BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment. METHODS: Gene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays. RESULTS: BUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t1/2, ~8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade. CONCLUSIONS: BUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

BUB1 regulates non-homologous end joining pathway to mediate radioresistance in triple-negative breast cancer.

Background: Triple-negative breast cancer (TNBC) is a highly aggressive form of breast cancer subtype often treated with radiotherapy (RT). Due to its intrinsic heterogeneity and lack of effective targets, it is crucial to identify novel molecular targets that would increase RT efficacy. Here we demonstrate the role of BUB1 (cell cycle Ser/Thr kinase) in TNBC radioresistance and offer a novel strategy to improve TNBC treatment. Methods: Gene expression analysis was performed to look at genes upregulated in TNBC patient samples compared to other subtypes. Cell proliferation and clonogenic survivals assays determined the IC 50 of BUB1 inhibitor (BAY1816032) and radiation enhancement ratio (rER) with pharmacologic and genomic BUB1 inhibition. Mammary fat pad xenografts experiments were performed in CB17/SCID. The mechanism through which BUB1 inhibitor sensitizes TNBC cells to radiotherapy was delineated by γ-H2AX foci assays, BLRR, Immunoblotting, qPCR, CHX chase, and cell fractionation assays. Results: BUB1 is overexpressed in BC and its expression is considerably elevated in TNBC with poor survival outcomes. Pharmacological or genomic ablation of BUB1 sensitized multiple TNBC cell lines to cell killing by radiation, although breast epithelial cells showed no radiosensitization with BUB1 inhibition. Kinase function of BUB1 is mainly accountable for this radiosensitization phenotype. BUB1 ablation also led to radiosensitization in TNBC tumor xenografts with significantly increased tumor growth delay and overall survival. Mechanistically, BUB1 ablation inhibited the repair of radiation-induced DNA double strand breaks (DSBs). BUB1 ablation stabilized phospho-DNAPKcs (S2056) following RT such that half-lives could not be estimated. In contrast, RT alone caused BUB1 stabilization, but pre-treatment with BUB1 inhibitor prevented stabilization (t 1/2 , ∼8 h). Nuclear and chromatin-enriched fractionations illustrated an increase in recruitment of phospho- and total-DNAPK, and KAP1 to chromatin indicating that BUB1 is indispensable in the activation and recruitment of non-homologous end joining (NHEJ) proteins to DSBs. Additionally, BUB1 staining of TNBC tissue microarrays demonstrated significant correlation of BUB1 protein expression with tumor grade. Conclusions: BUB1 ablation sensitizes TNBC cell lines and xenografts to RT and BUB1 mediated radiosensitization may occur through NHEJ. Together, these results highlight BUB1 as a novel molecular target for radiosensitization in women with TNBC.

AXL/WRNIP1 Mediates Replication Stress Response and Promotes Therapy Resistance and Metachronous Metastasis in HER2+ Breast Cancer.

Therapy resistance and metastatic progression are primary causes of cancer-related mortality. Disseminated tumor cells possess adaptive traits that enable them to reprogram their metabolism, maintain stemness, and resist cell death, facilitating their persistence to drive recurrence. The survival of disseminated tumor cells also depends on their ability to modulate replication stress in response to therapy while colonizing inhospitable microenvironments. In this study, we discovered that the nuclear translocation of AXL, a TAM receptor tyrosine kinase, and its interaction with WRNIP1, a DNA replication stress response factor, promotes the survival of HER2+ breast cancer cells that are resistant to HER2-targeted therapy and metastasize to the brain. In preclinical models, knocking down or pharmacologically inhibiting AXL or WRNIP1 attenuated protection of stalled replication forks. Furthermore, deficiency or inhibition of AXL and WRNIP1 also prolonged metastatic latency and delayed relapse. Together, these findings suggest that targeting the replication stress response, which is a shared adaptive mechanism in therapy-resistant and metastasis-initiating cells, could reduce metachronous metastasis and enhance the response to standard-of-care therapies. SIGNIFICANCE: Nuclear AXL and WRNIP1 interact and mediate replication stress response, promote therapy resistance, and support metastatic progression, indicating that targeting the AXL/WRNIP1 axis is a potentially viable therapeutic strategy for breast cancer.

The cancer testes antigen, HORMAD1, limits genomic instability in cancer cells by protecting stalled replication forks.

Tumors anomalously induce the expression of meiotic genes, which are otherwise restricted only to developing gametes. If and how these aberrantly expressed meiotic proteins influence DNA metabolism is not clear, but could have important implications for how tumors acquire and mitigate genomic instability. HORMAD1 is a highly conserved meiotic protein that is frequently expressed in lung adenocarincoma where its expression correlates with reduced patient survival and increased mutation burden. Here, we find that HORMAD1 associates with the replisome and is critical for protecting stalled DNA replication forks. Loss of HORMAD1 leads to nascent DNA strand degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. We find that these phenotypes are due to limited RAD51 loading onto stalled replication forks in the absence of HORMAD1. Ultimately, loss of HORMAD1 leads to increased DNA breaks and chromosomal defects, which is exacerbated dramatically by induction of replication stress. Tumor cells proliferate despite encountering chronic replication stress, placing them on the precipice of catastrophic genomic damage. Our data support the hypothesis that the aberrant expression of HORMAD1 is engaged to attenuate the accumulation of excessive DNA damage due to chronic replication stress, which may otherwise lead to accumulation of toxic levels of genomic instability.

DNA-PK is activated by SIRT2 deacetylation to promote DNA double-strand break repair by non-homologous end joining.

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. The interaction of the catalytic subunit of DNA-PK (DNA-PKcs) with the Ku70/Ku80 heterodimer on DSBs leads to DNA-PK activation; however, it is not known if upstream signaling events govern this activation. Here, we reveal a regulatory step governing DNA-PK activation by SIRT2 deacetylation, which facilitates DNA-PKcs localization to DSBs and interaction with Ku, thereby promoting DSB repair by NHEJ. SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR. SIRT2 deacetylase activity facilitates DNA-PKcs interaction with Ku and localization to DSBs and promotes DNA-PK activation and phosphorylation of downstream NHEJ substrates. Moreover, targeting SIRT2 with AGK2, a SIRT2-specific inhibitor, augments the efficacy of IR in cancer cells and tumors. Our findings define a regulatory step for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ. Furthermore, our data suggest that SIRT2 inhibition may be a promising rationale-driven therapeutic strategy for increasing the effectiveness of radiation therapy.

ATM phosphorylates the FATC domain of DNA-PKcs at threonine 4102 to promote non-homologous end joining.

Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double-stranded breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Ablating phosphorylation at T4102 attenuates DNA-PKcs kinase activity and this destabilizes the interaction between DNA-PKcs and the Ku-DNA complex, resulting in decreased assembly and stabilization of the NHEJ machinery at DSBs. Phosphorylation at T4102 promotes NHEJ, radioresistance, and increases genomic stability following DSB induction. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PKcs.

DNA-PKcs and ATM modulate mitochondrial ADP-ATP exchange as an oxidative stress checkpoint mechanism.

DNA-PKcs is a key regulator of DNA double-strand break repair. Apart from its canonical role in the DNA damage response, DNA-PKcs is involved in the cellular response to oxidative stress (OS), but its exact role remains unclear. Here, we report that DNA-PKcs-deficient human cells display depolarized mitochondria membrane potential (MMP) and reoriented metabolism, supporting a role for DNA-PKcs in oxidative phosphorylation (OXPHOS). DNA-PKcs directly interacts with mitochondria proteins ANT2 and VDAC2, and formation of the DNA-PKcs/ANT2/VDAC2 (DAV) complex supports optimal exchange of ADP and ATP across mitochondrial membranes to energize the cell via OXPHOS and to maintain MMP. Moreover, we demonstrate that the DAV complex temporarily dissociates in response to oxidative stress to attenuate ADP-ATP exchange, a rate-limiting step for OXPHOS. Finally, we found that dissociation of the DAV complex is mediated by phosphorylation of DNA-PKcs at its Thr2609 cluster by ATM kinase. Based on these findings, we propose that the coordination between the DAV complex and ATM serves as a novel oxidative stress checkpoint to decrease ROS production from mitochondrial OXPHOS and to hasten cellular recovery from OS.

ATM phosphorylates the FATC domain of DNA-PK cs at threonine 4102 to promote non-homologous end joining.

Ataxia-telangiectasia mutated (ATM) drives the DNA damage response via modulation of multiple signal transduction and DNA repair pathways. Previously, ATM activity was implicated in promoting the non-homologous end joining (NHEJ) pathway to repair a subset of DNA double strand breaks (DSBs), but how ATM performs this function is still unclear. In this study, we identified that ATM phosphorylates the DNA-dependent protein kinase catalytic subunit (DNA-PK cs ), a core NHEJ factor, at its extreme C-terminus at threonine 4102 (T4102) in response to DSBs. Phosphorylation at T4102 stabilizes the interaction between DNA-PK cs and the Ku-DNA complex and promotes assembly and stabilization of the NHEJ machinery at DSBs. Ablating phosphorylation at this site results in decreased NHEJ, radiosensitivity, and increased radiation-induced genomic instability. Collectively, these findings establish a key role for ATM in NHEJ-dependent repair of DSBs through positive regulation of DNA-PK cs .

The Cancer Testes Antigen, HORMAD1, is a Tumor-Specific Replication Fork Protection Factor.

Tumors frequently activate the expression of genes that are only otherwise required for meiosis. HORMAD1, which is essential for meiotic recombination in multiple species, is expressed in over 50% of human lung adenocarcinoma cells (LUAD). We previously found that HORMAD1 promotes DNA double strand break (DSB) repair in LUAD. Here, we report that HORMAD1 takes on an additional role in protecting genomic integrity. Specifically, we find HORMAD1 is critical for protecting stalled DNA replication forks in LUAD. Loss of HORMAD1 leads to nascent DNA degradation, an event which is mediated by the MRE11-DNA2-BLM pathway. Moreover, following exogenous induction of DNA replication stress, HORMAD1 deleted cells accumulate single stranded DNA (ssDNA). We find that these phenotypes are the result of a lack of RAD51 and BRCA2 loading onto stalled replication forks. Ultimately, loss of HORMAD1 leads to increased DSBs and chromosomal aberrations in response to replication stress. Collectively, our data support a model where HORMAD1 expression is selected to mitigate DNA replication stress, which would otherwise induce deleterious genomic instability.

DNA-PKcs-dependent phosphorylation of RECQL4 promotes NHEJ by stabilizing the NHEJ machinery at DNA double-strand breaks.

Non-homologous end joining (NHEJ) is the major pathway that mediates the repair of DNA double-strand breaks (DSBs) generated by ionizing radiation (IR). Previously, the DNA helicase RECQL4 was implicated in promoting NHEJ, but its role in the pathway remains unresolved. In this study, we report that RECQL4 stabilizes the NHEJ machinery at DSBs to promote repair. Specifically, we find that RECQL4 interacts with the NHEJ core factor DNA-PKcs and the interaction is increased following IR. RECQL4 promotes DNA end bridging mediated by DNA-PKcs and Ku70/80 in vitro and the accumulation/retention of NHEJ factors at DSBs in vivo. Moreover, interaction between DNA-PKcs and the other core NHEJ proteins following IR treatment is attenuated in the absence of RECQL4. These data indicate that RECQL4 promotes the stabilization of the NHEJ factors at DSBs to support formation of the NHEJ long-range synaptic complex. In addition, we observed that the kinase activity of DNA-PKcs is required for accumulation of RECQL4 to DSBs and that DNA-PKcs phosphorylates RECQL4 at six serine/threonine residues. Blocking phosphorylation at these sites reduced the recruitment of RECQL4 to DSBs, attenuated the interaction between RECQL4 and NHEJ factors, destabilized interactions between the NHEJ machinery, and resulted in decreased NHEJ. Collectively, these data illustrate reciprocal regulation between RECQL4 and DNA-PKcs in NHEJ.

Evaluation of the Response of HNSCC Cell Lines to γ-Rays and 12C Ions: Can Radioresistant Tumors Be Identified and Selected for 12C Ion Radiotherapy?

Head and neck squamous cell carcinoma (HNSCC) is the sixth most common malignancy worldwide. Thirty percent of patients will experience locoregional recurrence for which median survival is less than 1 year. Factors contributing to treatment failure include inherent resistance to X-rays and chemotherapy, hypoxia, epithelial to mesenchymal transition, and immune suppression. The unique properties of 12C radiotherapy including enhanced cell killing, a decreased oxygen enhancement ratio, generation of complex DNA damage, and the potential to overcome immune suppression make its application well suited to the treatment of HNSCC. We examined the 12C radioresponse of five HNSCC cell lines, whose surviving fraction at 3.5 Gy ranged from average to resistant when compared with a larger panel of 38 cell lines to determine if 12C irradiation can overcome X-ray radioresistance and to identify biomarkers predictive of 12C radioresponse. Cells were irradiated with 12C using a SOBP with an average LET of 80 keV/µm (CNAO: Pavia, Italy). RBE values varied depending upon endpoint used. A 37 gene signature was able to place cells in their respective radiosensitivity cohort with an accuracy of 86%. Radioresistant cells were characterized by an enrichment of genes associated with radioresistance and survival mechanisms including but not limited to G2/M Checkpoint MTORC1, HIF1α, and PI3K/AKT/MTOR signaling. These data were used in conjunction with an in silico-based modeling approach to evaluate tumor control probability after 12C irradiation that compared clinically used treatment schedules with fixed RBE values vs. the RBEs determined for each cell line. Based on the above analysis, we present the framework of a strategy to utilize biological markers to predict which HNSCC patients would benefit the most from 12C radiotherapy.

Restoration of DNA repair mitigates genome instability and increases productivity of Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells are the primary host for manufacturing of therapeutic proteins. However, productivity loss is a major problem and is associated with genome instability, as chromosomal aberrations reduce transgene copy number and decrease protein expression. We analyzed whole-genome sequencing data from 11 CHO cell lines and found deleterious single-nucleotide variants in DNA repair genes. Comparison with primary Chinese hamster cells confirmed DNA repair to be compromised in CHO. Correction of key DNA repair genes by single-nucleotide variant reversal or expression of intact complementary DNAs successfully improved DNA repair and mitigated karyotypic instability. Moreover, overexpression of intact copies of LIG4 and XRCC6 in a CHO cell line expressing secreted alkaline phosphatase mitigated transgene copy loss and improved protein titer retention. These results show that correction of DNA repair genes yields improvements in genome stability in CHO, and provide new opportunities for cell line development for sustainable protein expression.

Corrigendum: Hepatogenous photosensitisation in cows grazing turnips (Brassica rapa) in South Africa.

No abstract available.

Ablating putative Ku70 phosphorylation sites results in defective DNA damage repair and spontaneous induction of hepatocellular carcinoma.

Multiple pathways mediate the repair of DNA double-strand breaks (DSBs), with numerous mechanisms responsible for driving choice between the pathways. Previously, we reported that mutating five putative phosphorylation sites on the non-homologous end joining (NHEJ) factor, Ku70, results in sustained retention of human Ku70/80 at DSB ends and attenuation of DSB repair via homologous recombination (HR). In this study, we generated a knock-in mouse, in which the three conserved putative phosphorylation sites of Ku70 were mutated to alanine to ablate potential phosphorylation (Ku703A/3A), in order to examine if disrupting DSB repair pathway choice by modulating Ku70/80 dynamics at DSB ends results in enhanced genomic instability and tumorigenesis. The Ku703A/3A mice developed spontaneous and have accelerated chemical-induced hepatocellular carcinoma (HCC) compared to wild-type (Ku70+/+) littermates. The HCC tumors from the Ku703A/3A mice have increased γH2AX and 8-oxo-G staining, suggesting decreased DNA repair. Spontaneous transformed cell lines from Ku703A/3A mice are more radiosensitive, have a significant decrease in DNA end resection, and are more sensitive to the DNA cross-linking agent mitomycin C compared to cells from Ku70+/+ littermates. Collectively, these findings demonstrate that mutating the putative Ku70 phosphorylation sites results in defective DNA damage repair and disruption of this process drives genomic instability and accelerated development of HCC.

Hepatogenous photosensitisation in cows grazing turnips (Brassica rapa) in South Africa.

Holstein cows on a farm in the Humansdorp district, Eastern Cape province, South Africa, developed reddened, painful teat skin 3 days after grazing a mixed forage crop dominated by bulb turnip (Brassica rapa, Barkant cultivar). The crop was grazed 45 days after planting and 10% of the herd developed symptoms. More characteristic non-pigmented skin lesions started manifesting 1-2 days after the appearance of the teat lesions. Affected cows had elevated serum activities of gamma-glutamyl transferase, glutamate dehydrogenase and aspartate aminotransferase. These blood chemistry findings confirmed a secondary (hepatogenous) photosensitivity. As a result of the severity of the teat and skin lesions, seven cows were slaughtered and tissue samples from five of them were collected for histopathological examination. Liver lesions in cows that were culled 3 or more weeks after the onset of the outbreak showed oedematous concentric fibrosis around medium-sized bile ducts and inflammatory infiltrates in portal tracts. Characteristic lesions associated with other known hepatobiliary toxicities were not found. No new cases were reported 5 days after the cattle were removed from the turnips. The sudden introduction of the cows, without any period of transitioning or adaptation to grazing turnips, as well as the short latent period, clinical signs of photosensitisation, blood chemistry and histopathology, confirmed a diagnosis of Brassica-associated liver disease, a condition seen in New Zealand but not previously described in South Africa. Brassica forage crops are potentially toxic under certain conditions and farmers must be aware of these risks.

Human RecQ Helicases in DNA Double-Strand Break Repair.

RecQ DNA helicases are a conserved protein family found in bacteria, fungus, plants, and animals. These helicases play important roles in multiple cellular functions, including DNA replication, transcription, DNA repair, and telomere maintenance. Humans have five RecQ helicases: RECQL1, Bloom syndrome protein (BLM), Werner syndrome helicase (WRN), RECQL4, and RECQL5. Defects in BLM and WRN cause autosomal disorders: Bloom syndrome (BS) and Werner syndrome (WS), respectively. Mutations in RECQL4 are associated with three genetic disorders, Rothmund-Thomson syndrome (RTS), Baller-Gerold syndrome (BGS), and RAPADILINO syndrome. Although no genetic disorders have been reported due to loss of RECQL1 or RECQL5, dysfunction of either gene is associated with tumorigenesis. Multiple genetically independent pathways have evolved that mediate the repair of DNA double-strand break (DSB), and RecQ helicases play pivotal roles in each of them. The importance of DSB repair is supported by the observations that defective DSB repair can cause chromosomal aberrations, genomic instability, senescence, or cell death, which ultimately can lead to premature aging, neurodegeneration, or tumorigenesis. In this review, we will introduce the human RecQ helicase family, describe in detail their roles in DSB repair, and provide relevance between the dysfunction of RecQ helicases and human diseases.

MLH1 Deficiency-Triggered DNA Hyperexcision by Exonuclease 1 Activates the cGAS-STING Pathway.

Tumors with defective mismatch repair (dMMR) are responsive to immunotherapy because of dMMR-induced neoantigens and activation of the cGAS-STING pathway. While neoantigens result from the hypermutable nature of dMMR, it is unknown how dMMR activates the cGAS-STING pathway. We show here that loss of the MutLα subunit MLH1, whose defect is responsible for ~50% of dMMR cancers, results in loss of MutLα-specific regulation of exonuclease 1 (Exo1) during DNA repair. This leads to unrestrained DNA excision by Exo1, which causes increased single-strand DNA formation, RPA exhaustion, DNA breaks, and aberrant DNA repair intermediates. Ultimately, this generates chromosomal abnormalities and the release of nuclear DNA into the cytoplasm, activating the cGAS-STING pathway. In this study, we discovered a hitherto unknown MMR mechanism that modulates genome stability and has implications for cancer therapy.

Mechanism of efficient double-strand break repair by a long non-coding RNA.

Mechanistic studies in DNA repair have focused on roles of multi-protein DNA complexes, so how long non-coding RNAs (lncRNAs) regulate DNA repair is less well understood. Yet, lncRNA LINP1 is over-expressed in multiple cancers and confers resistance to ionizing radiation and chemotherapeutic drugs. Here, we unveil structural and mechanistic insights into LINP1's ability to facilitate non-homologous end joining (NHEJ). We characterized LINP1 structure and flexibility and analyzed interactions with the NHEJ factor Ku70/Ku80 (Ku) and Ku complexes that direct NHEJ. LINP1 self-assembles into phase-separated condensates via RNA-RNA interactions that reorganize to form filamentous Ku-containing aggregates. Structured motifs in LINP1 bind Ku, promoting Ku multimerization and stabilization of the initial synaptic event for NHEJ. Significantly, LINP1 acts as an effective proxy for PAXX. Collective results reveal how lncRNA effectively replaces a DNA repair protein for efficient NHEJ with implications for development of resistance to cancer therapy.

DNA-PKcs promotes chromatin decondensation to facilitate initiation of the DNA damage response.

The DNA damage response (DDR) encompasses the cellular response to DNA double-stranded breaks (DSBs), and includes recognition of the DSB, recruitment of numerous factors to the DNA damage site, initiation of signaling cascades, chromatin remodeling, cell-cycle checkpoint activation, and repair of the DSB. Key drivers of the DDR are multiple members of the phosphatidylinositol 3-kinase-related kinase family, including ataxia telangiectasia mutated (ATM), ataxia telangiectasia and Rad3-related (ATR), and the DNA-dependent protein kinase catalytic subunit (DNA-PKcs). ATM and ATR modulate multiple portions of the DDR, but DNA-PKcs is believed to primarily function in the DSB repair pathway, non-homologous end joining. Utilizing a human cell line in which the kinase domain of DNA-PKcs is inactivated, we show here that DNA-PKcs kinase activity is required for the cellular response to DSBs immediately after their induction. Specifically, DNA-PKcs kinase activity initiates phosphorylation of the chromatin factors H2AX and KAP1 following ionizing radiation exposure and drives local chromatin decondensation near the DSB site. Furthermore, loss of DNA-PKcs kinase activity results in a marked decrease in the recruitment of numerous members of the DDR machinery to DSBs. Collectively, these results provide clear evidence that DNA-PKcs activity is pivotal for the initiation of the DDR.