An autoinhibited state of 53BP1 revealed by small molecule antagonists and protein engineering.

The recruitment of 53BP1 to chromatin, mediated by its recognition of histone H4 dimethylated at lysine 20 (H4K20me2), is important for DNA double-strand break repair. Using a series of small molecule antagonists, we demonstrate a conformational equilibrium between an open and a pre-existing lowly populated closed state of 53BP1 in which the H4K20me2 binding surface is buried at the interface between two interacting 53BP1 molecules. In cells, these antagonists inhibit the chromatin recruitment of wild type 53BP1, but do not affect 53BP1 variants unable to access the closed conformation despite preservation of the H4K20me2 binding site. Thus, this inhibition operates by shifting the conformational equilibrium toward the closed state. Our work therefore identifies an auto-associated form of 53BP1-autoinhibited for chromatin binding-that can be stabilized by small molecule ligands encapsulated between two 53BP1 protomers. Such ligands are valuable research tools to study the function of 53BP1 and have the potential to facilitate the development of new drugs for cancer therapy.

SARS-CoV-2 Spike Impairs DNA Damage Repair and Inhibits V(D)J Recombination In Vitro.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to the coronavirus disease 2019 (COVID-19) pandemic, severely affecting public health and the global economy. Adaptive immunity plays a crucial role in fighting against SARS-CoV-2 infection and directly influences the clinical outcomes of patients. Clinical studies have indicated that patients with severe COVID-19 exhibit delayed and weak adaptive immune responses; however, the mechanism by which SARS-CoV-2 impedes adaptive immunity remains unclear. Here, by using an in vitro cell line, we report that the SARS-CoV-2 spike protein significantly inhibits DNA damage repair, which is required for effective V(D)J recombination in adaptive immunity. Mechanistically, we found that the spike protein localizes in the nucleus and inhibits DNA damage repair by impeding key DNA repair protein BRCA1 and 53BP1 recruitment to the damage site. Our findings reveal a potential molecular mechanism by which the spike protein might impede adaptive immunity and underscore the potential side effects of full-length spike-based vaccines.

Oncogenic KRAS drives radioresistance through upregulation of NRF2-53BP1-mediated non-homologous end-joining repair.

KRAS-activating mutations are oncogenic drivers and are correlated with radioresistance of multiple cancers, including colorectal cancer, but the underlying precise molecular mechanisms remain elusive. Herein we model the radiosensitivity of isogenic HCT116 and SW48 colorectal cancer cell lines bearing wild-type or various mutant KRAS isoforms. We demonstrate that KRAS mutations indeed lead to radioresistance accompanied by reduced radiotherapy-induced mitotic catastrophe and an accelerated release from G2/M arrest. Moreover, KRAS mutations result in increased DNA damage response and upregulation of 53BP1 with associated increased non-homologous end-joining (NHEJ) repair. Remarkably, KRAS mutations lead to activation of NRF2 antioxidant signaling to increase 53BP1 gene transcription. Furthermore, genetic silencing or pharmacological inhibition of KRAS, NRF2 or 53BP1 attenuates KRAS mutation-induced radioresistance, especially in G1 phase cells. These findings reveal an important role for a KRAS-induced NRF2-53BP1 axis in the DNA repair and survival of KRAS-mutant tumor cells after radiotherapy, and indicate that targeting NRF2, 53BP1 or NHEJ may represent novel strategies to selectively abrogate KRAS mutation-mediated radioresistance.

Mechanisms of BRCA1-BARD1 nucleosome recognition and ubiquitylation.

The BRCA1-BARD1 tumour suppressor is an E3 ubiquitin ligase necessary for the repair of DNA double-strand breaks by homologous recombination1-10. The BRCA1-BARD1 complex localizes to damaged chromatin after DNA replication and catalyses the ubiquitylation of histone H2A and other cellular targets11-14. The molecular bases for the recruitment to double-strand breaks and target recognition of BRCA1-BARD1 remain unknown. Here we use cryo-electron microscopy to show that the ankyrin repeat and tandem BRCT domains in BARD1 adopt a compact fold and bind to nucleosomal histones, DNA and monoubiquitin attached to H2A amino-terminal K13 or K15, two signals known to be specific for double-strand breaks15,16. We further show that RING domains17 in BRCA1-BARD1 orient an E2 ubiquitin-conjugating enzyme atop the nucleosome in a dynamic conformation, primed for ubiquitin transfer to the flexible carboxy-terminal tails of H2A and variant H2AX. Our work reveals a regulatory crosstalk in which recognition of monoubiquitin by BRCA1-BARD1 at the N terminus of H2A blocks the formation of polyubiquitin chains and cooperatively promotes ubiquitylation at the C terminus of H2A. These findings elucidate the mechanisms of BRCA1-BARD1 chromatin recruitment and ubiquitylation specificity, highlight key functions of BARD1 in both processes and explain how BRCA1-BARD1 promotes homologous recombination by opposing the DNA repair protein 53BP1 in post-replicative chromatin18-22. These data provide a structural framework to evaluate BARD1 variants and help to identify mutations that drive the development of cancer.

Discovery of a novel 53BP1 inhibitor through AlphaScreen-based high-throughput screening.

Tumor suppressor p53-binding protein 1 (53BP1), a tantem tudor domain (TTD) protein, takes part in DNA Damage Repair (DDR) pathways through the specific recognition of lysine methylation on histones. The dysregulation of 53BP1 is closely related to the development of many diseases including cancer. Moreover, recent studies found that deficiency of 53BP1 could increase the efficiency of precise CRISPR/Cas9 genome editing. Thus, discovery of inhibitor is beneficial to the study of biological functions of 53BP1 and the application of CRISPR/Cas9 genome editing. UNC2170 and its derivatives have been reported as 53BP1 targeted small molecular inhibitors with modest activities. Hence, to discover better 53BP1 inhibitors, we conducted an AlphaScreen assay based high-throughput screening (HTS) and identified a novel and effective 53BP1-TTD inhibitor DP308 which disrupts the binding between 53BP1 and H4K20me2 peptide with an IC50 value of 1.69 ± 0.73 µM. Both Microscale Themophoresis (MST) and Surface Plasmon Resonance (SPR) assays confirmed the direct binding between DP308 and 53BP1-TTD protein with binding affinity (Kd) of about 2.7 µM. Molecular docking studies further suggested that DP308 possibly occupies the H4K20me2 binding pocket of the 53BP1-TTD aromatic cage. These results demonstrated that DP308 is a promising small molecule inhibitor for further optimization towards a more potent chemical probe of 53BP1. Additionally, it could be a potential valuable tool for applying to gene editing therapy by increasing the efficiency of CRISPR/Cas9 genome editing.

Enhanced homology-directed repair for highly efficient gene editing in hematopoietic stem/progenitor cells.

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using Streptococcus pyogenes Cas9 mRNA and an oligodeoxynucleotide donor to repair genetic mutations showed the capability to restore physiological protein expression but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we report that transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology-directed repair to achieve highly efficient (80% gp91phox+ cells compared with healthy donor control subjects) long-term correction of X-CGD CD34+ cells.

FOXK1 Participates in DNA Damage Response by Controlling 53BP1 Function.

53BP1 plays a central role in dictating DNA repair choice between non-homologous end joining (NHEJ) and homologous recombination (HR), which is important for the sensitivity to poly(ADP-ribose) polymerase inhibitors (PARPis) of BRCA1-deficient cancers. In this study, we show that FOXK1 associates with 53BP1 and regulates 53BP1-dependent functions. FOXK1-53BP1 interaction is significantly enhanced upon DNA damage during the S phase in an ATM/CHK2-dependent manner, which reduces the association of 53BP1 with its downstream factors RIF1 and PTIP. Depletion of FOXK1 impairs DNA repair and induces compromised cell survival upon DNA damage. Overexpression of FOXK1 diminishes 53BP1 foci formation, which leads to resistance to PARPis and elevation of HR in BRCA1-deficient cells and decreased telomere fusion in TRF2-depleted cells. Collectively, our findings demonstrate that FOXK1 negatively regulates 53BP1 function by inhibiting 53BP1 localization to sites of DNA damage, which alters the DSB-induced protein complexes centering on 53BP1 and thus influences DNA repair choice.

Concurrent live imaging of DNA double-strand break repair and cell-cycle progression by CRISPR/Cas9-mediated knock-in of a tricistronic vector.

Cell-cycle progression can be arrested by ionizing radiation-induced DNA double-strand breaks (DSBs). Although DSBs are patched by DSB repair systems, which comprise proteins such as p53-binding protein 1 (53BP1), the relationship between DSB repair progression and cell-cycle status in living cells is unclear. The probe FUCCI (fluorescent ubiquitination-based cell-cycle indicator) was previously developed for visualizing cell-cycle status. Here, we established novel live-imaging probes based on custom-designed plasmids designated "Focicles" harboring a tricistronic compartment encoding distinct fluorescent proteins ligated to the murine 53BP1 foci-forming region (FFR) and two cell-cycle indicators that are known components of FUCCI (hCdt1 and hGmnn). We used CRISPR/Cas9-mediated genome editing to obtain Focicle knock-in cell lines in NIH3T3 cells, which were subject to X-ray irradiation that induced comparable numbers of Focicle and endogenous-53BP1 foci. In addition, the Focicle probes enabled the kinetic analysis of both DSB repair and cell-cycle arrest/progression after irradiation, demonstrating that the Focicle knock-in cells progressed to cell division after DNA damage elimination. These newly developed probes can help to gain a better understanding of the dynamics of DSB repair and cell-cycle control to in turn guide cancer treatment development and cancer-risk assessments.

DNA Repair Network Analysis Reveals Shieldin as a Key Regulator of NHEJ and PARP Inhibitor Sensitivity.

Repair of damaged DNA is essential for maintaining genome integrity and for preventing genome-instability-associated diseases, such as cancer. By combining proximity labeling with quantitative mass spectrometry, we generated high-resolution interaction neighborhood maps of the endogenously expressed DNA repair factors 53BP1, BRCA1, and MDC1. Our spatially resolved interaction maps reveal rich network intricacies, identify shared and bait-specific interaction modules, and implicate previously concealed regulators in this process. We identified a novel vertebrate-specific protein complex, shieldin, comprising REV7 plus three previously uncharacterized proteins, RINN1 (CTC-534A2.2), RINN2 (FAM35A), and RINN3 (C20ORF196). Recruitment of shieldin to DSBs, via the ATM-RNF8-RNF168-53BP1-RIF1 axis, promotes NHEJ-dependent repair of intrachromosomal breaks, immunoglobulin class-switch recombination (CSR), and fusion of unprotected telomeres. Shieldin functions as a downstream effector of 53BP1-RIF1 in restraining DNA end resection and in sensitizing BRCA1-deficient cells to PARP inhibitors. These findings have implications for understanding cancer-associated PARPi resistance and the evolution of antibody CSR in higher vertebrates.

Inhibition of 53BP1 favors homology-dependent DNA repair and increases CRISPR-Cas9 genome-editing efficiency.

Programmable nucleases, such as Cas9, are used for precise genome editing by homology-dependent repair (HDR). However, HDR efficiency is constrained by competition from other double-strand break (DSB) repair pathways, including non-homologous end-joining (NHEJ). We report the discovery of a genetically encoded inhibitor of 53BP1 that increases the efficiency of HDR-dependent genome editing in human and mouse cells. 53BP1 is a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR by suppressing end resection, which is the rate-limiting step in the initiation of HDR. We screened an existing combinatorial library of engineered ubiquitin variants for inhibitors of 53BP1. Expression of one variant, named i53 (inhibitor of 53BP1), in human and mouse cells, blocked accumulation of 53BP1 at sites of DNA damage and improved gene targeting and chromosomal gene conversion with either double-stranded DNA or single-stranded oligonucleotide donors by up to 5.6-fold. Inhibition of 53BP1 is a robust method to increase efficiency of HDR-based precise genome editing.

BRCA2 antagonizes classical and alternative nonhomologous end-joining to prevent gross genomic instability.

BRCA2-deficient cells exhibit gross genomic instability, but the underlying mechanisms are not fully understood. Here we report that inactivation of BRCA2 but not RAD51 destabilizes RPA-coated single-stranded DNA (ssDNA) structures at resected DNA double-strand breaks (DSBs) and greatly enhances the frequency of nuclear fragmentation following cell exposure to DNA damage. Importantly, these BRCA2-associated deficits are fueled by the aberrant activation of classical (c)- and alternative (alt)- nonhomologous end-joining (NHEJ), and rely on the well-defined DNA damage signaling pathway involving the pro-c-NHEJ factor 53BP1 and its downstream effector RIF1. We further show that the 53BP1-RIF1 axis promotes toxic end-joining events via the retention of Artemis at DNA damage sites. Accordingly, loss of 53BP1, RIF1, or Artemis prolongs the stability of RPA-coated DSB intermediates in BRCA2-deficient cells and restores nuclear integrity. We propose that BRCA2 antagonizes 53BP1, RIF1, and Artemis-dependent c-NHEJ and alt-NHEJ to prevent gross genomic instability in a RAD51-independent manner.

Combining 53BP1 with BRCA1 as a biomarker to predict the sensitivity of poly(ADP-ribose) polymerase (PARP) inhibitors.

Over half of patients with BRCA1-deficient cancers do not respond to treatment with poly(ADP-ribose) polymerase (PARP) inhibitors. In this study, we report that a combination of 53BP1 and BRCA1 may serve as a biomarker of PARP inhibitor sensitivity. Based on the mRNA levels of four homologous recombination repair (HR) genes and PARP inhibitor sensitivity, we selected BRCA1-deficient MDA-MB-436 cells to conduct RNA interference. Reducing expression of 53BP1, but not the other three HR genes, was found to lower simmiparib sensitivity. Additionally, we generated 53BP1-/-/BRCA1-/- clonal variants by the transcription activator-like effector nuclease (TALEN) technique and found that depleting 53BP1 impaired PARP inhibitor sensitivity with a 36.7-fold increase in their IC50 values. Consistent with its effect on PARP inhibitor sensitivity, 53BP1 loss alleviated cell cycle arrest and apoptosis and partially restored HR function. Importantly, 53BP1 depletion dramatically reduced the ability of PARP inhibitors to suppress tumor growth in vivo. The inhibition rate of simmiparib was 74.16% for BRCA1-deficient MDA-MB-436 xenografts, but only 7.79% for 53BP1/BRCA1-deficient xenografts. Re-expressing 53BP1 in the dual-deficient cells restored PARP inhibitor sensitivity and the levels of HR regulators. Considering that at least 10% of BRCA1-deficient breast and ovarian cancers have reduced expression of 53BP1, using a combination of 53BP1 with BRCA1 as a biomarker for patient selection should reduce the number of patients undergoing futile treatment with PARP inhibitors.

TIRR regulates 53BP1 by masking its histone methyl-lysine binding function.

P53-binding protein 1 (53BP1) is a multi-functional double-strand break repair protein that is essential for class switch recombination in B lymphocytes and for sensitizing BRCA1-deficient tumours to poly-ADP-ribose polymerase-1 (PARP) inhibitors. Central to all 53BP1 activities is its recruitment to double-strand breaks via the interaction of the tandem Tudor domain with dimethylated lysine 20 of histone H4 (H4K20me2). Here we identify an uncharacterized protein, Tudor interacting repair regulator (TIRR), that directly binds the tandem Tudor domain and masks its H4K20me2 binding motif. Upon DNA damage, the protein kinase ataxia-telangiectasia mutated (ATM) phosphorylates 53BP1 and recruits RAP1-interacting factor 1 (RIF1) to dissociate the 53BP1-TIRR complex. However, overexpression of TIRR impedes 53BP1 function by blocking its localization to double-strand breaks. Depletion of TIRR destabilizes 53BP1 in the nuclear-soluble fraction and alters the double-strand break-induced protein complex centring 53BP1. These findings identify TIRR as a new factor that influences double-strand break repair using a unique mechanism of masking the histone methyl-lysine binding function of 53BP1.

53BP1 loss induces chemoresistance of colorectal cancer cells to 5-fluorouracil by inhibiting the ATM-CHK2-P53 pathway.

PURPOSE: Loss of P53 binding protein 1 (53BP1) is considered a poor prognostic factor for colorectal cancer. However, its effect on chemosensitivity of colorectal cancer to 5-fluorouracil (5-FU) remains elusive. This study aimed to examine the association of 53BP1 expression with chemosensitivity of colorectal cancer cells to 5-FU. METHODS: Immunohistochemistry was performed on 30 metastatic colorectal cancer samples to assess the associations of 53BP1 levels with clinical therapeutic effects. In vitro, IC50 values for 5-FU and 53BP1 levels were determined by MTT assay and Western blot in 5 colorectal cancer cell lines. Then, 53BP1 was silenced in HCT116 and HT29 cells, and cell proliferation, apoptosis and cell cycle distribution were evaluated. Relative protein levels of ATM-CHK2-P53 pathway effectors and Bcl-2 family members were measured by Western blot. Finally, the effects of 53BP1 knockdown on tumor growth and 5-FU chemoresistance were investigated in vivo. RESULTS: 53BP1 expression was closely related to time to progression (TTP) after first-line chemotherapy. Namely, 53BP1 downregulation resulted in reduced TTP. In addition, 53BP1 silencing increased proliferation, inhibited apoptosis and induced S phase arrest in HCT116 and HT29 cells after 5-FU treatment. Moreover, 53BP1 knockdown also reduced the protein levels of ATM-CHK2-P53 apoptotic pathway effectors, caspase9 and caspase3, while increasing Bcl-2 expression. In vivo, 53BP1 silencing accelerated tumor proliferation in nude mice and enhanced resistance to 5-FU. CONCLUSIONS: These findings confirmed that 53BP1 loss might be a negative factor for chemotherapy efficacy, promoting cell proliferation and inhibiting apoptosis by suppressing ATM-CHK2-P53 signaling, and finally inducing 5-FU resistance.