Three Prime Repair Exonuclease 1 Research Articles

CGAS activation in classical dendritic cells causes autoimmunity in TREX1-deficient mice

Detection of cytosolic DNA by the cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway provides immune defense against pathogens and cancer but can also cause autoimmunity when overactivated. The exonuclease three prime repair exonuclease 1 (TREX1) degrades DNA in the cytosol and prevents cGAS activation by self-DNA. Loss-of-function mutations of the TREX1 gene are linked to autoimmune diseases such as Aicardi-Goutières syndrome, and mice deficient in TREX1 develop lethal inflammation in a cGAS-dependent manner. In order to determine the type of cells in which cGAS activation drives autoinflammation, we generated conditional cGAS knockout mice on the Trex1-/- background. Here, we show that genetic ablation of the cGAS gene in classical dendritic cells (cDCs), but not in macrophages, was sufficient to rescue Trex1-/- mice from all observed disease phenotypes including lethality, T cell activation, tissue inflammation, and production of antinuclear antibodies and interferon-stimulated genes. These results show that cGAS activation in cDC causes autoinflammation in response to self-DNA accumulated in the absence of TREX1.

Targeting TREX1 Induces Innate Immune Response in Drug-Resistant Small-Cell Lung Cancer.

Small-cell lung cancer (SCLC) is the most lethal type of lung cancer. Paradoxically, this tumor displays an initial exquisite response to chemotherapy; however, at relapse, the tumor is highly resistant to subsequent available therapies. Here, we report that the expression of three prime repair exonuclease 1 (TREX1) is strongly induced in chemoresistant SCLCs. Assay for transposase-accessible chromatin using sequencing and chromatin immunoprecipitation sequencing revealed a significant increase in chromatin accessibility and transcriptional activity of TREX1 gene locus in chemoresistant SCLCs. Analyses of human SCLC tumors and patient-derived xenografts (PDX) also showed an increase in TREX1 expression in postchemotherapy samples. TREX1 depletion caused the activation of cyclic GMP-AMP synthase stimulator of interferon gene pathway due to cytoplasmic accumulation of damage-associated double-stranded DNA, inducing immunogenicity and enhancing the sensitivity of drug-resistant cells to chemotherapy. These findings suggest TREX1 upregulation may partially contribute to the survival of resistant cells, and its inhibition may represent a promising therapeutic strategy to enhance antitumor immunity and potentiate the efficacy of chemotherapy and/or immunotherapy in chemoresistant SCLCs. Significance: In this study, we show that targeting TREX1 induces an innate immune response and resensitizes SCLC cells to chemotherapy, representing a promising novel target for "immunologically" cold tumors, such as SCLC.

T-Rex escaped from the cytosolic park: Re-thinking the impact of TREX1 exonuclease deficiencies on genomic stability.

The Three Prime Repair Exonuclease 1 (TREX1) has been implicated in several pathologies characterized by chronic and inborn inflammation. Aberrant innate immunity caused by DNA sensing through the cGAS-STING pathway has been proposed to play a major role in the etiology of these interferonopathies. However, the molecular source of this DNA sensing and the possible involvement of TREX1 in genome (in)stability remains poorly understood. Recent findings reignite the debate about the cellular functions performed by TREX1 nuclease, notably in chromosome biology and stability. Here I put into perspective recent findings that suggest that TREX1 is at the crossroads of DNA damage response and inflammation in different pathological contexts.

Three prime repair exonuclease 1 preferentially degrades the integration-incompetent HIV-1 DNA through favorable kinetics, thermodynamic, structural, and conformational properties

HIV-1 integration into the human genome is dependent on 3’-processing of the viral DNA. Recently, we reported that the cellular Three Prime Repair Exonuclease 1 (TREX1) enhances HIV-1 integration by degrading the unprocessed viral DNA, while the integration-competent 3’-processed DNA remained resistant. Here, we describe the mechanism by which the 3’-processed HIV-1 DNA resists TREX1-mediated degradation. Our kinetic studies revealed that the rate of cleavage (kcat) of the 3’-processed DNA was significantly lower (approximately 2-2.5-fold) than the unprocessed HIV-1 DNA by TREX1. The kcat values of human TREX1 for the processed U5 and U3 DNA substrates were 3.8 s-1 and 4.5 s-1, respectively. In contrast, the unprocessed U5 and U3 substrates were cleaved at 10.2 s-1 and 9.8 s-1, respectively. The efficiency of degradation (kcat/Km) of the 3’-processed DNA (U5-70.2 and U3-28.05 pM-1s-1) was also significantly lower than the unprocessed DNA (U5-103.1 and U3-65.3 pM-1s-1). Furthermore, the binding affinity (Kd) of TREX1 was markedly lower (∼ 2-fold) for the 3’-processed DNA compared to the unprocessed DNA. Molecular docking and dynamics studies revealed distinct conformational binding modes of TREX1 with the 3’-processed and unprocessed HIV-1 DNA. Particularly, the unprocessed DNA was favorably positioned in the active site with polar interactions with the catalytic residues of TREX1. Additionally, a stable complex was formed between TREX1 and the unprocessed DNA compared the 3’-processed DNA. These results pinpoint the mechanism by which TREX1 preferentially degrades the integration-incompetent HIV-1 DNA and reveal the unique structural and conformational properties of the integration-competent 3’-processed HIV-1 DNA.

Inactivation of Myostatin Delays Senescence via TREX1-SASP in Bovine Skeletal Muscle Cells.

The myostatin (MSTN) gene also regulates the developmental balance of skeletal muscle after birth, and has long been linked to age-related muscle wasting. Many rodent studies have shown a correlation between MSTN and age-related diseases. It is unclear how MSTN and age-associated muscle loss in other animals are related. In this study, we utilized MSTN gene-edited bovine skeletal muscle cells to investigate the mechanisms relating to MSTN and muscle cell senescence. The expression of MSTN was higher in older individuals than in younger individuals. We obtained consecutively passaged senescent cells and performed senescence index assays and transcriptome sequencing. We found that senescence hallmarks and the senescence-associated secretory phenotype (SASP) were decreased in long-term-cultured myostatin inactivated (MT-KO) bovine skeletal muscle cells (bSMCs). Using cell signaling profiling, MSTN was shown to regulate the SASP, predominantly through the cycle GMP-AMP synthase-stimulator of antiviral genes (cGAS-STING) pathway. An in-depth investigation by chromatin immunoprecipitation (ChIP) analysis revealed that MSTN influenced three prime repair exonuclease 1 (TREX1) expression through the SMAD2/3 complex. The downregulation of MSTN contributed to the activation of the MSTN-SMAD2/3-TREX1 signaling axis, influencing the secretion of SASP, and consequently delaying the senescence of bSMCs. This study provided valuable new insight into the role of MSTN in cell senescence in large animals.

Abstract LB361: The exonuclease TREX1 constitutes an innate immune checkpoint limiting cGAS/STING-mediated anti-tumor immunity

Abstract The DNA exonuclease TREX1 (Three-prime repair exonuclease 1) is critical for preventing autoimmunity in mice and humans by degrading endogenous cytosolic DNA, which otherwise triggers activation of the innate cGAS/STING pathway leading to the production of type I IFNs. Since tumor cells are prone to aberrant cytosolic DNA accumulation, we hypothesized that they are critically dependent on TREX1 activity to limit their immunogenicity. Here we show, that in tumor cells TREX1 indeed restricts the spontaneous activation of the cGAS/STING pathway and the subsequent induction of a type I IFN response. As a result, TREX1 deficiency compromised in vivo tumor growth in mice. This delay depended on a functional immune system, systemic type I IFN signaling, and tumor-intrinsic cGAS expression. Mechanistically, we show that tumor TREX1 loss drove activation of CD8 T cells and NK cells, prevented CD8 T cell exhaustion and remodeled an immunosuppressive myeloid compartment. Consequently, TREX1 deficiency synergized with T cell directed immune checkpoint blockade. Collectively, we conclude that TREX1 is essential to limit tumor immunogenicity, and that targeting this innate immune checkpoint remodels the tumor microenvironment and enhances anti-tumor immunity by itself and in combination with T cell-targeted therapies. Citation Format: Junghyun Lim, Ryan Rodriguez, Katherine Williams, John Silva, Alan Gutierrez, Paul Tyler, Faezzah Baharom, Tao Sun, Eva Lin, Scott Martin, Brandon Kayser, Robert J. Johnston, Ira Mellman, Lélia Delamarre, Nathaniel West, Sören Müller, Yan Qu, Klaus Heger. The exonuclease TREX1 constitutes an innate immune checkpoint limiting cGAS/STING-mediated anti-tumor immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB361.

Abstract 4345: Evaluation of the prevalence and impact of molecular variation in the three-prime repair exonuclease 1 TREX1 across mammalian species and in human malignancies

Abstract The three-prime repair exonuclease 1, TREX1, degrades cytosolic DNA to prevent aberrant immune activation. Inactivation of TREX1 results in DNA accumulation in the cytosol and subsequent induction of the cGAS-STING DNA sensing pathway, downstream interferon-mediated innate immune signaling, and inflammation. Correspondingly, germline pathogenic mutational defects in the TREX1 gene lead to hereditary autoimmune and autoinflammatory disorders. While TREX1 mutations are also reported in some tumors, their consequences in cancer remain poorly understood. To assess the functional importance of amino acid residues in TREX1, we analyzed protein sequences of the functional TREX1 isoform, TREX1b, from 168 mammalian species. We also conducted a survey of genomic variant databases COSMIC, ClinVar, TCGA, LOVD, Decipher, and gnomAD for disease-associated TREX1 mutations. Finally, we modeled TREX1 mutations occurring in human tumors to evaluate predicted impact on protein stability and function using structure-based approaches FoldX and INPS-3D, and compared results to available predictions from AlphaMissense and COSMIC-3D. In the Cancer Genome Atlas (TCGA), 16 out of 32 cancer histologies have tumor samples bearing protein-altering TREX1 variants, with bladder cancer and melanoma having the highest frequency of TREX1 variants at 2.7% of tumor samples. Moreover, all 32 cancer types have some samples with loss of one or both copies of TREX1; frequencies of heterozygous loss range from 0.6% (thymoma) to 86% (kidney clear cell and lung squamous cell carcinomas). We also observed TREX1 copy gain with lower frequency than instances of copy loss, but also occurring in 31 out of 32 studies. Multiple tumors have a combination of a TREX1 protein sequence variant and copy number gain or loss, suggesting that their potential interplay may increase functional impact of TREX1 variants. Out of 15 cancer-associated TREX1 variants, 5 (33%) were predicted to be pathogenic by AlphaMissense. For FoldX and INPS-3D mutagenesis modeling, we used the published experimental 3D crystal structure of the wild type (WT) human TREX1 (PDB: 7TQQ). The changes in Gibbs free energy of folding ΔΔG between WT and mutant predicted by FoldX and INPS-3D both correlated strongly with the AlphaMissense pathogenic probability scores (Pearson r=0.69 and 0.73, respectively). We will discuss implications of TREX1 variation and conservation across mammalian species and potential effects of germline TREX1 variants in hereditary autoimmune and autoinflammatory disorders and on somatic and germline TREX1 variants in human cancer cells and tumor samples. Citation Format: Mariam M. Konate, Marwa Shekfeh, Julia Krushkal. Evaluation of the prevalence and impact of molecular variation in the three-prime repair exonuclease 1 TREX1 across mammalian species and in human malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4345.

The Exonuclease TREX1 Constitutes an Innate Immune Checkpoint Limiting cGAS/STING-Mediated Antitumor Immunity.

The DNA exonuclease three-prime repair exonuclease 1 (TREX1) is critical for preventing autoimmunity in mice and humans by degrading endogenous cytosolic DNA, which otherwise triggers activation of the innate cGAS/STING pathway leading to the production of type I IFNs. As tumor cells are prone to aberrant cytosolic DNA accumulation, we hypothesized that they are critically dependent on TREX1 activity to limit their immunogenicity. Here, we show that in tumor cells, TREX1 restricts spontaneous activation of the cGAS/STING pathway, and the subsequent induction of a type I IFN response. As a result, TREX1 deficiency compromised in vivo tumor growth in mice. This delay in tumor growth depended on a functional immune system, systemic type I IFN signaling, and tumor-intrinsic cGAS expression. Mechanistically, we show that tumor TREX1 loss drove activation of CD8+ T cells and NK cells, prevented CD8+ T-cell exhaustion, and remodeled an immunosuppressive myeloid compartment. Consequently, TREX1 deficiency combined with T-cell-directed immune checkpoint blockade. Collectively, we conclude that TREX1 is essential to limit tumor immunogenicity, and that targeting this innate immune checkpoint remodels the tumor microenvironment and enhances antitumor immunity by itself and in combination with T-cell-targeted therapies. See related article by Toufektchan et al., p. 673.

TREX1 is required for microglial cholesterol homeostasis and oligodendrocyte terminal differentiation in human neural assembloids

Three Prime Repair Exonuclease 1 (TREX1) gene mutations have been associated with Aicardi-Goutières Syndrome (AGS) – a rare, severe pediatric autoimmune disorder that primarily affects the brain and has a poorly understood etiology. Microglia are brain-resident macrophages indispensable for brain development and implicated in multiple neuroinflammatory diseases. However, the role of TREX1 – a DNase that cleaves cytosolic nucleic acids, preventing viral- and autoimmune-related inflammatory responses – in microglia biology remains to be elucidated. Here, we leverage a model of human embryonic stem cell (hESC)-derived engineered microglia-like cells, bulk, and single-cell transcriptomics, optical and transmission electron microscopy, and three-month-old assembloids composed of microglia and oligodendrocyte-containing organoids to interrogate TREX1 functions in human microglia. Our analyses suggest that TREX1 influences cholesterol metabolism, leading to an active microglial morphology with increased phagocytosis in the absence of TREX1. Notably, regulating cholesterol metabolism with an HMG-CoA reductase inhibitor, FDA-approved atorvastatin, rescues these microglial phenotypes. Functionally, TREX1 in microglia is necessary for the transition from gliogenic intermediate progenitors known as pre-oligodendrocyte precursor cells (pre-OPCs) to precursors of the oligodendrocyte lineage known as OPCs, impairing oligodendrogenesis in favor of astrogliogenesis in human assembloids. Together, these results suggest routes for therapeutic intervention in pathologies such as AGS based on microglia-specific molecular and cellular mechanisms.

Molecular insight into the specific enzymatic properties of TREX1 revealing the diverse functions in processing RNA and DNA/RNA hybrids.

In various autoimmune diseases, dysfunctional TREX1 (Three prime RepairExonuclease 1) leads to accumulation of endogenous single-stranded DNA (ssDNA), double-stranded DNA (dsDNA) and DNA/RNA hybrids in the cytoplasm and triggers immune activation through the cGAS-STING pathway. Although inhibition of TREX1 could be a useful strategy for cancer immunotherapy, profiling cellular functions in terms of its potential substrates is a key step. Particularly important is the functionality of processing DNA/RNA hybrids and RNA substrates. The exonuclease activity measurements conducted here establish that TREX1 can digest both ssRNA and DNA/RNA hybrids but not dsRNA. The newly solved structures of TREX1-RNA product and TREX1-nucleotide complexes show that 2'-OH does not impose steric hindrance or specific interactions for the recognition of RNA. Through all-atom molecular dynamics simulations, we illustrate that the 2'-OH-mediated intra-chain hydrogen bonding in RNA would affect the binding with TREX1 and thereby reduce the exonuclease activity. This notion of higher conformational rigidity in RNA leading TREX1 to exhibit weaker catalytic cleavage is further validated by the binding affinity measurements with various synthetic DNA-RNA junctions. The results of this work thus provide new insights into the mechanism by which TREX1 processes RNA and DNA/RNA hybrids and contribute to the molecular-level understanding of the complex cellular functions of TREX1 as an exonuclease.

Manganese facilitated cGAS-STING-IFNI pathway activation induced by ionizing radiation in glioma cells

Purpose After irradiation, double-stranded DNA leaked into the cytoplasm activates the cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) pathway, leading to the production of type I interferon (IFNI). In this study, we sought to probe the effect of ionizing radiation on activity of cGAS-STING-IFNI pathway in normoxic or hypoxic glioma cells and explore a more effective method to activate the signaling pathway, thereby activating the anti-tumor immune response and improving the therapeutic effect of radiotherapy for glioma. Materials and methods Human glioma cells U251 and T98G cultured in normoxia or hypoxia (1% O2) were irradiated with different doses of X-ray. The relative expressions of cGAS, IFN-I stimulated genes (ISGs), and three-prime repair exonuclease 1 (TREX1) were detected by qPCR. The expression levels of interferon regulatory factor 3 (IRF3) and p-IRF3 proteins were detected by Western blot. The production of cGAMP and IFN-β in the supernatant was detected by ELISA assay. U251 and T98G cell lines with stable knockdown of TREX1 were established after transfection with lentivirus vectors. EdU cell proliferation assay was used to screen suitable metal ions concentrations. The phagocytosis of DCs was observed by immunofluorescence microscope. The phenotype of DCs was detected by flow cytometry. The migration ability of DCs was detected by a transwell experiment. Results We found that cytosolic dsDNA, 2′3′-cGAMP, cGAS and ISGs expression, and IFN-β in cell supernatant were all increased with the doses of X-ray in the range of 0–16 Gy in normoxic glioma cells. Nevertheless, hypoxia significantly inhibited the radiation-induced dose-dependent activation of cGAS-STING-IFNI pathway. Furthermore, manganese (II) ion (Mn2+) significantly improved cGAS-STING-IFNI pathway activation induced by X-ray in both normoxic and hypoxic glioma cells, thereby promoting the maturation and migration of DCs. Conclusions The responses of cGAS-STING-IFNI pathway to ionizing radiation were mainly investigated under normoxic condition, but the experiments described here indicated that hypoxia could hinder the pathway activation. However, Mn2+ showed radiosensitizing effects on the pathway under either normoxic or hypoxic conditions demonstrating its potential as a radiosensitizer for glioma through activating an anti-tumor immune response.

Role of three-prime repair exonuclease (TREX1) variants in the susceptibility to systemic lupus erythematosus patients

Role of three-prime repair exonuclease (TREX1) variants in the susceptibility to systemic lupus erythematosus patients

Multiple RNA- and DNA-binding proteins exhibit direct transfer of polynucleotides with implications for target-site search

We previously demonstrated that the polycomb repressive complex 2 chromatin-modifying enzyme can directly transfer between RNA and DNA without a free-enzyme intermediate state. Simulations suggested that such a direct transfer mechanism may be generally necessary for RNA to recruit proteins to chromatin, but the prevalence of direct transfer capability is unknown. Herein, we used fluorescence polarization assays and observed direct transfer for several well-characterized nucleic acid-binding proteins: three-prime repair exonuclease 1, heterogeneous nuclear ribonucleoprotein U, Fem-3-binding factor 2, and MS2 bacteriophage coat protein. For TREX1, the direct transfer mechanism was additionally observed in single-molecule assays, and the data suggest that direct transfer occurs through an unstable ternary intermediate with partially associated polynucleotides. Generally, direct transfer could allow many DNA- and RNA-binding proteins to conduct a one-dimensional search for their target sites. Furthermore, proteins that bind both RNA and DNA might be capable of readily translocating between those ligands.

Synthesis and Excision Repair of Site-Specific 3'-End DNA-Histone Cross-Links Derived from Abasic Sites.

Histones catalyze the DNA strand incision at apurinic/apyrimidinic (AP) sites accompanied by formation of reversible but long-lived DNA-protein cross-links (DPCs) at 3'-DNA termini within single-strand breaks. These DPCs need to be removed because 3'-hydroxyl is required for gap-filling DNA repair synthesis but are challenging to study because of their reversible nature. Here we report a chemical approach to synthesize stable and site-specific 3'-histone-DPCs and their repair by three nucleases, human AP endonuclease 1, tyrosyl-DNA phosphodiesterase 1, and three-prime repair exonuclease 1. Our method employs oxime ligation to install an alkyne to 3'-DNA terminus, genetic incorporation of an azidohomoalanine to histone H4 at a defined position, and click reaction to conjugate DNA to H4 site-specifically. Using these model DPC substrates, we demonstrated that the DPC repair efficiency is highly affected by the local protein environment, and prior DPC proteolysis facilitates the repair.

TREX1 cytosolic DNA degradation correlates with autoimmune disease and cancer immunity.

Three Prime Repair Exonuclease 1 (TREX1) is a major 3'-5' exonuclease in mammalian cells that primarily degrades DNA substrates in the cytoplasm. TREX1 deficiency results in an aberrant accumulation of self-DNA in the cytosol, predominantly activating the innate immune response via the cGAS-STING-IFN pathways. Characterized by interferon production, autoimmune diseases, such as Aicardi-Goutieres syndrome (AGS), systemic lupus erythematosus (SLE), familial chilblain lupus (FCL), and retinal vasculopathy and leukodystrophy (RVCL), are associated with TREX1 loss of function. Besides, abnormal expression of TREX1 also appears in different types of tumor cells. In this review, we described the structure and mechanisms of TREX1 in degrading self-DNA and discussed the potential sources of these aberrant nucleic acids. We also summarized the major research advances in TREX1-related diseases, including autoimmune diseases and cancer. These contents will serve as a foundation for further study on the function of TREX1 and provide advances in the diagnosis, treatment, and prognosis of related disorders.

NMR applications to find and progress TREX1 binders

Inhibition of the exonuclease activity of Three-prime Repair EXonuclease 1 (TREX1) has emerged as a potential novel immunotherapeutic strategy. Herein we describe our screening approach to find initial low-affinity fragment hits for TREX1 from our 19F library and the use of both ligand- and target-based NMR methods for hit confirmation and validation. In a further step to support the early hit expansion stage we describe our setup of a 19F competition assay for KI determination of analogs.

A case of retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations: Long‐term treatment with repeated courses of immunotherapy

AbstractWe present here the case of a 48‐year‐old man who first developed retinopathy, and motor deficit and aphasia occurred 8 years later. A tumor‐like lesion was seen by brain MRI. He was diagnosed with retinal vasculopathy with cerebral leukoencephalopathy and systemic manifestations (RVCL‐S) because a three prime repair exonuclease 1 (TREX1) gene abnormality was identified. After intravenous methylprednisolone pulse (IVMP) and intravenous immunoglobulin (IVIg) therapy, cerebrospinal fluid (CSF) test results and clinical manifestations improved. However, a new lesion appeared 3 years later. Immunotherapy became ineffective, and repeated relapses occurred. We evaluated the results of laboratory tests and brain MRI for 6 years after the first treatment. Coagulation tests, including measurements of vWF activity, remained high, so immunotherapy was thought to have a limited effect.

Human TREX1 Repairs 3'-End DNA Lesions in Vitro.

Human three-prime repair exonuclease 1 (TREX1) is the major 3' to 5' exonuclease that functions to deplete the cytosolic DNA to prevent the autoimmune response. TREX1 is upregulated and translocates from cytoplasm to the nucleus in response to genotoxic stress, but the function of nuclear TREX1 is not well understood. Herein, we wish to report our in vitro finding that TREX1 efficiently excises 3'-phospho-α,β-unsaturated aldehyde and 3'-deoxyribose phosphate that are commonly produced as base excision repair intermediates and also from the nonenzymatic strand incision at abasic sites.

Human TDP1, APE1and TREX1 repair 3'-DNA-peptide/protein cross-links arising from abasic sites in vitro.

Histones and many other proteins react with abundant endogenous DNA lesions, apurinic/apyrimidinic (abasic, AP) sites and/or 3′-phospho-α,β-unsaturated aldehyde (3′-PUA), to form unstable but long-lived Schiff base DNA–protein cross-links at 3′-DNA termini (3′-PUA–protein DPCs). Poly (ADP-ribose) polymerase 1 (PARP1) cross-links to the AP site in a similar manner but the Schiff base is reduced by PARP1’s intrinsic redox capacity, yielding a stable 3′-PUA–PARP1 DPC. Eradicating these DPCs is critical for maintaining the genome integrity because 3′-hydroxyl is required for DNA synthesis and ligation. But how they are repaired is not well understood. Herein, we chemically synthesized 3′-PUA-aminooxylysine-peptide adducts that closely resemble the proteolytic 3′-PUA–protein DPCs, and found that they can be repaired by human tyrosyl-DNA phosphodiesterase 1 (TDP1), AP endonuclease 1 (APE1) and three-prime repair exonuclease 1 (TREX1). We characterized these novel repair pathways by measuring the kinetic constants and determining the effect of cross-linked peptide length, flanking DNA structure, and the opposite nucleobase. We further found that these nucleases can directly repair 3′-PUA–histone DPCs, but not 3′-PUA–PARP1 DPCs unless proteolysis occurs initially. Collectively, we demonstrated that in vitro 3′-PUA–protein DPCs can be repaired by TDP1, APE1, and TREX1 following proteolysis, but the proteolysis is not absolutely required for smaller DPCs.

Proceedings to the 7th Annual Conference of the Particle Therapy Cooperative Group North America (PTCOG-NA)

Proceedings to the 7th Annual Conference of the Particle Therapy Cooperative Group North America (PTCOG-NA)