active site with ATP mimetics. We have taken the novel and innovative approach to inhibiting DNA-PK via blocking the Ku 70/80 heterodimer interaction with DNA, a necessary step in DNA-PK activation. Exploiting this unique mechanism of kinase activation, we have identified a series of highly potent and specific DNA-PK inhibitors that impart their inhibitory activity via disruption of the binding of Ku protein to DNA ends. This novel approach affords significant advantages to current approaches in kinase inhibition. Novel derivatives of our initial hit inhibit DNA-PK catalytic activity at nanomolar concentrations and potentiate cellular sensitivity to DSB-inducing agents like etoposide and bleomycin. Data demonstrate that the cellular effects observed are a function of Ku inhibition and that this novel class of DNA-PK inhibitors can be further developed as anti-cancer therapeutics that can be used as an adjuvant to, or concomitant with radiotherapy and other cancer therapies that induce DNA damage. Citation Format: John J. Turchi, Navnath Gavande, Pamela S. VanderVere-Carozza, Tyler Vernon, Katherine S. Pawelczak. Targeting DNA-PK and the DNA damage response via small molecule Ku inhibitors [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr LB-A11.

Inhibitor of nonhomologous end joining can inhibit proliferation of diffuse large B-Cell lymphoma cells and potentiate the effect of ionization radiation

Aim: Diffuse large B-cell lymphoma (DLBCL) is the most common and aggressive type of non-Hodgkin's lymphoma that accounts for ~40% of all lymphomas. DLBCL is considered to be clinically heterogeneous with highest mortality rate. Recent advances in gene expression profiling helped in identifying different subtypes of DLBCL, and since then, many therapeutic options have been explored to treat DLBCL patients. Although it is effective, a significant proportion of the patients suffer due to drug resistance. One of the potential causes for this could be elevated DNA repair in the resistant cancer cells. Thus, the present study is aimed at investigating the potential of SCR7, a DNA repair inhibitor in inducing cytotoxicity on a DLBCL cell line, and to study its ability to potentiate effect when used in combination with ionizing radiation. Materials and Methods: DLBCL cell line, Standford University Diffuse Histiocytic Lymphoma 8 (SUDHL8) was treated with various concentrations of SCR7, a DNA repair inhibitor that targets nonhomologous DNA end joining. While cytotoxicity induced by SCR7 was evaluated through trypan blue assay and flow cytometry analysis, 5,5',6,6 tetrachloro-1,1',3,3'-tetraethyl benzimidazol-carbocyanine iodide and annexin V-FITC/propidium iodide [PI] double-staining assays were used to study the mechanism of cell death. Modulation in the level of DNA repair and apoptotic proteins following treatment with SCR7 was examined by immunoblotting. Effect of SCR7 on sensitizing radiotherapy was further investigated in the SUDHL8 cells. Results: SCR7 induced cytotoxicity in the DLBCL cell line in a concentration- and time-dependent manner. Cell cycle analysis and annexin V/PI double-staining assay confirmed apoptosis in cells without interfering with cell cycle progression. Change in mitochondrial membrane potential in conjunction with alterations in the levels of apoptotic proteins suggested activation of both intrinsic and extrinsic pathways of apoptosis. Importantly, administration of SCR7 potentiated the effect of radiation upon combination therapy in DLBCL. Conclusion: Our results suggest that SCR7 could be developed as an alternative chemotherapeutic approach against DLBCL and is also effective along with radiotherapy.

ELL2 regulates DNA non-homologous end joining (NHEJ) repair in prostate cancer cells

ELL2 is an androgen-responsive gene that is expressed by prostate epithelial cells and is frequently down-regulated in prostate cancer. Deletion of Ell2 in the murine prostate induced murine prostatic intraepithelial neoplasia and ELL2 knockdown enhanced proliferation and migration in C4-2 prostate cancer cells. Here, knockdown of ELL2 sensitized prostate cancer cells to DNA damage and overexpression of ELL2 protected prostate cancer cells from DNA damage. Knockdown of ELL2 impaired non-homologous end joining repair but not homologous recombination repair. Transfected ELL2 co-immunoprecipitated with both Ku70 and Ku80 proteins. ELL2 could bind to and co-accumulate with Ku70/Ku80 proteins at sites of DNA damage. Knockdown of ELL2 dramatically inhibited Ku70 and Ku80 recruitment and retention at DNA double-strand break sites in prostate cancer cells. The impaired recruitment of Ku70 and Ku80 proteins to DNA damage sites upon ELL2 knockdown was rescued by re-expression of an ELL2 transgene insensitive to siELL2. This study suggests that ELL2 is required for efficient NHEJ repair via Ku70/Ku80 in prostate cancer cells.

Homologous recombination-mediated repair of DNA double-strand breaks operates in mammalian mitochondria.

Mitochondrial DNA is frequently exposed to oxidative damage, as compared to nuclear DNA. Previously, we have shown that while microhomology-mediated end joining can account for DNA deletions in mitochondria, classical nonhomologous DNA end joining, the predominant double-strand break (DSB) repair pathway in nucleus, is undetectable. In the present study, we investigated the presence of homologous recombination (HR) in mitochondria to maintain its genomic integrity. Biochemical studies revealed that HR-mediated repair of DSBs is more efficient in the mitochondria of testes as compared to that of brain, kidney and spleen. Interestingly, a significant increase in the efficiency of HR was observed when a DSB was introduced. Analyses of the clones suggest that most of the recombinants were generated through reciprocal exchange, while~30% of recombinants were due to gene conversion in testicular extracts. Colocalization and immunoblotting studies showed the presence of RAD51 and MRN complex proteins in the mitochondria and immunodepletion of MRE11, RAD51 or NIBRIN suppressed the HR-mediated repair. Thus, our results reveal importance of homologous recombination in the maintenance of mitochondrial genome stability.

Structural step forward for NHEJ.

In a recent paper published in Cell Research, a cryo-EM structure reveals the interface between DNA-PKcs and the Ku70/80:DNA complex, together forming the DNA-dependent protein kinase holoenzyme in non-homologous DNA end joining. Insight from this structure suggests how an allosteric rearrangement of DNA-PKcs driven by Ku70/80:DNA binding regulates kinase activity in this largest member of a family of structurally homologous phosphoinositide 3-kinase-related protein kinases that includes mTOR, ATR, and ATM.

Effects of DNA end configuration on XRCC4-DNA ligase IV and its stimulation of Artemis activity

In humans, nonhomologous DNA end-joining (NHEJ) is the major pathway by which DNA double-strand breaks are repaired. Recognition of each broken DNA end by the DNA repair protein Ku is the first step in NHEJ, followed by the iterative binding of nucleases, DNA polymerases, and the XRCC4-DNA ligase IV (X4-LIV) complex in an order influenced by the configuration of the two DNA ends at the break site. The endonuclease Artemis improves joining efficiency by functioning in a complex with DNA-dependent protein kinase, catalytic subunit (DNA-PKcs) that carries out endonucleolytic cleavage of 5' and 3' overhangs. Previously, we observed that X4-LIV alone can stimulate Artemis activity on 3' overhangs, but this DNA-PKcs-independent endonuclease activity of Artemis awaited confirmation. Here, using in vitro nuclease and ligation assays, we find that stimulation of Artemis nuclease activity by X4-LIV and the efficiency of blunt-end ligation are determined by structural configurations at the DNA end. Specifically, X4-LIV stimulated Artemis to cut near the end of 3' overhangs without the involvement of other NHEJ proteins. Of note, this ligase complex is not able to stimulate Artemis activity at hairpins or at 5' overhangs. We also found that X4-LIV and DNA-PKcs interfere with one another with respect to stimulating Artemis activity at 3' overhangs, favoring the view that these NHEJ proteins are sequentially rather than concurrently recruited to DNA ends. These data suggest specific functional and positional relationships among these components that explain genetic and molecular features of NHEJ and V(D)J recombination within cells.

Saccharomyces cerevisiae Red1 protein exhibits nonhomologous DNA end–joining activity and potentiates Hop1-promoted pairing of double-stranded DNA

Elucidation of the function of synaptonemal complex (SC) in Saccharomyces cerevisiae has mainly focused on in vivo analysis of recombination-defective meiotic mutants. Consequently, significant gaps remain in the mechanistic understanding of the activities of various SC proteins and the functional relationships among them. S. cerevisiae Hop1 and Red1 are essential structural components of the SC axial/lateral elements. Previous studies have demonstrated that Hop1 is a structure-selective DNA-binding protein exhibiting high affinity for the Holliday junction and promoting DNA bridging, condensation, and pairing between double-stranded DNA molecules. However, the exact mode of action of Red1 remains unclear, although it is known to interact with Hop1 and to suppress the spore viability defects of hop1 mutant alleles. Here, we report the purification and functional characterization of the full-length Red1 protein. Our results revealed that Red1 forms a stable complex with Hop1 in vitro and provided quantitative insights into their physical interactions. Mechanistically, Red1 preferentially associated with the Holliday junction and 3-way junction rather than with single- or double-stranded DNA with overhangs. Although Hop1 and Red1 exhibited similar binding affinities toward several DNA substrates, the two proteins displayed some significant differences. Notably, Red1, by itself, lacked DNA-pairing ability; however, it potentiated Hop1-promoted intermolecular pairing between double-stranded DNA molecules. Moreover, Red1 exhibited nonhomologous DNA end-joining activity, thus revealing an unexpected role for Red1 in recombination-based DNA repair. Collectively, this study presents the first direct insights into Red1's mode of action and into the mechanism underlying its role in chromosome synapsis and recombination.

Inactivation of Pol \u03b8 and C-NHEJ eliminates off-target integration of exogenous DNA

Off-target or random integration of exogenous DNA hampers precise genomic engineering and presents a safety risk in clinical gene therapy strategies. Genetic definition of random integration has been lacking for decades. Here, we show that the A-family DNA polymerase θ (Pol θ) promotes random integration, while canonical non-homologous DNA end joining plays a secondary role; cells double deficient for polymerase θ and canonical non-homologous DNA end joining are devoid of any integration events, demonstrating that these two mechanisms define random integration. In contrast, homologous recombination is not reduced in these cells and gene targeting is improved to 100% efficiency. Such complete reversal of integration outcome, from predominately random integration to exclusively gene targeting, provides a rational way forward to improve the efficacy and safety of DNA delivery and gene correction approaches.

Abstract 2212: Protein engineering of the decapping enzyme NudT16 enhances its ability to hydrolyze protein-conjugated ADP-ribose for post-translational site identification via mass spectrometry

Abstract During nonhomologous end joining of DNA follow double-stranded breaks, one common signaling response is the adenosine diphosphate ribosylation (ADP-ribosylation) of recruiter scaffold proteins. ADP-ribosylation is a post-translational modification (PTM) that occurs on aspartate, glutamate, lysine, arginine and cysteine on proteins and is mediated chiefly by polyadenosine ribose polymerases (PARP). Using NAD+ as its substrate, PARP transfers an ADP-ribose onto another protein, releasing a nicotinamide ring in the process; this modification presents as a mono-ADP-ribosylation (MARylation) or poly-ADP-ribosylation (PARylation). While site identification of MARylated protein can be achieved through tandem MS/MS and searching for peptides whose spectra have been shifted by 541.0611 Da, a similar approach for PARylated protein is confounded by the variable length and branching of its PTM. To this end, ADP-ribosylated protein is normally incubated with a snake venom phosphodiesterase (SVP) to leave a standardized phosphoribose tag of 212.0086 Da. It has been shown that a recombinantly-expressed Nudix protein NudT16 provides a cost-effective alternative to SVP to process ADP-ribosylated protein. NudT16 in the cell normally decaps small nucleolar RNA. Our studies here sought to utilize the x-ray crystallographic structure of NudT16 in complex with ADP-ribose in guiding rational design mutations to enhance binding of our desired ADP-ribose substrate and to widen the hydrolase’s active site to better accommodate a protein-conjugated ADP-ribose. Mutants have demonstrated improved catalytic efficiency for ADP-ribose hydrolysis. Radiolabeled immunoassays of PARylated protein concur with these kinetic experiments by showing a reduction of radiolabeled PARP. Rational design of a ADP-ribose-hydrolyzing NudT16 with comparable catalytic efficiency to SVP eases lead discovery of biological mechanisms that are mediated through ADP-ribosylation such as DNA repair. Citation Format: Puchong Thirawatananond, Robert L. McPherson, Jasmine K. Malhi, Anthony K. Leung, Sandra B. Gabelli. Protein engineering of the decapping enzyme NudT16 enhances its ability to hydrolyze protein-conjugated ADP-ribose for post-translational site identification via mass spectrometry [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2212. doi:10.1158/1538-7445.AM2017-2212

Abstract 3002: Therapeutic targeting of PGBD5-induced DNA repair dependence in pediatric solid tumors

Abstract Despite intense efforts, the cure rates of childhood solid tumors are not satisfactory. Resistance to intensive chemotherapy is common, and targets for molecular therapies are largely undefined, particularly for tumors caused by aberrant developmental and epigenetic factors. We have now found that the majority of childhood solid tumors, rhabdoid tumors, neuroblastoma, medulloblastoma and Ewing sarcoma, express an active DNA transposase PGBD5. Using functional genetic approaches, we observed that mouse and human cells deficient in non-homologous end joining (NHEJ) DNA repair cannot tolerate the expression of PGBD5. In a chemical screen of DNA repair inhibitors, we identified the ATR/ATM inhibitor AZD6738 as a specific sensitizer of PGBD5-dependent DNA damage and apoptosis. Ectopic expression of PGBD5 was sufficient to induce hypersensitivity to AZD6738, and endogenous PGBD5 was necessary for its anti-tumor effects. We found that PGBD5-expressing tumor cells accumulate unrepaired DNA damage in response to AZD6738 treatment, and undergo apoptosis in both dividing and G1 phase cells in the absence of measureable DNA replication stress. Accordingly, AZD6738 exhibits nanomolar potency against the majority of neuroblastoma, medulloblastoma, Ewing sarcoma and rhabdoid tumor cells tested, while sparing non-transformed human and mouse embryonic fibroblasts in vitro. Finally, AZD6738 exhibits excellent single-agent activity against high-risk neuroblastoma and medulloblastoma xenografts, with induction of apoptosis and tumor regression in vivo. These findings delineate a therapeutically actionable synthetic lethality for the majority of childhood solid tumors. Citation Format: Anton Henssen, Casie Reed, Eileen Jiang, Wong Wai, Elisa de Stanchina, Yasumichi Kuwahara, Hajime Hosoi, Johannes H. Schulte, Neil Ganem, John Petrini, Alex Kentsis. Therapeutic targeting of PGBD5-induced DNA repair dependence in pediatric solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3002. doi:10.1158/1538-7445.AM2017-3002

Abstract LB-119: Targeting DNA-PK via small molecule inhibitors of the Ku-DNA interaction

Abstract DNA-PK, the DNA-dependent protein kinase, is a validated target for cancer therapeutics that drives the DNA damage response and plays a critical role in the nonhomologous end joining (NHEJ) DNA repair pathway. Inhibition of DNA-PK activity can elicit anticancer activity and also sensitize cancer cells to DNA damaging therapy. NHEJ is responsible for the repair of DNA double strand breaks (DSB), particularly those induced by ionizing radiation (IR). The generation of DNA DSBs is the mechanism of clinical efficacy of radiation therapy and numerous DNA damaging chemotherapeutic drugs used to treat various cancers. Modulating the pathway responsible for repairing these breaks has been shown to have a profound impact on the efficacy of IR or chemotherapy in the clinic. We have taken a completely unique and novel approach to inhibiting DNA-PK that is based on extensive knowledge regarding DNA-PK kinase activation and affords considerable advantages to current approaches in DNA-PK inhibition. DNA-PK activation and NHEJ pathway engagement requires binding of the Ku70/80 heterodimer to DNA ends and we are targeting this protein-DNA interaction. We have discovered and developed a series of highly potent and selective DNA-PK inhibitors that act via blocking the binding of Ku70/80 to DNA ends. The lead compound from this series of molecules inhibits DNA-PK catalytic activity at nanomolar concentrations, has single-agent anticancer activity in cancer cell lines, and potentiates cellular sensitivity to IR treatment and DSB-producing chemotherapeutic agents. These data demonstrate the utility of inhibiting DNA-PK via targeting the Ku-DNA interaction. Citation Format: John J. Turchi, Navnath Gavande, Pamela VanderVere-Carozza, Tyler Vernon, Katherine Pawelczak. Targeting DNA-PK via small molecule inhibitors of the Ku-DNA interaction [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr LB-119. doi:10.1158/1538-7445.AM2017-LB-119

Non-homologous DNA end joining and alternative pathways to double-strand break repair.

DNA double-strand breaks (DSBs) are the most dangerous type of DNA damage because they can result in the loss of large chromosomal regions. In all mammalian cells, DSBs that occur throughout the cell cycle are repaired predominantly by the non-homologous DNA end joining (NHEJ) pathway. Defects in NHEJ result in sensitivity to ionizing radiation and the ablation of lymphocytes. The NHEJ pathway utilizes proteins that recognize, resect, polymerize and ligate the DNA ends in a flexible manner. This flexibility permits NHEJ to function on a wide range of DNA-end configurations, with the resulting repaired DNA junctions often containing mutations. In this Review, we discuss the most recent findings regarding the relative involvement of the different NHEJ proteins in the repair of various DNA-end configurations. We also discuss the shunting of DNA-end repair to the auxiliary pathways of alternative end joining (a-EJ) or single-strand annealing (SSA) and the relevance of these different pathways to human disease.

Structural and functional characterization of the PNKP-XRCC4-LigIV DNA repair complex.

Non-homologous end joining (NHEJ) repairs DNA double strand breaks in non-cycling eukaryotic cells. NHEJ relies on polynucleotide kinase/phosphatase (PNKP), which generates 5΄-phosphate/3΄-hydroxyl DNA termini that are critical for ligation by the NHEJ DNA ligase, LigIV. PNKP and LigIV require the NHEJ scaffolding protein, XRCC4. The PNKP FHA domain binds to the CK2-phosphorylated XRCC4 C-terminal tail, while LigIV uses its tandem BRCT repeats to bind the XRCC4 coiled-coil. Yet, the assembled PNKP-XRCC4–LigIV complex remains uncharacterized. Here, we report purification and characterization of a recombinant PNKP–XRCC4–LigIV complex. We show that the stable binding of PNKP in this complex requires XRCC4 phosphorylation and that only one PNKP protomer binds per XRCC4 dimer. Small angle X-ray scattering (SAXS) reveals a flexible multi-state complex that suggests that both the PNKP FHA and catalytic domains contact the XRCC4 coiled-coil and LigIV BRCT repeats. Hydrogen-deuterium exchange indicates protection of a surface on the PNKP phosphatase domain that may contact XRCC4–LigIV. A mutation on this surface (E326K) causes the hereditary neuro-developmental disorder, MCSZ. This mutation impairs PNKP recruitment to damaged DNA in human cells and provides a possible disease mechanism. Together, this work unveils multipoint contacts between PNKP and XRCC4–LigIV that regulate PNKP recruitment and activity within NHEJ.

Non-homologous End Joining Inhibitor SCR-7 to Exacerbate Low-dose Doxorubicin Cytotoxicity in HeLa Cells.

Among the genotoxic drug regimens, doxorubicin (DOX) is known for its high-dose side effects in several carcinomas, including cervical cancer. This study reports on testing the combined use of a DOX genotoxic drug and SCR-7 non-homologous end joining (NHEJ) inhibitor for HeLa cells. An in vitro DNA damaging assay of DOX was performed on plasmid and genomic DNA substrate. In vitro cytotoxicity was investigated using trypan blue dye exclusion, DNA metabolizing, and propidium iodide-based flow cytometric assays. DOX (between 20–100 μM) displayed clear DNA binding and interaction, such as the shearing and smearing of plasmid and genomic DNA. DNA metabolizing assay data indicate that HeLa lysate with DOX and SCR-7 treatment exhibited better in vitro plasmid DNA stability compared with DOX treatment alone. SCR-7 augmented the effects of low-dose DOX by demonstrating enhanced cell death from 15% to 50%. The flow cytometric data also supported that the combination of SCR-7 with DOX lead to a 23% increase in propidium iodide-based HeLa staining, thus indicating enhanced death. In summary, the inhibition of NHEJ DNA repair pathway can potentiate low-dose DOX to produce appreciable cytotoxicity in HeLa cells.

Regulation of human polλ by ATM-mediated phosphorylation during non-homologous end joining

DNA double strand breaks (DSBs) trigger a variety of cellular signaling processes, collectively termed the DNA-damage response (DDR), that are primarily regulated by protein kinase ataxia-telangiectasia mutated (ATM). Among DDR activated processes, the repair of DSBs by non-homologous end joining (NHEJ) is essential. The proper coordination of NHEJ factors is mainly achieved through phosphorylation by an ATM-related kinase, the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), although the molecular basis for this regulation has yet to be fully elucidated. In this study we identify the major NHEJ DNA polymerase, DNA polymerase lambda (Polλ), as a target for both ATM and DNA-PKcs in human cells. We show that Polλ is efficiently phosphorylated by DNA-PKcs in vitro and predominantly by ATM after DSB induction with ionizing radiation (IR) in vivo. We identify threonine 204 (T204) as a main target for ATM/DNA-PKcs phosphorylation on human Polλ, and establish that its phosphorylation may facilitate the repair of a subset of IR-induced DSBs and the efficient Polλ-mediated gap-filling during NHEJ. Molecular evidence suggests that Polλ phosphorylation might favor Polλ interaction with the DNA-PK complex at DSBs. Altogether, our work provides the first demonstration of how Polλ is regulated by phosphorylation to connect with the NHEJ core machinery during DSB repair in human cells.

Ferulic acid (FA) abrogates γ-radiation induced oxidative stress and DNA damage by up-regulating nuclear translocation of Nrf2 and activation of NHEJ pathway

The present study was aimed to evaluate the radioprotective effect of ferulic acid (FA), a naturally occurring plant flavonoid in terms of DNA damage and damage related alterations of repair pathways by gamma radiation. FA was administered at a dose of 50 mg/kg body weight for five consecutive days prior to exposing the swiss albino mice to a single dose of 10 Gy gamma radiation. Ionising radiation induces oxidative damage manifested by decreased expression of Cu, Zn-SOD (SOD stands for super oxide dismutase), Mn-SOD and catalase. Gamma radiation promulgated reactive oxygen species (ROS) mediated DNA damage and modified repair pathways. ROS enhanced nuclear translocation of p53, activated ATM (ataxia telangiectasia-mutated protein), increased expression of GADD45a (growth arrest and DNA-damage-inducible protein) gene and inactivated Non homologous end joining (NHEJ) repair pathway. The comet formation in irradiated mice peripheral blood mononuclear cells (PBMC) reiterated the DNA damage in IR exposed groups. FA pretreatment significantly prevented the comet formation and regulated the nuclear translocation of p53, inhibited ATM activation and expression of GADD45a gene. FA promoted the nuclear translocation of nuclear factor (erythroid-derived 2)-like 2 (Nrf2) and activated NHEJ repair pathway to overcome ROS mediated oxidative stress and DNA damage. Therefore, the current study stated that FA can challenge the oxidative stress by (i) inducing nuclear translocation of Nrf2, (ii) scavenging ROS, and (iii) activating NHEJ DNA repair process.

Cloning, localization and focus formation at DNA damage sites of canine Ku70

Understanding the molecular mechanisms of DNA double-strand break (DSB) repair machinery, specifically non-homologous DNA-end joining (NHEJ), is crucial for developing next-generation radiotherapies and common chemotherapeutics for human and animal cancers. The localization, protein-protein interactions and post-translational modifications of core NHEJ factors, might play vital roles for regulation of NHEJ activity. The human Ku heterodimer (Ku70/Ku80) is a core NHEJ factor in the NHEJ pathway and is involved in sensing of DSBs. Companion animals, such as canines, have been proposed to be an excellent model for cancer research, including development of chemotherapeutics. However, the post-translational modifications, localization and complex formation of canine Ku70 have not been clarified. Here, we show that canine Ku70 localizes in the nuclei of interphase cells and that it is recruited quickly at laser-microirradiated DSB sites. Structurally, two DNA-PK phosphorylation sites (S6 and S51), an ubiquitination site (K114), two canonical sumoylation consensus motifs, a CDK phosphorylation motif, and a nuclear localization signal (NLS) in the human Ku70 are evolutionarily conserved in canine and mouse species, while the acetylation sites in human Ku70 are partially conserved. Intriguingly, the primary candidate nucleophile (K31) required for 5’dRP/AP lyase activity of human and mouse Ku70 is not conserved in canines, suggesting that canine Ku does not possess this activity. Our findings provide insights into the molecular mechanisms of Ku-dependent NHEJ in a canine model and form a platform for the development of next-generation common chemotherapeutics for human and animal cancers.