Ku protein is a complex of two subunits, Ku70 and Ku80. Ku is suspected to participate in both DNA double-strand break repair and transcription. Since both of these processes take place in the cell nucleus, we have been investigating the subcellular localization and nuclear transport of Ku proteins. In the present study, we analyzed the subcellular localization and nuclear localization signal (NLS) of Ku70. Fusion proteins of Ku70 and green fluorescent protein (GFP) transiently expressed in cells were clearly localized in the nuclei of interphase cells. Ku70 staining was distributed throughout both the nucleus and the cytoplasm in late telophase to early G1 phase cells. The NLS of Ku70 was located at the region composed of 18 amino acid residues (positions 539 to 556). This region overlapped with the Ku80-independent DNA-binding domain reported previously. The Ku70 NLS consisted of two basic subregions and a nonbasic intervening region. All the subregions were necessary for complete NLS activity. The amino acids in the nonbasic intervening region of Ku70 might be important for full NLS activity not only to provide sufficient length between the two separated clusters of basic amino acids but also to have an adequate amino acid sequence. All of the basic amino acid residues in the basic subregions were conserved among mammalian and avian homologues, confirming their importance in the nuclear translocation of Ku70. The structure of the Ku70 NLS resembled the consensus of a bipartite-type NLS. The Ku70 NLS was mediated to target to the nuclear rim by two components of the nuclear pore-targeting complex, PTAC58 and PTAC97.

Prostate cancer cell lines were examined for proteins that partnered with the transcription factor C/EBPalpha by use of a pull-down assay with S-tagged C/EBPalpha combined with matrix-assisted laser desorption ionization time-of-flight mass spectroscopy analysis. Ku70, Ku80, and poly(ADP-ribose) polymerase-1 (PARP-1) were identified as proteins that associated with C/EBPalpha. The physical interaction of C/EBPalpha with these partner proteins was further demonstrated by glutathione S-transferase (GST) pull-downs using purified protein expressed in Escherichia coli. The strongest binding was between C/EBPalpha and PARP-1. Immunoprecipitation of C/EBPalpha expressed in prostate cancer cells co-precipitated Ku70, Ku80, and PARP-1. Deletion analysis of C/EBPalpha indicated that the C terminus of C/EBPalpha was essential for the interaction of C/EBPalpha with Ku70, Ku80, and PARP-1. Functional analysis of the interaction between C/EBPalpha and the Ku proteins as well as PARP-1 showed that cells exhibiting these interactions had increased radiation sensitivity and decreased ability to repair double strand DNA breaks. Deficient DNA repair was dependent on the prostate cancer cell line tested, suggesting a complex process. We conclude that the association of C/EBPalpha with Ku proteins and PARP-1 raises the likelihood that C/EBPalpha-expressing prostate cancer cells may be more sensitive to DNA-damaging agents and may be important in the design of new prostate cancer therapies.

High linear energy transfer (LET) ionising radiation (IR) such as radon-derived alpha particles and high mass, high energy (HZE) particles of cosmic radiation are the predominant forms of IR to which humanity is exposed throughout life. High-LET forms of IR are established carcinogens relevant to human cancer, and their potent mutagenicity is believed, in part, to be due to a greater incidence of clustered DNA double strand breaks (DSBs) and associated lesions, as ionization events occur within a more confined genomic space. The repair of such DNA damage is now well-documented to occur with slower kinetics relative to that induced by low-LET IR, and to be more reliant upon homology-directed repair pathways. Underlying these phenomena is the relative inability of non-homologous end-joining (NHEJ) to adequately resolve high-LET IR-induced DSBs. Current findings suggest that the functionality of the DNA-dependent protein kinase (DNA-PK), comprised of the Ku70-Ku80 heterodimer and the DNA-PK catalytic subunit (DNA-PKcs), is particularly perturbed by high-LET IR-induced clustered DSBs, rendering DNA-PK dependent NHEJ less relevant to resolving these lesions. By contrast, the NHEJ-associated DNA processing endonuclease Artemis shows a greater relevance to high-LET IR-induced DSB repair. Here, we will review the cellular response to high-LET irradiation, the implications of the chronic, low-dose modality of this exposure and molecular pathways that respond to high-LET irradiation induced DSBs, with particular emphasis on NHEJ factors.

BACKGROUND

DNA double-strand breaks (DSB) are the major lethal lesions induced by ionizing radiation. The capability for DNA DSB repair is crucial for inherent radiosensitivity of tumor and normal cells. DNA-PKcs, Ku 70, Ku 85, Xrcc4, and Nbs1 play a critical role in DNA DSB repair.

METHODS

We immunohistochemically investigated the expression of DNA-PKcs, Ku 70, Ku85, Xrcc4, and Nbs1 in 134 specimens from various normal and tumor tissues with different radiosensitivity.

CONCLUSIONS

Immunopositivity to Ku70, Ku85, DNA-PKcs, Xrcc4, and Nbs1 was found in all tumor tissues examined. The staining for Ku70, Ku85, and DNA-PKcs was nuclear; but, for Xrcc4 and Nbs1, it was nuclear and cytoplasmic. There were no apparent differences in the expression of these five proteins among cancerous tissues and the corresponding normal tissues. No apparent differences in nuclear staining intensity were detected in the expression of these five proteins among tumor tissues with different radiosensitivity, although non-Hodgkins' lymphoma (B or T cell) tended to show a lower expression than the others. The stromal cells generally expressed these five proteins at much lower frequency than either tumor or epithelial cells in both tumor and normal tissues.

RNase H1-dependent antisense oligonucleotides (ASOs) are chemically modified to enhance pharmacological properties. Major modifications include phosphorothioate (PS) backbone and different 2'-modifications in 2-5 nucleotides at each end (wing) of an ASO. Chemical modifications can affect protein binding and understanding ASO-protein interactions is important for better drug design. Recently we identified many intracellular ASO-binding proteins and found that protein binding could affect ASO potency. Here, we analyzed the structure-activity-relationships of ASO-protein interactions and found 2'-modifications significantly affected protein binding, including La, P54nrb and NPM. PS-ASOs containing more hydrophobic 2'-modifications exhibit higher affinity for proteins in general, although certain proteins, e.g. Ku70/Ku80 and TCP1, are less affected by 2'-modifications. We found that Hsp90 protein binds PS-ASOs containing locked-nucleic-acid (LNA) or constrained-ethyl-bicyclic-nucleic-acid ((S)-cEt) modifications much more avidly than 2'-O-methoxyethyl (MOE). ASOs bind the mid-domain of Hsp90 protein. Hsp90 interacts with more hydrophobic 2' modifications, e.g. (S)-cEt or LNA, in the 5'-wing of the ASO. Reduction of Hsp90 protein decreased activity of PS-ASOs with 5'-LNA or 5'-cEt wings, but not with 5'-MOE wing. Together, our results indicate Hsp90 protein enhances the activity of PS/LNA or PS/(S)-cEt ASOs, and imply that altering protein binding of ASOs using different chemical modifications can improve therapeutic performance of PS-ASOs.

Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the "model" fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of M. robertsii.

OBJECTIVE

Ku70 and Ku86, multifunctional DNA repair proteins, bind to broken DNA ends, including double-strand breaks, and trigger a DNA repair pathway. To investigate the involvement of these proteins in DNA fragmentation after ischemia/reperfusion, Ku protein expression was examined before and after transient focal cerebral ischemia (FCI) in mice.

METHODS

Adult male CD-1 mice were subjected to 60 minutes of FCI by intraluminal suture blockade of the middle cerebral artery. Ku protein expression was studied by immunohistochemistry and Western blot analysis. DNA fragmentation was evaluated by gel electrophoresis and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL). The spatial relationship between Ku expression and DNA fragmentation was examined by double labeling with Ku and TUNEL after reperfusion.

RESULTS

Immunohistochemistry showed constitutive expression of Ku proteins in control brains. The number of Ku-expressing cells was decreased in the entire middle cerebral artery territory as early as 4 hours after reperfusion and remained reduced until 24 hours. Western blot analyses confirmed the significant reduction of these proteins (59.4% and 57.7% reduction in optical density at 4 hours of reperfusion from the normal level of Ku70 and Ku86 bands, respectively; P<0.001). DNA gel electrophoresis demonstrated DNA laddering 24 hours after reperfusion, but not at 4 hours. Double staining with Ku and TUNEL showed a concomitant loss of Ku immunoreactivity and TUNEL-positive staining.

CONCLUSIONS

These results suggest that the early reduction of Ku proteins and the loss of defense against DNA damage may underlie the mechanism of DNA fragmentation after FCI.

Oxidative DNA damage is produced by reactive oxygen species (ROS) which are generated by exogenous and endogenous sources and continuously challenge the cell. One of the most severe DNA lesions is the double-strand break (DSB), which is mainly repaired by nonhomologous end joining (NHEJ) pathway in mammals. NHEJ directly joins the broken ends, without using the homologous template. Ku70/86 heterodimer, also known as Ku, is the first component of NHEJ as it directly binds DNA and recruits other NHEJ factors to promote the repair of the broken ends. Neurons are particularly metabolically active, displaying high rates of transcription and translation, which are associated with high metabolic and mitochondrial activity as well as oxygen consumption. In such a way, excessive oxygen radicals can be generated and constantly attack DNA, thereby producing several lesions. This condition, together with defective DNA repair systems, can lead to a high accumulation of DNA damage resulting in neurodegenerative processes and defects in neurodevelopment. In light of recent findings, in this paper, we will discuss the possible implication of Ku in neurodevelopment and in mediating the DNA repair dysfunction observed in certain neurodegenerations.

Steroidogenic factor 1 (SF-1 or NR5A1) is a nuclear receptor that controls adrenogenital cell growth and differentiation. Adrenogenital primordial cells from SF-1 knockout mice die of apoptosis, but the mechanism by which SF-1 regulates cell survival is not entirely clear. Besides functioning in the nucleus, SF-1 also resides in the centrosome and controls centrosome homeostasis. Here, we show that SF-1 restricts centrosome overduplication by inhibiting aberrant activation of DNA-dependent protein kinase (DNA-PK) in the centrosome. SF-1 was found to be associated with Ku70/Ku80 only in the centrosome, sequestering them from the catalytic subunit of DNA-PK (DNA-PKcs). In the absence of SF-1, DNA-PKcs was recruited to the centrosome and activated, causing aberrant activation of centrosomal Akt and cyclin-dependent kinase 2 (CDK2)/cyclin A and leading to centrosome overduplication. Centrosome overduplication caused by SF-1 depletion was averted by the elimination of DNA-PKcs, Ku70/80, or cyclin A or by the inhibition of CDK2 or Akt. In the nucleus, SF-1 did not interact with Ku70/80, and SF-1 depletion did not activate a nuclear DNA damage response. Centriole biogenesis was also unaffected. Thus, centrosomal DNA-PK signaling triggers centrosome overduplication, and this centrosomal event, but not the nuclear DNA damage response, is controlled by SF-1.

Dacarbazine (DTIC) is one of the most popular alkylating agents used for the treatment of malignant melanoma. DTIC induces apoptosis of melanoma cells via double-strand breaks (DSBs). Melanoma cells, however, tend to increase their expression of DNA repair molecules in order to be resistant to DTIC. Here, we show that DTIC increases expression of Rad51, but not Ku70, in a cultured B16-F10 mouse melanoma cell line in dose- and time-dependent manners. On introducing Rad51 short interfering RNA (siRNA) with the hemagglutinating virus of Japan envelope (HVJ-E) to B16-F10 cells, DSBs induced by DTIC treatment were not efficiently repaired and resulted in enhanced apoptotic cell death. Colony formation of B16-F10 cells that received Rad51 siRNA was significantly decreased by DTIC treatment as compared with cells that received scramble siRNA. In melanoma-bearing mice, the combination of three intratumoral injections of HVJ-E containing Rad51 siRNA and five intraperitoneal injections of DTIC at a clinical dose synergistically suppressed the tumors. Moreover, HVJ-E demonstrated anti-tumor immunity by inducing cytotoxic T lymphocytes to B16-F10 cells on administration of DTIC. These results suggest that the combination of chemotherapy with HVJ-E containing therapeutic molecules will provide a promising therapeutic strategy for patients bearing malignant tumors resistant to chemotherapeutic agents.

HIV-1 integrase (IN) is a key viral enzyme during HIV-1 replication that catalyzes the insertion of viral DNA into the host genome. Recent studies have provided important insights into the multiple posttranslational modifications (PTMs) of IN (e.g., ubiquitination, SUMOylation, acetylation and phosphorylation), which regulate its multifaceted functions. A number of host cellular proteins, including Lens Epithelium-derived Growth factor (LEDGF/p75), p300 and Ku70 have been shown to interact with IN and be involved in the PTM process of IN, either facilitating or counteracting the IN PTMs. Although previous studies have revealed much about the important roles of IN PTMs, how IN functions are fine-tuned by these PTMs under the physiological setting still needs to be determined. Here, we review the advances in the understanding of the mechanisms and roles of multiple IN PTMs.

Spermatogenesis is a complex process that generates haploid germ cells or spores and implements meiosis, a succession of two special cell divisions that are required for homologous chromosome segregation. During prophase to the first meiotic division, homologous recombination (HR) repairs Spo11-dependent DNA double-strand breaks (DSBs) in the presence of telomere movements to allow for chromosome pairing and segregation at the meiosis I division. In contrast to HR, non-homologous end joining (NHEJ), the major DSB repair mechanism during the G1 cell cycle phase, is downregulated during early meiotic prophase. At somatic mammalian telomeres, the NHEJ factor Ku70/80 inhibits HR, as does the Rap1 component of the shelterin complex. Here, we investigated the role of Ku70 and Rap1 in meiotic telomere redistribution and genome protection in spermatogenesis by studying single and double knockout mice. Ku70(-/-) mice display reduced testis size and compromised spermatogenesis, whereas meiotic telomere dynamics and chromosomal bouquet formation occurred normally in Ku70(-/-) and Ku70(-/-)Rap1(Δ/Δ) knockout spermatocytes. Elevated mid-preleptotene frequencies were associated with significantly increased DNA damage in Ku-deficient B spermatogonia, and in differentiated Sertoli cells. Significantly elevated levels of γH2AX foci in Ku70(-/-) diplotene spermatocytes suggest compromised progression of DNA repair at a subset of DSBs. This might explain the elevated meiotic metaphase apoptosis that is present in Ku70-deficient stage XII testis tubules, indicating spindle assembly checkpoint activation. In summary, our data indicate that Ku70 is important for repairing DSBs in somatic cells and in late spermatocytes of the testis, thereby assuring the fidelity of spermatogenesis.

The melanoma differentiation-associated gene-7 (mda-7) was identified by virtue of its enhanced expression in human melanoma cells induced into terminal differentiation. Enforced expression of mda-7 in human cancer cell lines of diverse origins results in the suppression of growth and induction of apoptosis. We have shown that adenoviral-mediated mda-7 (Ad-mda7) radiosensitizes non-small-cell lung cancer (NSCLC) cells by enhancing the apoptotic pathway. To identify the mechanism of this radiosensitization, we examined the level of proteins involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair. Western blot analysis indicated that the expression of NHEJ pathway components Ku70, XRCC4, and DNA ligase IV was downregulated in NSCLC cells--A549 with Ad-mda7 treatment. No such change was observed in normal human CCD16 fibroblasts previously shown not to be radiosensitized by Ad-mda7. The biological significance of these changes of expression of proteins critical for repair of radiation-induced DSBs was confirmed via the analysis of DSB rejoining kinetics using pulsed field gel electrophoresis and assessment of host cell reactivation capacity following Ad-mda7 treatment. Based on these results, we hypothesize that Ad-mda7 sensitizes NSCLC cells to ionizing radiation by suppressing the activity of NHEJ, a pathway essential for repair of radiation-induced DSBs.

Persistent DNA damage triggers cellular senescence, which may play an important role in the pathogenesis of cigarette smoke (CS)-induced lung diseases. Both p21(CDKN1A) (p21) and poly(ADP-ribose) polymerase-1 (PARP-1) are involved in DNA damage and repair. However, the role of p21-PARP-1 axis in regulating CS-induced lung DNA damage and cellular senescence remains unknown. We hypothesized that CS causes DNA damage and cellular senescence through a p21-PARP-1 axis. To test this hypothesis, we determined the levels of γH2AX (a marker for DNA double-strand breaks) as well as non-homologous end joining proteins (Ku70 and Ku80) in lungs of mice exposed to CS. We found that the level of γH2AX was increased, whereas the level of Ku70 was reduced in lungs of CS-exposed mice. Furthermore, p21 deletion reduced the level of γH2AX, but augmented the levels of Ku70, Ku80, and PAR in lungs by CS. Administration of PARP-1 inhibitor 3-aminobenzamide increased CS-induced DNA damage, but lowered the levels of Ku70 and Ku80, in lungs of p21 knockout mice. Moreover, 3-aminobenzamide increased senescence-associated β-galactosidase activity, but decreased the expression of proliferating cell nuclear antigen in mouse lungs in response to CS. Interestingly, 3-aminobenzamide treatment had no effect on neutrophil influx into bronchoalveolar lavage fluid by CS. These results demonstrate that the p21-PARP-1 pathway is involved in CS-induced DNA damage and cellular senescence.

OBJECTIVE

Radiotherapy is integrated into the multimodal treatment of localized hepatocellular carcinoma (HCC) refractory to conventional treatment. Tumor control remains unsatisfactory and the sublethal effect associates with secondary spread. The use of an effective molecularly targeted agent in combination with radiotherapy is a potential therapeutic approach. Our aim was to assess the effect of combining a phenylbutyrate-derived histone deacetylase (HDAC) inhibitor, AR-42, with radiotherapy in in vitro and in vivo models of human HCC.

METHODS

Human HCC cell lines (Huh-7 and PLC-5) were used to evaluate the in vitro synergism of combining AR-42 with irradiation. Flow cytometry analyzed the cell cycle changes, whereas Western blot investigated the protein expressions after the combined treatment. Severe combined immunodeficient (SCID) mice bearing ectopic and orthotopic HCC xenografts were treated with AR-42 and/or radiotherapy for the in vivo response.

RESULTS

AR-42 significantly enhanced radiation-induced cell death by the inhibition of the DNA end-binding activity of Ku70, a highly versatile regulatory protein for DNA repair, telomere maintenance, and apoptosis. In ectopic xenografts of Huh-7 and PLC-5, pretreatment with AR-42 significantly enhanced the tumor-suppressive effect of radiotherapy by 48% and 66%, respectively. A similar combinatorial effect of AR-42 (10 and 25 mg/kg) and radiotherapy was observed in Huh-7 orthotopic model of tumor growth by 52% and 82%, respectively. This tumor suppression was associated with inhibition of intratumoral Ku70 activity as well as reductions in markers of HDAC activity and proliferation, and increased apoptosis.

CONCLUSIONS

AR-42 is a potent, orally bioavailable inhibitor of HDAC with therapeutic value as a radiosensitizer of HCC.

Integrin alpha M (ITGAM; CD11b) is a component of the macrophage-1 antigen complex, which mediates leukocyte adhesion, migration and phagocytosis as part of the immune system. We previously identified a missense polymorphism, rs1143679 (R77H), strongly associated with systemic lupus erythematosus (SLE). However, the molecular mechanisms of this variant are incompletely understood. A meta-analysis of published and novel data on 28 439 individuals with European, African, Hispanic and Asian ancestries reinforces genetic association between rs1143679 and SLE [Pmeta = 3.60 × 10(-90), odds ratio (OR) = 1.76]. Since rs1143679 is in the most active region of chromatin regulation and transcription factor binding in ITGAM, we quantitated ITGAM RNA and surface protein levels in monocytes from patients with each rs1143679 genotype. We observed that transcript levels significantly decreased for the risk allele ('A') relative to the non-risk allele ('G'), in a dose-dependent fashion: ('AA' < 'AG' < 'GG'). CD11b protein levels in patients' monocytes were directly correlated with RNA levels. Strikingly, heterozygous individuals express much lower (average 10- to 15-fold reduction) amounts of the 'A' transcript than 'G' transcript. We found that the non-risk sequence surrounding rs1143679 exhibits transcriptional enhancer activity in vivo and binds to Ku70/80, NFKB1 and EBF1 in vitro, functions that are significantly reduced with the risk allele. Mutant CD11b protein shows significantly reduced binding to fibrinogen and vitronectin, relative to non-risk, both in purified protein and in cellular models. This two-pronged contribution (nucleic acid- and protein-level) of the rs1143679 risk allele to decreasing ITGAM activity provides insight into the molecular mechanisms of its potent association with SLE.

Long non-coding RNAs (lncRNAs) have been regarded as the primary genetic regulators of several important biological processes. However, the biological functions of lncRNAs in radiation-induced lung damage remain largely unknown. The present study aimed to investigate the potential effects of lncRNAs on radiation-induced lung injury (RILI). Female C57BL/6 mice were exposed to 12 Gy single doses of total body irradiation (TBI). LncRNA microarray screening was conducted at 24 h post-irradiation (IR) to investigate the differentially-expressed lncRNAs during RILI. Following the subsequent bioinformatics analysis and reverse transcription-polymerase chain reaction (RT-PCR) validation, one of the verified differentially-expressed long intergenic radiation-responsive ncRNAs (LIRRs), LIRR1, was selected for further functional study. The normal human bronchial epithelial BEAS-2B cell line was used as the cell model. The recombinant eukaryotic expression vector for the lncRNA was designed, constructed and transfected using lipofectamine. RT-PCR, clonogenic and flow cytometry assays, immunofluorescence detection and western blot analysis were performed to reveal the role of the lncRNA in the radiosensitivity regulation of the RILI target cells. In lung tissues 24 h after 12 Gy TBI, six of the identified differentially-expressed LIRRs near the coding genes were validated using quantitative (q)PCR. The upregulation of two LIRRs was observed and confirmed using qPCR. LIRR1 was chosen for further functional study. Following the stable transfection of LIRR1, identified through G418 screening, increased radiosensitivity, evident cell cycle G1 phase arrest and increased γ-H2AX foci formation were observed in the bronchial epithelial BEAS-2B cell line subsequent to IR. LIRR1 overexpression also led to decreased expression of the KU70, KU80 and RAD50 DNA repair proteins, marked activation of p53, decreased mouse double minute 2 homolog (MDM2) expression, and substantially induced p21 and suppressed cyclin-dependent kinase 2 in BEAS-2B following IR. Subsequent to the use of Pifithrin-α, a specific inhibitor of p53 activation, increased MDM2 expression was observed in the LIRR1-overexpressing cells, suggesting that LIRR1 could mediate the DNA damage response (DDR) signaling in a p53-dependent manner. The present study provides a novel mechanism for RILI, using the concept of lncRNAs.

During the evolution of plant genomes, sequence inversions occurred repeatedly making the respective regions inaccessible for meiotic recombination and thus for breeding. Therefore, it is important to develop technologies that allow the induction of inversions within chromosomes in a directed and efficient manner. Using the Cas9 nuclease from Staphylococcus aureus (SaCas9), we were able to obtain scarless heritable inversions with high efficiency in the model plant Arabidopsis thaliana. Via deep sequencing, we defined the patterns of junction formation in wild-type and in the non-homologous end-joining (NHEJ) mutant ku70-1. Surprisingly, in plants deficient of KU70, inversion induction is enhanced, indicating that KU70 is required for tethering the local broken ends together during repair. However, in contrast to wild-type, most junctions are formed by microhomology-mediated NHEJ and thus are imperfect with mainly deletions, making this approach unsuitable for practical applications. Using egg-cell-specific expression of Cas9, we were able to induce heritable inversions at different genomic loci and at intervals between 3 and 18 kb, in the percentage range, in the T1 generation. By screening individual lines, inversion frequencies of up to the 10% range were found in T2. Most of these inversions had scarless junctions and were without any sequence change within the inverted region, making the technology attractive for use in crop plants. Applying our approach, it should be possible to reverse natural inversions and induce artificial ones to break or fix linkages between traits at will.

The modulator of retrovirus infection (MRI or CYREN) is a 30-kDa protein with a conserved N-terminal Ku-binding motif (KBM) and a C-terminal XLF-like motif (XLM). We show that MRI is intrinsically disordered and interacts with many DNA damage response (DDR) proteins, including the kinases ataxia telangiectasia mutated (ATM) and DNA-PKcs and the classical non-homologous end joining (cNHEJ) factors Ku70, Ku80, XRCC4, XLF, PAXX, and XRCC4. MRI forms large multimeric complexes that depend on its N and C termini and localizes to DNA double-strand breaks (DSBs), where it promotes the retention of DDR factors. Mice deficient in MRI and XLF exhibit embryonic lethality at a stage similar to those deficient in the core cNHEJ factors XRCC4 or DNA ligase IV. Moreover, MRI is required for cNHEJ-mediated DSB repair in XLF-deficient lymphocytes. We propose that MRI is an adaptor that, through multivalent interactions, increases the avidity of DDR factors to DSB-associated chromatin to promote cNHEJ.

Bacterial infection influences genomic stability and integrity by causing DNA damage, which increases the possibility of tumor initiation and development. We aimed to investigate whether Fusobacterium nucleatum, one of the periodontal pathogens, promoted oral squamous cell carcinoma (OSCC) by causing DNA double-strand break (DSB). Tca8113 tongue squamous cell carcinoma cells were infected with F. nucleatum. The expression of γH2AX was detected by western blots and immunofluorescence. The proliferation and cell cycle alterations were tested by CCK8 and flow cytometry, respectively. The expression levels of Ku70, p53, and p27 were evaluated by quantitative real-time polymerase chain reaction and western blots. A plasmid was used for the overexpression of Ku70 to verify the possible relationship between Ku70 and p53. We confirmed the presence of DSBs in the response to F. nucleatum by detecting the expression of γH2AX. The cell proliferation ability was increased with an accelerated cell cycle while the expression of p27 was decreased. Meanwhile, the expression of Ku70 and wild p53 was downregulated. When Ku70 was overexpressed, the expression of wild p53 in response to F. nucleatum infection was upregulated and cell proliferation was accordingly inhibited. We concluded that F. nucleatum infection promoted the proliferation ability of Tca8113 by causing DNA damage via the Ku70/p53 pathway.

Antibody class-switch DNA recombination (CSR) is initiated by AID-introduced DSBs in the switch (S) regions targeted for recombination, as effected by Ku70/Ku86-mediated NHEJ. Ku-deficient B cells, however, undergo (reduced) CSR through an alternative(A)-NHEJ pathway, which introduces microhomologies in S-S junctions. As microhomology-mediated end-joining requires annealing of single-strand DNA ends, we addressed the contribution of single-strand annealing factors HR Rad52 and translesion DNA polymerase θ to CSR. Compared with their Rad52+/+ counterparts, which display normal CSR, Rad52-/- B cells show increased CSR, fewer intra-Sμ region recombinations, no/minimal microhomologies in S-S junctions, decreased c-Myc/IgH translocations and increased Ku70/Ku86 recruitment to S-region DSB ends. Rad52 competes with Ku70/Ku86 for binding to S-region DSB ends. It also facilitates a Ku-independent DSB repair, which favours intra-S region recombination and mediates, particularly in Ku absence, inter-S-S recombination, as emphasized by the significantly greater CSR reduction in Rad52-/- versus Rad52+/+ B cells on Ku86 knockdown.

DNA repair deficiency results in neurodegenerative disease and increased susceptibility to excitotoxic cell death, suggesting a critical but undefined role for DNA damage in neurodegeneration. We compared DNA damage, Ku70-Bax interaction, and Bax-dependent excitotoxic cell death in kainic acid-treated primary cortical neurons derived from both wild-type mice and mice deficient in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) encoded by the Prkdc gene. In both wild-type and Prkdc(-/-) neurons, kainic acid treatment resulted in rapid induction of DNA damage (53BP1 foci formation) followed by nuclear pyknosis. Bax deficiency, by either Bax shRNA-mediated knockdown or gene deletion, protected wild-type and heterozygous but not Prkdc(-/-) neurons from kainate-induced excitotoxicity. Cotransfection of DNA-PKcs with Bax shRNA restored Bax shRNA-mediated neuroprotection in Prkdc(-/-) neurons, suggesting that DNA-PKcs is required for kainate-induced activation of the pro-apoptotic Bax pathway. Immunoprecipitation studies revealed that the DNA-PKcs-nonphosphorylatable Ku70 (S6A/S51A) bound 3- to 4-fold greater Bax than wild-type Ku70, suggesting that DNA-PKcs-mediated Ku70 phosphorylation causes release of Bax from Ku70. In support of this, kainic acid induced translocation of a Bax-EGFP fusion protein to the mitochondria in the presence of a cotransfected wild-type, but not mutant Ku70 (S6A/S51A) gene when examined at 4 and 8 h following kainate addition. We conclude that DNA-PKcs links DNA damage to Bax-dependent excitotoxic cell death, by phosphorylating Ku70 on serines 6 and/or 51, to initiate Bax translocation to the mitochondria and directly activate a pro-apoptotic Bax-dependent death cascade.

The presence of DNA double-stranded breaks in a mammalian cell typically activates the Non-Homologous End Joining (NHEJ) pathway to repair the damage and signal to downstream systems that govern cellular decisions such as apoptosis or senescence. The signalling system also stimulates effects such as the generation of reactive oxygen species (ROS) which in turn feed back into the damage response. Although the overall process of NHEJ is well documented, we know little of the dynamics and how the system operates as a whole. We have developed a computational model which includes DNA Protein Kinase (DNA-PK) dependent NHEJ (D-NHEJ) and back-up NHEJ mechanisms (B-NHEJ) and use it to explain the dynamic response to damage induced by different levels of gamma irradiation in human fibroblasts. Our work suggests that the observed shift from fast to slow repair of DNA damage foci at higher levels of damage cannot be explained solely by inherent stochasticity in the NHEJ system. Instead, our model highlights the importance of Ku oxidation which leads to increased Ku dissociation rates from DNA damage foci and shifts repair in favour of the less efficient B-NHEJ system.

BACKGROUND

The Malaysian Tualang honey (TH) is not only cytotoxic to human breast cancer cell lines but it has recently been reported to promote the anticancer activity induced by tamoxifen in MCF-7 and MDA-MB-231 cells suggesting its potential as an adjuvant for the chemotherapeutic agent. However, tamoxifen produces adverse effects that could be due to its ability to induce cellular DNA damage. Therefore, the study is undertaken to determine the possible modulation of the activity of 4-hydroxytamoxifen (OHT), an active metabolite of tamoxifen, by TH in non-cancerous epithelial cell line, MCF-10A, in comparison with MCF-7 cells.

METHODS

MCF-7 and MCF-10A cells were treated with TH, OHT or the combination of both and cytotoxicity and antiproliferative activity were determined using LDH and MTT assays, respectively. The effect on cellular DNA integrity was analysed by comet assay and the expression of DNA repair enzymes was determined by Western blotting.

RESULTS

OHT exposure was cytotoxic to both cell lines whereas TH was cytotoxic to MCF-7 cells only. TH also significantly decreased the cytotoxic effect of OHT in MCF-10A but not in MCF-7 cells. TH induced proliferation of MCF10A cells but OHT caused growth inhibition that was abrogated by the concomitant treatment with TH. While TH enhanced the OHT-induced DNA damage in the cancer cells, it dampened the genotoxic effect of OHT in the non-cancerous cells. This was supported by the increased expression of DNA repair proteins, Ku70 and Ku80, in MCF-10A cells by TH.

CONCLUSIONS

The findings indicate that TH could afford protection of non-cancerous cells from the toxic effects of tamoxifen by increasing the efficiency of DNA repair mechanism in these cells.