High-complexity of DNA double-strand breaks is key for alternative end-joining choice.

The repair of DNA double-strand breaks (DSBs) through alternative non-homologous end-joining (alt-NHEJ) pathway significantly contributes to genetic instability. However, the mechanism governing alt-NHEJ pathway choice, particularly its association with DSB complexity, remains elusive due to the absence of a suitable reporter system. In this study, we established a unique Escherichia coli reporter system for detecting complex DSB-initiated alternative end-joining (A-EJ), an alt-NHEJ-like pathway. By utilizing various types of ionizing radiation to generate DSBs with varying degrees of complexity, we discovered that high complexity of DSBs might be a determinant for A-EJ choice. To facilitate efficient repair of high-complexity DSBs, A-EJ employs distinct molecular patterns such as longer micro-homologous junctions and non-templated nucleotide addition. Furthermore, the A-EJ choice is modulated by the degree of homology near DSB loci, competing with homologous recombination machinery. These findings further enhance the understanding of A-EJ/alt-NHEJ pathway choice.

PD-L1 deglycosylation promotes its nuclear translocation and accelerates DNA double-strand-break repair in cancer.

Resistance to radiotherapy is a major barrier during cancer treatment. Here using genome-scale CRISPR/Cas9 screening, we identify CD274 gene, which encodes PD-L1, to confer lung cancer cell resistance to ionizing radiation (IR). Depletion of endogenous PD-L1 delays the repair of IR-induced DNA double-strand breaks (DSBs) and PD-L1 loss downregulates non-homologous end joining (NHEJ) while overexpression of PD-L1 upregulates NHEJ. IR induces translocation of PD-L1 from the membrane into nucleus dependent on deglycosylation of PD-L1 at N219 and CMTM6 and leads to PD-L1 recruitment to DSBs foci. PD-L1 interacts with Ku in the nucleus and enhances Ku binding to DSB DNA. The interaction between the IgC domain of PD-L1 and the core domain of Ku is required for PD-L1 to accelerate NHEJ-mediated DSB repair and produce radioresistance. Thus, PD-L1, in addition to its immune inhibitory activity, acts as mechanistic driver for NHEJ-mediated DSB repair in cancer.

Enhancing homology-directed repair efficiency with HDR-boosting modular ssDNA donor.

Despite the potential of small molecules and recombinant proteins to enhance the efficiency of homology-directed repair (HDR), single-stranded DNA (ssDNA) donors, as currently designed and chemically modified, remain suboptimal for precise gene editing. Here, we screen the biased ssDNA binding sequences of DNA repair-related proteins and engineer RAD51-preferred sequences into HDR-boosting modules for ssDNA donors. Donors with these modules exhibit an augmented affinity for RAD51, thereby enhancing HDR efficiency across various genomic loci and cell types when cooperated with Cas9, nCas9, and Cas12a. By combining with an inhibitor of non-homologous end joining (NHEJ) or the HDRobust strategy, these modular ssDNA donors achieve up to 90.03% (median 74.81%) HDR efficiency. The HDR-boosting modules targeting an endogenous protein enable a chemical modification-free strategy to improve the efficacy of ssDNA donors for precise gene editing.

The Fanconi anemia core complex promotes CtIP-dependent end resection to drive homologous recombination at DNA double-strand breaks.

During the repair of interstrand crosslinks (ICLs) a DNA double-strand break (DSB) is generated. The Fanconi anemia (FA) core complex, which is recruited to ICLs, promotes high-fidelity repair of this DSB by homologous recombination (HR). However, whether the FA core complex also promotes HR at ICL-independent DSBs, for example induced by ionizing irradiation or nucleases, remains controversial. Here, we identified the FA core complex members FANCL and Ube2T as HR-promoting factors in a CRISPR/Cas9-based screen. Using isogenic cell line models, we further demonstrated an HR-promoting function of FANCL and Ube2T, and of their ubiquitination substrate FANCD2. We show that FANCL and Ube2T localize at DSBs in a FANCM-dependent manner, and are required for the DSB accumulation of FANCD2. Mechanistically, we demonstrate that FANCL ubiquitin ligase activity is required for the accumulation of CtIP at DSBs, thereby promoting end resection and Rad51 loading. Together, these data demonstrate a dual genome maintenance function of the FA core complex and FANCD2 in promoting repair of both ICLs and DSBs.

Cyclin F-EXO1 axis controls cell cycle-dependent execution of double-strand break repair.

Ubiquitination is a crucial posttranslational modification required for the proper repair of DNA double-strand breaks (DSBs) induced by ionizing radiation (IR). DSBs are mainly repaired through homologous recombination (HR) when template DNA is present and nonhomologous end joining (NHEJ) in its absence. In addition, microhomology-mediated end joining (MMEJ) and single-strand annealing (SSA) provide backup DSBs repair pathways. However, the mechanisms controlling their use remain poorly understood. By using a high-resolution CRISPR screen of the ubiquitin system after IR, we systematically uncover genes required for cell survival and elucidate a critical role of the E3 ubiquitin ligase SCFcyclin F in cell cycle-dependent DSB repair. We show that SCFcyclin F-mediated EXO1 degradation prevents DNA end resection in mitosis, allowing MMEJ to take place. Moreover, we identify a conserved cyclin F recognition motif, distinct from the one used by other cyclins, with broad implications in cyclin specificity for cell cycle control.

Dual activities of an X-family DNA polymerase regulate CRISPR-induced insertional mutagenesis across species.

The canonical non-homologous end joining (c-NHEJ) repair pathway, generally viewed as stochastic, has recently been shown to produce predictable outcomes in CRISPR-Cas9 mutagenesis. This predictability, mainly in 1-bp insertions and small deletions, has led to the development of in-silico prediction programs for various animal species. However, the predictability of CRISPR-induced mutation profiles across species remained elusive. Comparing CRISPR-Cas9 repair outcomes between human and plant species reveals significant differences in 1-bp insertion profiles. The high predictability observed in human cells links to the template-dependent activity of human Polλ. Yet plant Polλ exhibits dual activities, generating 1-bp insertions through both templated and non-templated manners. Polλ knockout in plants leads to deletion-only mutations, while its overexpression enhances 1-bp insertion rates. Two conserved motifs are identified to modulate plant Polλ's dual activities. These findings unveil the mechanism behind species-specific CRISPR-Cas9-induced insertion profiles and offer strategies for predictable, precise genome editing through c-NHEJ.

Identification and Validation of New DNA-PKcs Inhibitors through High-Throughput Virtual Screening and Experimental Verification.

DNA-PKcs is a crucial protein target involved in DNA repair and response pathways, with its abnormal activity closely associated with the occurrence and progression of various cancers. In this study, we employed a deep learning-based screening and molecular dynamics (MD) simulation-based pipeline, identifying eight candidates for DNA-PKcs targets. Subsequent experiments revealed the effective inhibition of DNA-PKcs-mediated cell proliferation by three small molecules (5025-0002, M769-1095, and V008-1080). These molecules exhibited anticancer activity with IC50 (inhibitory concentration at 50%) values of 152.6 µM, 30.71 µM, and 74.84 µM, respectively. Notably, V008-1080 enhanced homology-directed repair (HDR) mediated by CRISPR/Cas9 while inhibiting non-homologous end joining (NHEJ) efficiency. Further investigations into the structure-activity relationships unveiled the binding sites and critical interactions between these small molecules and DNA-PKcs. This is the first application of DeepBindGCN_RG in a real drug screening task, and the successful discovery of a novel DNA-PKcs inhibitor demonstrates its efficiency as a core component in the screening pipeline. Moreover, this study provides important insights for exploring novel anticancer therapeutics and advancing the development of gene editing techniques by targeting DNA-PKcs.

Non-homologous end joining shapes the genomic rearrangement landscape of chromothripsis from mitotic errors.

Mitotic errors generate micronuclei entrapping mis-segregated chromosomes, which are susceptible to catastrophic fragmentation through chromothripsis. The reassembly of fragmented chromosomes by error-prone DNA double-strand break (DSB) repair generates diverse genomic rearrangements associated with human diseases. How specific repair pathways recognize and process these lesions remains poorly understood. Here we use CRISPR/Cas9 to systematically inactivate distinct DSB repair pathways and interrogate the rearrangement landscape of fragmented chromosomes. Deletion of canonical non-homologous end joining (NHEJ) components substantially reduces complex rearrangements and shifts the rearrangement landscape toward simple alterations without the characteristic patterns of chromothripsis. Following reincorporation into the nucleus, fragmented chromosomes localize within sub-nuclear micronuclei bodies (MN bodies) and undergo ligation by NHEJ within a single cell cycle. In the absence of NHEJ, chromosome fragments are rarely engaged by alternative end-joining or recombination-based mechanisms, resulting in delayed repair kinetics, persistent 53BP1-labeled MN bodies, and cell cycle arrest. Thus, we provide evidence supporting NHEJ as the exclusive DSB repair pathway generating complex rearrangements from mitotic errors.

HLTF disrupts Cas9-DNA post-cleavage complexes to allow DNA break processing.

The outcome of CRISPR-Cas-mediated genome modifications is dependent on DNA double-strand break (DSB) processing and repair pathway choice. Homology-directed repair (HDR) of protein-blocked DSBs requires DNA end resection that is initiated by the endonuclease activity of the MRE11 complex. Using reconstituted reactions, we show that Cas9 breaks are unexpectedly not directly resectable by the MRE11 complex. In contrast, breaks catalyzed by Cas12a are readily processed. Cas9, unlike Cas12a, bridges the broken ends, preventing DSB detection and processing by MRE11. We demonstrate that Cas9 must be dislocated after DNA cleavage to allow DNA end resection and repair. Using single molecule and bulk biochemical assays, we next find that the HLTF translocase directly removes Cas9 from broken ends, which allows DSB processing by DNA end resection or non-homologous end-joining machineries. Mechanistically, the activity of HLTF requires its HIRAN domain and the release of the 3'-end generated by the cleavage of the non-target DNA strand by the Cas9 RuvC domain. Consequently, HLTF removes the H840A but not the D10A Cas9 nickase. The removal of Cas9 H840A by HLTF explains the different cellular impact of the two Cas9 nickase variants in human cells, with potential implications for gene editing.

DSB-induced oxidative stress: Uncovering crosstalk between DNA damage response and cellular metabolism.

While that ROS causes DNA damage is well documented, there has been limited investigation into whether DNA damages and their repair processes can conversely induce oxidative stress. By generating a site-specific DNA double strand break (DSB) via I-SceI endonuclease expression in S. cerevisiae without damaging other cellular components, this study demonstrated that DNA repair does trigger oxidative stress. Deleting genes participating in the initiation of the resection step of homologous recombination (HR), like the MRX complex, resulted in stimulation of ROS. In contrast, deleting genes acting downstream of HR resection suppressed ROS levels. Additionally, blocking non-homologous end joining (NHEJ) also suppressed ROS. Further analysis identified Rad53 as a key player that relays DNA damage signals to alter redox metabolism in an HR-specific manner. These results suggest both HR and NHEJ can drive metabolism changes and oxidative stress, with NHEJ playing a more prominent role in ROS stimulation. Further analysis revealed a correlation between DSB-induced ROS increase and enhanced activity of NADPH oxidase Yno1 and various antioxidant enzymes. Deleting the antioxidant gene SOD1 induced synthetic lethality in HR-deficient mutants like mre11Δ and rad51Δ upon DSB induction. These findings uncover a significant interplay between DNA repair mechanisms and cellular metabolism, providing insights into understanding the side effects of genotoxic therapies and potentially aiding development of more effective cancer treatment strategies.

Current status and prospect of the DNA double-strand break repair pathway in colorectal cancer development and treatment.

Colorectal cancer (CRC) is one of the most common malignancies worldwide. Double-strand break (DSB) is the most severe type of DNA damage. However, few reviews have thoroughly examined the involvement of DSB in CRC. Latest researches demonstrated that DSB repair plays an important role in CRC. For example, DSB-related genes such as BRCA1, Ku-70 and DNA polymerase theta (POLQ) are associated with the occurrence of CRC, and POLQ even showed to affect the prognosis and resistance for radiotherapy in CRC. This review comprehensively summarizes the DSB role in CRC, explores the mechanisms and discusses the association with CRC treatment. Four pathways for DSB have been demonstrated. 1. Nonhomologous end joining (NHEJ) is the major pathway. Its core genes including Ku70 and Ku80 bind to broken ends and recruit repair factors to form a complex that mediates the connection of DNA breaks. 2. Homologous recombination (HR) is another important pathway. Its key genes including BRCA1 and BRCA2 are involved in finding, pairing, and joining broken ends, and ensure the restoration of breaks in a normal double-stranded DNA structure. 3. Single-strand annealing (SSA) pathway, and 4. POLθ-mediated end-joining (alt-EJ) is a backup pathway. This paper elucidates roles of the DSB repair pathways in CRC, which could contribute to the development of potential new treatment approaches and provide new opportunities for CRC treatment and more individualized treatment options based on therapeutic strategies targeting these DNA repair pathways.

DNA-PK: A synopsis beyond synapsis.

Given its central role in life, DNA is remarkably easy to damage. Double strand breaks (DSBs) are the most toxic form of DNA damage, and DSBs pose the greatest danger to genomic integrity. In higher vertebrates, the non-homologous end joining pathway (NHEJ) is the predominate pathway that repairs DSBs. NHEJ has three steps: 1) DNA end recognition by the DNA dependent protein kinase [DNA-PK], 2) DNA end-processing by numerous NHEJ accessory factors, and 3) DNA end ligation by the DNA ligase IV complex (LX4). Although this would appear to be a relatively simple mechanism, it has become increasingly apparent that it is not. Recently, much insight has been derived regarding the mechanism of non-homologous end joining through a proliferation of cryo-EM studies, structure-function mutational experiments informed by these new structural data, and novel single-molecule imaging approaches. An emerging consensus in the field is that NHEJ progresses from initial DSB end recognition by DNA-PK to synapsis of the two DNA ends in a long-range synaptic complex where ends are held too far apart (115 Å) for ligation, and then progress to a short-range synaptic complex where ends are positioned close enough for ligation. What was surprising from these structural studies was the observation of two distinct types of DNA-PK dimers that represent NHEJ long-range complexes. In this review, we summarize current knowledge about the function of the distinct NHEJ synaptic complexes and align this new information with emerging cellular single-molecule microscopy studies as well as with previous studies of DNA-PK's function in repair.

ATM and 53BP1 regulate alternative end joining-mediated V(D)J recombination.

G0-G1 phase alternative end joining (A-EJ) is a recently defined mutagenic pathway characterized by resected deletion and translocation joints that are predominantly direct and are distinguished from A-EJ in cycling cells that rely much more on microhomology-mediated end joining (MMEJ). Using chemical and genetic approaches, we systematically evaluate potential A-EJ factors and DNA damage response (DDR) genes to support this mechanism by mapping the repair fates of RAG1/2-initiated double-strand breaks in the context of Igκ locus V-J recombination and chromosome translocation. Our findings highlight a polymerase theta-independent Parp1-XRCC1/LigIII axis as central A-EJ components, supported by 53BP1 in the context of an Ataxia-telangiectasia mutated (ATM)-activated DDR. Mechanistically, we demonstrate varied changes in short-range resection, MMEJ, and translocation, imposed by compromising specific DDR activities, which include polymerase alpha, Ataxia-telangiectasia and Rad3-related (ATR), DNA2, and Mre11. This study advances our understanding of DNA damage repair within the 53BP1 regulatory domain and the RAG1/2 postcleavage complex.

A Genomewide Evolution-Based CRISPR/Cas9 with Donor-Free (GEbCD) for Developing Robust and Productive Industrial Yeast.

Developing more robust and productive industrial yeast is crucial for high-efficiency biomanufacturing. However, the challenges posed by the long time required and the low abundance of mutations generated through genomewide evolutionary engineering hinder the development and optimization of desired hosts for industrial applications. To address these issues, we present a novel solution called the Genomewide Evolution-based CRISPR/Cas with Donor-free (GEbCD) system, in which nonhomologous-end-joining (NHEJ) repair can accelerate the acquisition of highly abundant yeast mutants. Together with modified rad52 of the DNA double-strand break repair in Saccharomyces cerevisiae, a hypermutation host was obtained with a 400-fold enhanced mutation ability. Under multiple environmental stresses the system could rapidly generate millions of mutants in a few rounds of iterative evolution. Using high-throughput screening, an industrial S. cerevisiae SISc-Δrad52-G4-72 (G4-72) was obtained that is strongly robust and has higher productivity. G4-72 grew stably and produced ethanol efficiently in multiple-stress environments, e.g. high temperature and high osmosis. In a pilot-scale fermentation with G4-72, the fermentation temperature was elevated by 8 °C and ethanol production was increased by 6.9% under the multiple stresses posed by the industrial fermentation substrate. Overall, the GEbCD system presents a powerful tool to rapidly generate abundant mutants and desired hosts, and offers a novel strategy for optimizing microbial chassis with regard to demands posed in industrial applications.

PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks.

DNA polymerase theta (Polθ)-mediated end-joining (TMEJ) repairs DNA double-strand breaks and confers resistance to genotoxic agents. How Polθ is regulated at the molecular level to exert TMEJ remains poorly characterized. We find that Polθ interacts with and is PARylated by PARP1 in a HPF1-independent manner. PARP1 recruits Polθ to the vicinity of DNA damage via PARylation dependent liquid demixing, however, PARylated Polθ cannot perform TMEJ due to its inability to bind DNA. PARG-mediated de-PARylation of Polθ reactivates its DNA binding and end-joining activities. Consistent with this, PARG is essential for TMEJ and the temporal recruitment of PARG to DNA damage corresponds with TMEJ activation and dissipation of PARP1 and PAR. In conclusion, we show a two-step spatiotemporal mechanism of TMEJ regulation. First, PARP1 PARylates Polθ and facilitates its recruitment to DNA damage sites in an inactivated state. PARG subsequently activates TMEJ by removing repressive PAR marks on Polθ.

Up-Regulation of Non-Homologous End-Joining by MUC1.

Ionizing radiation (IR) and chemotherapy with DNA-damaging drugs such as cisplatin are vital cancer treatment options. These treatments induce double-strand breaks (DSBs) as cytotoxic DNA damage; thus, the DSB repair activity in each cancer cell significantly influences the efficacy of the treatments. Pancreatic cancers are known to be resistant to these treatments, and the overexpression of MUC1, a member of the glycoprotein mucins, is associated with IR- and chemo-resistance. Therefore, we investigated the impact of MUC1 on DSB repair. This report examined the effect of the overexpression of MUC1 on homologous recombination (HR) and non-homologous end-joining (NHEJ) using cell-based DSB repair assays. In addition, the therapeutic potential of NHEJ inhibitors including HDAC inhibitors was also studied using pancreatic cancer cell lines. The MUC1-overexpression enhances NHEJ, while partially suppressing HR. Also, MUC1-overexpressed cancer cell lines are preferentially killed by a DNA-PK inhibitor and HDAC1/2 inhibitors. Altogether, MUC1 induces metabolic changes that create an imbalance between NHEJ and HR activities, and this imbalance can be a target for selective killing by HDAC inhibitors. This is a novel mechanism of MUC1-mediated IR-resistance and will form the basis for targeting MUC1-overexpressed pancreatic cancer.

Polymerase theta is a synthetic lethal target for killing Epstein-Barr virus lymphomas.

Treatment options for Epstein-Barr virus (EBV)-cancers are limited, underscoring the need for new therapeutic approaches. We have previously shown that EBV-transformed cells and cancers lack homologous recombination (HR) repair, a prominent error-free pathway that repairs double-stranded DNA breaks; instead, EBV-transformed cells demonstrate genome-wide scars of the error-prone microhomology-mediated end joining (MMEJ) repair pathway. This suggests that EBV-cancers are vulnerable to synthetic lethal therapeutic approaches that target MMEJ repair. Indeed, we have previously found that targeting PARP, an enzyme that contributes to MMEJ, results in the death of EBV-lymphoma cells. With the emergence of clinical resistance to PARP inhibitors and the recent discovery of inhibitors of Polymerase theta (POLθ), the polymerase essential for MMEJ, we investigated the role of POLθ in EBV-lymphoma cells. We report that EBV-transformed cell lines, EBV-lymphoma cell lines, and EBV-lymphomas in AIDS patients demonstrate greater abundance of POLθ, driven by the EBV protein EBNA1, compared to EBV-uninfected primary lymphocytes and EBV-negative lymphomas from AIDS patients (a group that also abundantly expresses POLθ). We also find POLθ enriched at cellular DNA replication forks and exposure to the POLθ inhibitor Novobiocin impedes replication fork progress, impairs MMEJ-mediated repair of DNA double-stranded breaks, and kills EBV-lymphoma cells. Notably, cell killing is not due to Novobiocin-induced activation of the lytic/replicative phase of EBV. These findings support a role for POLθ not just in DNA repair but also DNA replication and as a therapeutic target in EBV-lymphomas and potentially other EBV-cancers as EBNA1 is expressed in all EBV-cancers.IMPORTANCEEpstein-Barr virus (EBV) contributes to ~2% of the global cancer burden. With a recent estimate of >200,000 deaths a year, identifying molecular vulnerabilities will be key to the management of these frequently aggressive and treatment-resistant cancers. Building on our earlier work demonstrating reliance of EBV-cancers on microhomology-mediated end-joining repair, we now report that EBV lymphomas and transformed B cell lines abundantly express the MMEJ enzyme POLθ that likely protects cellular replication forks and repairs replication-related cellular DNA breaks. Importantly also, we show that a newly identified POLθ inhibitor kills EBV-cancer cells, revealing a novel strategy to block DNA replication and repair of these aggressive cancers.

Di- and tri-methylation of histone H3K36 play distinct roles in DNA double-strand break repair.

Histone H3 Lys36 (H3K36) methylation and its associated modifiers are crucial for DNA double-strand break (DSB) repair, but the mechanism governing whether and how different H3K36 methylation forms impact repair pathways is unclear. Here, we unveil the distinct roles of H3K36 dimethylation (H3K36me2) and H3K36 trimethylation (H3K36me3) in DSB repair via non-homologous end joining (NHEJ) or homologous recombination (HR). Yeast cells lacking H3K36me2 or H3K36me3 exhibit reduced NHEJ or HR efficiency. yKu70 and Rfa1 bind H3K36me2- or H3K36me3-modified peptides and chromatin, respectively. Disrupting these interactions impairs yKu70 and Rfa1 recruitment to damaged H3K36me2- or H3K36me3-rich loci, increasing DNA damage sensitivity and decreasing repair efficiency. Conversely, H3K36me2-enriched intergenic regions and H3K36me3-enriched gene bodies independently recruit yKu70 or Rfa1 under DSB stress. Importantly, human KU70 and RPA1, the homologs of yKu70 and Rfa1, exclusively associate with H3K36me2 and H3K36me3 in a conserved manner. These findings provide valuable insights into how H3K36me2 and H3K36me3 regulate distinct DSB repair pathways, highlighting H3K36 methylation as a critical element in the choice of DSB repair pathway.

Characterization and regulation of cell cycle-independent noncanonical gene targeting.

Homology-dependent targeted DNA integration, generally referred to as gene targeting, provides a powerful tool for precise genome modification; however, its fundamental mechanisms remain poorly understood in human cells. Here we reveal a noncanonical gene targeting mechanism that does not rely on the homologous recombination (HR) protein Rad51. This mechanism is suppressed by Rad52 inhibition, suggesting the involvement of single-strand annealing (SSA). The SSA-mediated gene targeting becomes prominent when DSB repair by HR or end-joining pathways is defective and does not require isogenic DNA, permitting 5% sequence divergence. Intriguingly, loss of Msh2, loss of BLM, and induction of a target-site DNA break all significantly and synergistically enhance SSA-mediated targeted integration. Most notably, SSA-mediated integration is cell cycle-independent, occurring in the G1 phase as well. Our findings provide unequivocal evidence for Rad51-independent targeted integration and unveil multiple mechanisms to regulate SSA-mediated targeted as well as random integration.

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.