DNA-PKcs Protein Research Articles

The interplay of DNA repair context with target sequence predictably biases Cas9-generated mutations.

Mutagenic outcomes of CRISPR/Cas9-generated double-stranded breaks depend on both the sequence flanking the cut and cellular DNA damage repair. The interaction of these features has been largely unexplored, limiting our ability to understand and manipulate the outcomes. Here, we measured how the absence of 18 repair genes changed frequencies of 83,680 unique mutational outcomes generated by Cas9 double-stranded breaks at 2,838 synthetic target sequences in mouse embryonic stem cells. This large scale survey allowed us to classify the outcomes in an unbiased way, generating hypotheses about new modes of double-stranded break repair. Our data indicate a specialised role for Prkdc (DNA-PKcs protein) and Polm (Polμ) in creating 1bp insertions that match the nucleotide on the proximal side of the Cas9 cut with respect to the protospacer-adjacent motif (PAM), a variable involvement of Nbn (NBN) and Polq (Polθ) in the creation of different deletion outcomes, and a unique class of uni-directional deletion outcomes that are dependent on both end-protection gene Xrcc5 (Ku80) and the resection gene Nbn (NBN). We used the knowledge of the reproducible variation across repair milieus to build predictive models of the mutagenic outcomes of Cas9 scission that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of CRISPR/Cas9-generated mutations.

CRL4ADTL degrades DNA-PKcs to modulate NHEJ repair and induce genomic instability and subsequent malignant transformation

Genomic instability induced by DNA damage and improper DNA damage repair is one of the main causes of malignant transformation and tumorigenesis. DNA double strand breaks (DSBs) are the most detrimental form of DNA damage, and nonhomologous end-joining (NHEJ) mechanisms play dominant and priority roles in initiating DSB repair. A well-studied oncogene, the ubiquitin ligase Cullin 4A (CUL4A), is reported to be recruited to DSB sites in genomic DNA, but whether it regulates NHEJ mechanisms of DSB repair is unclear. Here, we discovered that the CUL4A-DTL ligase complex targeted the DNA-PKcs protein in the NHEJ repair pathway for nuclear degradation. Overexpression of either CUL4A or DTL reduced NHEJ repair efficiency and subsequently increased the accumulation of DSBs. Moreover, we demonstrated that overexpression of either CUL4A or DTL in normal cells led to genomic instability and malignant proliferation. Consistent with the in vitro findings, in human precancerous lesions, CUL4A expression gradually increased with increasing malignant tendency and was negatively correlated with DNA-PKcs and positively correlated with γ-H2AX expression. Collectively, this study provided strong evidence that the CUL4A-DTL axis increases genomic instability and enhances the subsequent malignant transformation of normal cells by inhibiting NHEJ repair. These results also suggested that CUL4A may be a prognostic marker of precancerous lesions and a potential therapeutic target in cancer.

Abstract 4040: A cell-based screening assay to identify novel kinase inhibitors

Abstract The majority of the FDA-approved kinase inhibitors target ATP binding sites of kinases. Despite their activity against tumorigenesis, these therapies are often non-specific and are susceptible to resistance mechanisms. One method of enhancing the selectivity of these compounds and overcoming potential chemoresistance is to better identify novel compounds that are highly specific. In this study, we introduce one such strategy by identifying kinase inhibitors that target kinase-substrate interactions. c-MYC overexpression and deregulation has been implicated in the development and progression of a number of malignancies. We have previously identified that OCT4 binds to the MYC promoter/enhancer region to transcriptionally activate c-MYC. Novel OCT4 binding sites in the c-MYC promoter region were identified, and kinases (DNA-PKcs for the current study) that mediate the phosphorylation of specific amino acid residues of OCT4 were determined. We then identified the domains of DNA-PKcs that are critical to bind and phosphorylate OCT4. The goal of the current project is to develop an assay to identify the compounds that interferes the interaction between OCT4 and DNA-PKcs and to validate the identified "hits." In order to selectively inhibit the interaction of protein-kinase related to c-MYC transcriptional activation, we co-expressed the fragments of OCT4 required for c-MYC expression and the fragments DNA-PKcs required to activate OCT4 to 1) confirm the interaction between DNA-PKcs and OCT4, and 2) develop a cell-based assay that can screen novel compounds that prevent this interaction. After confirming the kinase-substrate interaction, we co-transduced the crucial fragments of DNA-PKcs and OCT4 tagged with luminescent probes in HEK 293FT cells. We then screened a chemical library of compounds to identify hits that inhibited luminescence, and thus our kinase-substrate protein interaction. In our screening assay, 67 compounds were found to inhibit the luminescence interaction between the tagged OCT4 and DNA-PKcs protein fragments. After the initial screening, we conducted a two-step hit validation for the 67 hits identified that consisted of: 1) the decrease in pOCT4S93 expression along with the reduction in c-MYC expression: 2) and the inhibition of binding between DNA-PKcs and OCT4 by co-immunoprecipitation. Additional experiments to validate the hits include kinase activity assays and immunoblotting to determine if the screened hits inhibited DNA-PKcs kinase activity. In conclusion, our screening assay and validation experiments identified 7 novel compounds that demonstrated: 1) impairment of DNA-PKcs-mediated phosphorylation of OCT4; and 2) inhibition of the DNA-PKcs-OCT4 kinase-substrate interaction. These newly identified compounds have the potential to oppose aberrant c-MYC expression and warrant further investigation in order to determine their activity in high c-MYC-expressing malignancies. Citation Format: Ismail S. Mohiuddin, Sung J. Wei, Inhyoung Yang, Gloria Martinez, Hwangeui Cho, Min H. Kang. A cell-based screening assay to identify novel kinase inhibitors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4040.

Cells Deficient in the Shwachman-Diamond Syndrome Protein SBDS or the Diamond-Blackfan Anemia Protein RPS19 Have Impaired Homologous Recombination

The inherited bone marrow failure syndromes (IBMFS) are rare genetic disorders caused by mutations in critical components of fundamental cellular processes such as ribosome biogenesis, DNA repair, and telomere maintenance. The IBMFS Shwachman-Diamond syndrome(SDS) and Diamond-Blackfan anemia (DBA) are classified as ribosomopathies due to etiologic mutations in genes encoding factors involved in ribosome biogenesis (SBDSin the majority of patients with SDS) or ribosomal proteins (RPS19most commonly in patients with DBA). Although these disorders can be distinguished clinically and from the other IBMFS, they share with each other and with other IBMFS increased predisposition to myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Whereas genomic instability due to defective DNA repair or telomere maintenance is thought to underlie cancer predisposition in the IBMFS Fanconi anemia and dyskeratosis congenita, respectively, the molecular mechanisms driving cancer in SDS and DBA are not fully understood. Our research has focused on DNA repair in SDS and DBA. A prior report suggested lymphoblastoid cell lines (LCLs) derived from patients with SDS arehypersensitive to ionizing radiation (IR). Consistent with this, we found SDS-LCLs had decreased survival following IR compared to control-LCLsin colony survival assays. To determine if this cellular phenotype was unique to SDS or present in the other IBMFS ribosomopathy, DBA, we examined LCLs derived from patients with DBA, including those with mutations in RPS19, RPS26, RPL5and RPL11. We found that the DBA-LCLs were similarly hypersensitive to IR as compared to control-LCLs. Further examination of γ-H2AX, a DNA damage response (DDR) factor and marker of DNA double strand breaks (DSBs), revealed that SDS- and DBA-LCLs had delayed resolution of γ-H2AX foci and increased protein levels at 24 hrs after IR as compared to control LCLs. p53, phospho-ATM, and DNA-PKcs protein levels were also higher in SDS-LCL compared to controls. The decreased survival and increased and sustained DDR following IR led us to hypothesize that SDS and DBA cells have a defect in DSB repair. There are two major pathways of DSB repair in mammals, nonhomologous end-joining (NHEJ) and homology-directed repair (HDR), and loss of either results in hypersensitivity to IR. To examine each pathway, we employed U2OS (human osteosarcoma) and HCT116 (human colon cancer) cells containing an integrated green fluorescent protein HDR or NHEJ reporter transgene. Interestingly, we found that knockdown of either SBDS or RPS19 proteins resulted in an approximately 50% reduction in HDR efficiency but no change in NHEJefficiency compared to the scrambled control in both cell lines. We next sought to determine the mechanism underlying the effect of SBDS and RPS19 deficiency on HDR. A survey of proteins required for HDR revealed a reduction in the recombinase RAD51 in SDS-LCLs and in SBDS-depleted HCT116 and U2OS cells, whereas, an initial survey in SDS-LCLs[e1] of factors involved in NHEJ did not reveal a specific NHEJ factor deficiency. Knockdown of eiF6 is known to rescue the defect in 40S and 60S ribosome subunit joining that manifests in SDS patient cells. However, we found eIF6 depletion failed to rescue the level of RAD51 protein and had no impact on HDR in SBDS-deficient cells. We conclude that decreased RAD51 levels in SBDS-deficient cells might contribute to impaired HDR, however, this decrease is independent of the ribosome subunit joining defect. Similarly, RPS19 knock down resulted in a reduction in RAD51 protein level, suggesting a potentially common pathway. We also asked whether SBDS or RPS19 might be more directly involved in the DDR or repair of DSBs. Consistent with this, we found SBDS and RPS19 recruited to chromatin surrounding an I-Sce1 site following DSB induction in chromatin immunoprecipitation assays. Collectively, these findings provide evidence that SBDS and RPS19 may be directly involved in the DDR or DSB repair and raise the possibility that loss of this function may contribute to MDS/AML predisposition in SDS and DBA patients. Disclosures No relevant conflicts of interest to declare.

Knockdown of microRNA-1323 restores sensitivity to radiation by suppression of PRKDC activity in radiation-resistant lung cancer cells.

Resistance to radiation is a major problem in cancer treatment. The mechanisms of radioresistance remain poorly understood; however, mounting evidence supports a role for microRNAs (miRNAs) in the modulation of key cellular pathways mediating the response to radiation. The present study aimed to identify specific miRNAs and their effect on radioresistant cells. The global miRNA profile of an established radioresistant lung cancer cell line and the corresponding control cells was determined. Differential expression of the miRNAs was confirmed by quantitative real-time PCR (qRT-PCR). The binding effect of identical novel miRNAs and target mRNAs was determined by luciferase assay. Lung cancer cells were transfected with miRNA-specific mimics or inhibitors. The DNA-dependent protein kinase (DNA-PKcs) protein level was tested by western blot analysis. Radiosensitivity of cancer cells was determined using colony formation assay. Among the differentially expressed miRNAs, 25 miRNAs were overexpressed while 18 were suppressed in the radioresistant cells, both basally and in response to radiation compared to their control. An miRNA signature miR-1323 exhibited a >5-fold increase in the radioresistant cells. miR-1323 was demonstrated to bind to PRKDC 3'UTR, which is involved in DNA repair. Ectopic expression of miR-1323 significantly increased the survival fraction of irradiated cancer cells. Inhibition of miR-1323 reversed the radioresistance of cancer cells and subsequently suppressed the expression of miR-1323-regulated DNA-PKcs protein. The present study indicated that miRNAs are involved in the radioresistance of human lung cancer cells. A possible mechanism for resistance to radiation was via enhanced DNA repair. The present study demonstrated a role for miR-1323 in modulating radioresistance and highlights the need for further study investigating the potential role of miR-1323 as both a predictive marker of response and a novel therapeutic agent with which to enhance the efficacy of radiotherapy.

Differential Phosphorylation of DNA-PKcs Regulates the Interplay between End-Processing and End-Ligation during Nonhomologous End-Joining

Nonhomologous end-joining (NHEJ) is a major DNA double-strand break repair pathway that is conserved in eukaryotes. In vertebrates, NHEJ further acquires end-processing capacities (e.g., hairpin opening) in addition to direct end-ligation. The catalytic subunit of DNA-PK (DNA-PKcs) is a vertebrate-specific NHEJ factor that can be autophosphorylated or transphosphorylated by ATM kinase. Using a mouse model expressing a kinase-dead (KD) DNA-PKcs protein, we show that ATM-mediated transphosphorylation of DNA-PKcs regulates end-processing at the level of Artemis recruitment, while strict autophosphorylation of DNA-PKcs is necessary to relieve the physical blockage on end-ligation imposed by the DNA-PKcs protein itself. Accordingly, DNA-PKcs(KD/KD) mice and cells show severe end-ligation defects and p53- and Ku-dependent embryonic lethality, but open hairpin-sealed ends normally in the presence of ATM kinase activity. Together, our findings identify DNA-PKcs as the molecular switch that coordinates end-processing and end-ligation at the DNA ends through differential phosphorylations.

Predictive factors for the sensitivity of radiotherapy and prognosis of esophageal squamous cell carcinoma

Purpose: To identify predictive biomarkers for radiosensitization and prognosis of esophageal squamous cell carcinoma (ESCC).Materials and methods: A total of 150 advanced stage ESCC patients were treated with preoperative radiotherapy. The protein levels of Dicer 1, DNA methyltransferase 1 (Dnmt1), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) and the mRNA levels of Dicer 1, Dnmt1, and let-7b microRNA (miRNA) were measured in ESCC tumor tissues before and after radiotherapy. Global DNA methylation was measured and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining was performed.Results: Negative Dicer 1, Dnmt1, and DNA-PKcs protein expression were observed in 72%, 67.3%, and 50.7% of ESCC patients, respectively. Primary Dicer 1 and Dnmt1 expression positively correlated with radiation sensitization and longer survival of ESCC patients, while increased Dicer 1 and Dnmt1 expression after radiation correlated with increased apoptosis in residual tumor tissues. Dicer 1 and Dnmt1 expression correlated with let-7b miRNA expression and global DNA methylation levels, respectively. In contrast, positive DNA-PKcs expression negatively correlated with radiation-induced pathological reactions, and increased DNA-PKcs expression correlated with increased apoptosis after radiation.Conclusion: Global DNA hypomethylation and low miRNA expression are involved in the sensitization of ESCC to radiotherapy and prognosis of patients with ESCC.

Effects of Ligustrazine on DNA damage and apoptosis induced by irradiation

Ligustrazine has been used to treat heart and blood vessel disease in China. In the present study, we investigated the potential action of Ligustrazine as a component of chuanxiong (a Chinese herb) in scavenging hydroxyl radical and superoxide radical as indicated in the ESR spin-trapping measurement. Treatment of Ligustrazine in mice decreased mortality after whole body γ-irradiation. The anti-radiation action of Ligustrazine was studied by measuring DNA damage (Comet assay and γ-H2AX formation) and apoptosis induced by irradiation. It was triggered by altering the level of DNA-PKcs protein, a critical component of DNA double-strand break (DSB) repair pathways in mice after irradiation. Consistently, the phosphorylation of Akt protein, a mediator of survival signaling, was concurrently increased by Ligustrazine treatment. Additionally, the cytokines along with the phosphorylation of the p38 protein which is activated by a variety of environmental stresses and inflammatory cytokines decreased in the Ligustrazine-treated group as compared to irradiation group. Our results suggest that Ligustrazine has radioprotective effect through its capabilities as a powerful antioxidant, in reducing reactive oxygen species (ROS) level induced by irradiation, minimizing DNA damage and apoptosis, and activating survival signal Akt pathways. This study will be of value in the development of novel radioprotective compounds.

Abstract 4680: DNA dependent protein kinase as a new molecular target for treatment of non-small cell lung cancer

Abstract Prognosis with non-small cell lung cancer (NSCLC) has improved with the recent introduction of molecular targeted therapy. However, it is limited to patients with epidermal growth factor receptor (EGFR) mutations, who benefit from EGFR tyrosine kinase inhibitors (EGFR-TKI). A new molecular target is needed with other NSCLC patients. In general, chemotherapeutic agents cause DNA double-strand breaks (DNA DSB), leading to apoptosis of cancer cells; DNA repair reverses their effect, resulting in resistance to chemotherapy. DNA DSB repair can be mediated via two major pathways: homologous recombination (HR) and non-homologous end-joining (NHEJ). More than 90% of DSBs in mammalian cells are repaired by NHEJ, which is accomplished by core protein components including Ku70/Ku86, DNA-dependent protein kinase (DNA-PK), XRCC4, DNA ligase IV, Artemis, and Cernunnos-XLF. We recently reported that NK314, a dual inhibitor of topoisomerase IIα (Top2β) and DNA-PK, evidences a potent anti-tumor effect in adult T-cell leukemia-lymphoma (ATL). NK314 induced DNA DSBs and inhibited DNA repair mediated through degradation of the DNA-PK catalytic subunit (DNA-PKcs). In the present study, we examined the effect of NK314 in NSCLC cell lines and assessed DNA-PK as a possible molecular target for NSCLC. Twenty separate NSCLC cell lines were used, with IC50 varying from 46-193 nM. Levels of DNA-PKcs protein varied among the cell lines, whereas those of Ku70 and Ku86 did not. The inhibitory effect of cell growth was closely related to level of DNA-PKcs protein, but not to levels of other DNA-damage-activated protein kinases such as ATM or ATR. Establishment of predictive markers concomitantly with the development of a novel molecular target is indispensable for clinical application. Because detection of DNA-PKcs in peripheral blood is difficult, we investigated an alternative marker for predicting DNA-PKcs inhibitor. We previously found that heterogeneous nuclear ribonucleoprotein B1 (hnRNP B1), an RNA binding protein, interacts with DNA-PK, and we established a system for detecting hnRNP B1 mRNA in peripheral blood. We therefore examined the suitability of hnRNP B1 as an alternative biomarker for DNA-PK inhibitors. Close correlation between levels of DNA-PKcs and hnRNP B1 proteins was established using Western blot analysis with NSCLC cell lines (p=0.995). These results suggest that DNA-PK is a novel molecular target for NSCLC, and detection of hnRNP B1 might serve as an alternative molecular marker for the amount of DNA-PK inhibitors. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4680. doi:1538-7445.AM2012-4680

DNA-PKcs expression in hepatoma and normal mouse liver tissues of various developmental stages and its influence on cell proliferation

Objective:To observe the expression of DNA dependent protein kinase catalytic subunit(DNA-PKcs) in the hepatoma tissues and mouse liver tissues of different developmental stages,so as to understand the role of DNA-PKcs in cell proliferation and tumorigenesis.Methods: Immunohistochemistry and Western blotting assay were used to observe the protein expression of DNA-PKcs in liver tissues and hepatoma tissues.The siRNA technique was used to silence the expression of DNA-PKcs in the HepG2 cells;cell proliferation assay and tumor transplantation test in nude mice were performed to evaluate the changes of the proliferation ability and tumorigenesis.Western blotting assay was also conducted to examine the expression of proliferation related proteins p-GSK3β and c-myc.Results: DNA-PKcs expression decreased in the liver tissues with the decrease of cell proliferation ability during the development of mice,and the expression level of DNA-PKcs was weak in liver tissues of adult mouse;but the DNA-PKcs protein level in the hepatoma tissues was significantly elevated(P0.01).Cell growth curve showed that the proliferation of HepG2 cells was significantly decreased after suppression of DNA-PKcs with siRNA(P0.01),accompanied by a suppressed tumorigenesis ability.The expression of signal pathway related protein p-GSK3β and c-myc was inhibited after DNA-PKcs silencing in HepG2 cells(P0.01).Conclusion: DNA-PKcs expression level is closely related to the proliferation ability of liver cells.Overexpression of DNA-PKcs may participate in the development and progression of hepatoma through mediating cell proliferation via the Wnt/GSK/c-myc related signal pathway.

Significance of Phosphatase and Tensin Homologue (PTEN), O6-Methylguanine-DNA Methyltransferase (MGMT), and DNA-dependent Protein Kinase Catalytic Subunit (DNA-PKcs) Protein Expression in Gynaecomastia

This study was designed to investigate the pathogenesis of gynaecomastia by measuring phosphatase and tensin homologue (PTEN), O(6)-methylguanine-DNA methyltransferase (MGMT) and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) protein in breast tissue specimens from 68 patients with gynaecomastia and 24 normal male controls using immunohistochemical staining. The gynaecomastia cases were divided into three different histological types: florid, intermediate and fibrous. The PTEN, MGMT and DNA-PKcs proteins were detected in both gynaecomastia and normal breast tissue, but the levels of immunohistochemical staining of each protein were significantly lower in gynaecomastia breast tissue than in normal breast tissue. There were also significant differences in the levels of immunohistochemical staining for the three proteins according to gynaecomastia histological type. These results suggest that abnormally low levels of PTEN, MGMT and DNA-PKcs protein in gynaecomastia breast tissue may play a role in the development of gynaecomastia. Further research is required to elucidate fully their individual roles in the pathophysiology of gynaecomastia.

Correlativity study between expression of DNA double-strand break repair protein and radiosensitivity of tumor cells

DNA double-strand break (DSB) is generally regarded as the most lethal of all DNA lesions after radiation. Ku80, DNA-PK catalytic subunit (DNA-PKcs) and ataxia telangiectasia mutated (ATM) proteins are major DSB repair proteins. In this study, survival fraction at 2Gy (SF2) values of eight human tumor cell lines (including four human cervical carcinoma cell lines HeLa, SiHa, C33A, Caski, three human breast carcinoma cell lines MCF-7, MDA-MB-231, MDA-MB-453, and one human lung carcinoma cell line A549) were acquired by clone formation assay, and western blot was applied to detect the expressions of Ku80, DNA-PKcs and ATM protein. The correlativity of protein expression with SF2 value was analyzed by Pearson linear correlation analysis. We found that the expression of the same protein in different cell lines and the expression of three proteins in the same cell line had a significant difference. The SF2 values were also different in eight tumor cell lines and there was a positive correlativity between the expression of DNA-PKcs and SF2 (r =0.723, P = 0.043), but Ku80 and ATM expression had no correlation with SF2 (P > 0.05). These findings suggest that the expression level of DNAPKcs protein can be an indicator for predicting the radiosensitivity of tumor cells.

AG825 increases radiosensitivity of breast cancer cell

To observe the radiosensitivity effect of AG825 on breast cancer cell line with high expression of ERBB2 in vitro. MTT and clone formation assay were used to observe the effect of AG825 on proliferation and radiosensitivity of breast cancer line MDA-MB-453. After MDA-MB-453 was exposed to AG825 and radiation, comet assay and Western blotting were applied to detect double strand break and expressions DNA-PKcs protein, respectively. AG825 inhibited proliferation rate and decrease survival fraction of MDA-MB-453. After radiation, compared with control group, expression of DNA-PKcs was lower in group with AG825 presence but double strand break was higher. AG825 could increase radiosensitivity of breast cancer cell line MDA-MB-453, and it may associate with its inhibition of radiation induced expression of DNA-PKcs and double strand break repair.

Inhibition of histone acetyltransferase activity by anacardic acid sensitizes tumor cells to ionizing radiation

Histone acetyltransferases (HATs) regulate transcription, chromatin structure and DNA repair. Here, we utilized a novel HAT inhibitor, anacardic acid, to examine the role of HATs in the DNA damage response. Anacardic acid inhibits the Tip60 HAT in vitro, and blocks the Tip60-dependent activation of the ATM and DNA–PKcs protein kinases by DNA damage in vivo. Further, anacardic acid sensitizes human tumor cells to the cytotoxic effects of ionizing radiation. These results demonstrate a central role for HATs such as Tip60 in regulating the DNA damage response. HAT inhibitors provide a novel therapeutic approach for increasing the sensitivity of tumors to radiation therapy.

643. Prolonged Expression of Human Alpha-1-Antitrypsin in C3H/Hej Mice with Helper-Dependent Adenoviral Vectors Irrespective of Tissue Distribution of Gene Expression

We have recently constructed a novel HAC vector called 21ΔqHAC, generated by deletion of a distal portion of the long arm of human chromosome 21 and insertion of a loxP sequence to facilitate insertion of any desired gene construct. We intend to use this HAC vector to rescue T and B cell development and the radiosensitive phenotype in the DNA-PKcs-deficient SCID (severe combined immunodeficiency) mice. Our approach will involve introduction of 21ΔqHAC containing the DNA-PKcs gene into DNA-PKcs-deficient ES cells, differentiation of corrected ES cells into blood stem cells and transplantation of these blood stem cells into SCID mice. To evaluate the usefulness of 21ΔqHAC vector for this purpose, we first attempted in vitro studies using CHO cells (V3) exhibiting radiosensitive phenotype due to a DNA-PKcs deficiency. In this experiment, we inserted a ~21 kb doxycycline-inducible DNA-PKcs expression cassette into 21ΔqHAC vector by Cre-mediated recombination. The resulting 21ΔqHAC/DNA-PKcs construct was then introduced into the V3 cells. RT-PCR results from cells containing the 21ΔqHAC/DNA-PKcs vector showed doxycycline-dependent regulation of the DNA-PKcs gene. Furthermore, radiosensitivity assays showed significant phenotype rescue in these cells due to the production of functional DNA-PKcs protein. To our knowledge, our study is the first to report successful artificial control of gene expression by placing the multiple regulatory elements of the tetracycline system in a single expression cassette within a HAC vector. Our results also show that 21ΔqHAC is a very useful tool in the introduction of a therapeutic gene and the correction of a defective phenotype.

Structural analysis of DNA–PKcs: modelling of the repeat units and insights into the detailed molecular architecture

DNA-dependent protein kinase (DNA–PK) is part of the eukaryotic DNA double strand break repair pathway and as such is crucial for maintenance of genomic stability, as well as for V(D)J (variable-diversity-joining) recombination. The catalytic subunit of DNA–PK (DNA–PKcs) belongs to the phosphatidylinositol-3 (PI-3) kinase-like kinase (PIKK) superfamily and is comprised of ∼4100 amino acids. We have used a novel repeat detection method to analyse this enormous protein and have identified two different types of helical repeat motifs in the N-terminal region of the sequence, as well as other previously unreported features in this repeat region. A comparison with the ATMs, ATRs, and TORs show that the features identified are likely to be conserved throughout the PIKK superfamily. Homology modelling of parts of the DNA–PKcs sequence has been undertaken and we have been able to fit the models to previously obtained electron microscopy data. This work provides an insight into the overall architecture of the DNA–PKcs protein and identifies regions of interest for further experimental studies.

Protein-DNA Complexes Containing DNA-Dependent Protein Kinase in Crude Extracts from Human and Rodent Cells

The DNA-dependent protein kinase (DNA-PK) is composed of a large catalytic subunit (DNA-PKcs) and a DNA-binding protein, Ku. Cells lacking DNA-PK activity are radiosensitive and are defective in DNA double-strand break repair and V(D)J recombination. Although much information regarding the interactions of Ku with DNA ends is available, relatively little is known about the interaction of DNA-PKcs with DNA-bound Ku. Here we show, using electrophoretic mobility shift assays, that chemical crosslinkers enhance the formation of protein-DNA complexes containing DNA-PKcs, Ku and other proteins in extracts from cells of normal human cell lines. Extracts from cells of the radiosensitive human cell line M059J, which lacks DNA-PKcs, are not competent to form these protein-DNA complexes, while addition of purified DNA-PKcs protein restores complex formation. This assay may be useful for screening for DNA-PK function in cells of human cell lines and for identifying proteins that interact with the DNA-PK-DNA complex. We also show that Ku protein in rodent cells can interact with human DNA-PKcs; however, this assay may be less useful for studying Ku/DNA-PKcs interactions in cells of rodent cell lines due to the low abundance of DNA-PKcs in these cells.

Cryo-EM imaging of the catalytic subunit of the DNA-dependent protein kinase

The DNA-dependent protein kinase (DNA-PK) plays an important role in mammalian DNA double-strand break repair and immunoglobulin gene rearrangement. The DNA-PK holoenzyme is activated by assembly at DNA ends and is comprised of DNA-PKcs, a 460 kDa protein kinase catalytic subunit, and Ku, a 70 kDa/80 kDa heterodimeric DNA-targeting component. We have solved the three-dimensional structure of DNA-PKcs to ∼21 Å resolution by analytically combining images of nearly 9500 individual particles extracted from cryo-electron micrographs. The DNA-PKcs protein has an open, pseudo 2-fold symmetric structure with a gap separating a crown-shaped top from a rounded base. Columns of density are observed to protrude into the gap from both the crown and the base. Measurements of the enclosed volume indicate that the interior of the protein is largely hollow. The structure of DNA-PKcs suggests that its association with DNA may involve the internalization of double-stranded ends.

Molecular and biochemical characterisation of DNA-dependent protein kinase-defective rodent mutant irs-20

The catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs) is a member of a sub-family of phosphatidylinositol (PI) 3-kinases termed PIK-related kinases. A distinguishing feature of this sub-family is the presence of a conserved C-terminal region downstream of a PI 3-kinase domain. Mutants defective in DNA-PKcs are sensitive to ionising radiation and are unable to carry out V(D)J recombination. Irs-20 is a DNA-PKcs-defective cell line with milder gamma-ray sensitivity than two previously characterised mutants, V-3 and mouse scid cells. Here we show that the DNA-PKcs protein from irs-20 cells can bind to DNA but is unable to function as a protein kinase. To verify the defect in irs-20 cells and provide insight into the function and expression of DNA-PKcs in double-strand break repair and V(D)J recombination we introduced YACs encoding human and mouse DNA-PKcs into defective mutants and achieved complementation of the defective phenotypes. Furthermore, in irs-20 we identified a mutation in DNA-PKcs that causes substitution of a lysine for a glutamic acid in the fourth residue from the C-terminus. This represents a strong candidate for the inactivating mutation and provides supportive evidence that the extreme C-terminal motif is important for protein kinase activity.