Caffeine inhibits nonhomologous end joining by impairing ligase IV/XRCC4 function
Susmita Kumari, Divya Sathees, Prashant Kumar Rai, Lipsa Rani Sahu, Sathees C Raghavan
TL;DR
Caffeine inhibits DNA repair by blocking the function of a key protein complex involved in fixing DNA breaks.
Contribution
This study reveals that caffeine directly inhibits DNA ligase IV/XRCC4 function, a novel mechanism for its DNA repair suppression.
Findings
Caffeine inhibits nonhomologous end joining (NHEJ) in a concentration-dependent manner.
Caffeine directly binds to XRCC4, disrupting its interaction with DNA ligase IV and impairing DNA repair.
Disruption of caffeine's interaction site in XRCC4 partially restores DNA end joining in caffeine-treated cells.
Abstract
DNA double-strand breaks (DSBs), the most lethal DNA lesions, are repaired primarily by homologous recombination (HR) or nonhomologous end joining (NHEJ). Caffeine is known to inhibit HR by displacing Rad51 from single-stranded DNA, but its impact on NHEJ was unclear. Here, we show that caffeine inhibits NHEJ in a concentration-dependent manner using biochemical and cellular assays. Increased 53BP1 and γ-H2AX foci upon caffeine exposure indicate inhibition of chromosomal NHEJ, leading to accumulation of DSBs. γ-H2AX immunofluorescence, neutral comet, and TUNEL assays revealed persistent DNA breaks and reduced repair. Mechanistically, in silico, biophysical, and biochemical analyses demonstrate that caffeine directly binds to XRCC4, disrupting its interaction with DNA ligase IV and thereby inhibiting repair. Biolayer interferometry confirmed caffeine–XRCC4 binding, with mutation of…
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Figure 10- —Department of Atomic Energy, India10.13039/501100001502
- —Indian Council of Medical Research10.13039/501100001411
- —Department of Biotechnology10.13039/501100001407
- —Department of Science & Technology-Fund for Improvement of S&T
- —Central Facility of Biochemistry, IISc. S.C.R
- —JC Bose National Fellow
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Taxonomy
TopicsDNA Repair Mechanisms · PARP inhibition in cancer therapy · Genetic factors in colorectal cancer
Introduction
Among various DNA lesions, double-strand breaks (DSBs) are the most lethal type, which can lead to cell death if left unrepaired [1]. Mammalian cells have evolved different double-strand break repair pathways to tackle DSBs inside the cell, which include homologous recombination (HR), nonhomologous end joining (NHEJ), and microhomology-mediated end joining (MMEJ) [2–5]. HR utilizes a template from the sister chromatid and occurs in the S and G2 phases of the mammalian cell cycle [6], while NHEJ is an error-prone repair pathway but active throughout the cell cycle [5]. Alternative NHEJ (Alt-NHEJ) is a backup pathway to classical NHEJ and utilizes a small region of microhomology of 5–25 nt (referred to as MMEJ) for repair [2, 7–9].
Caffeine belongs to the methylxanthine class and is an analog of adenosine. It is extensively used as a tool to study DNA repair protein signaling and cell cycle checkpoint responses [10–13]. At the molecular level, caffeine is known to inhibit the in vitro protein kinase activity of ATM (Ataxia-telangiectasia-mutated) and ATR (Ataxia-telangiectasia and RAD3-related) [14]. There are several reports suggesting that caffeine can inhibit HR-mediated repair. Using budding yeast as the model system, it has been shown that caffeine treatment impaired HR by inhibiting 5′ to 3′ end resection due to the rapid loss of nucleases, Sae2 and Dna2 [10]. In addition to this, it was reported that caffeine treatment in irradiated HeLa cells blocked the formation of Rad51 foci that depend on 5′ to 3′ resection of broken chromosome ends; however, there was no significant difference in the number of RPA foci [10]. In an accompanying study, the authors demonstrated that caffeine treatment inhibited gene conversion by dose-dependent eviction of Rad51 from ssDNA. Further, it was noted that this inhibition was independent of the Mec1^ATR^/Tel1^ATM^ inhibition or caffeine’s inhibition of 5′ to 3′ resection of DSB ends [11]. It was concluded that caffeine treatment can disrupt gene conversion by disrupting the formation of the Rad51 recombinase filament that forms on single-stranded DNA (ssDNA) created at DSB ends [11]. Yet another study reported that caffeine inhibited homology-directed repair (HDR) by increasing the interactions of the RAD51 nucleoprotein filament with nonhomologous DNA and thus interfered with the homologous joint molecule formation by inhibiting RAD51 catalyzed strand invasion process [13]. The results of this study showed that caffeine inhibited the homology search by the RAD51 nucleoprotein filament. Besides, a significant reduction in the inhibition of HDR in XRCC3-deficient cells as compared to the wild-type cells upon caffeine treatment was also reported [12]. A recent report demonstrated that prolonged caffeine exposure activates a strong DNA damage response, leading to G2/M arrest, depletion of key HR proteins like Rad51 and BRCA2, and p53-dependent apoptosis. Despite this, caffeine enhances early HR-associated DNA synthesis but fails to increase complete HR repair events [15].
Another member of the PI3KK family, DNA-PKcs, a key protein involved in NHEJ, was shown to be inhibited in vitro by caffeine at concentrations as high as 10 mM through a mixed noncompetitive mechanism with respect to ATP [16]. However, it weakly inhibited DNA-PK autophosphorylation and failed to inhibit DNA-PKcs dependent double-strand break repair in vivo [16]. Thus, although caffeine has been shown to inhibit the HR pathway for DSB repair by independent mechanisms [10–13]; very little is known about its impact on the NHEJ-mediated DSB repair pathway. Here, we report that caffeine inhibited joining through NHEJ using in vitro, ex vivo, and in vivo assay systems. Our study shows that inhibition of NHEJ-mediated repair by caffeine leads to the accumulation of DSBs within the cells. Finally, we report that caffeine inhibits NHEJ by binding to XRCC4 and thus blocking the ligase IV/XRCC4 mediated sealing of DNA breaks.
Materials and methods
Enzymes, chemicals, and reagents
Chemicals and reagents used in the experiments were purchased from Merck (USA), SRL (India), and Himedia (India). DNA-modifying enzymes were purchased from New England Biolabs (USA). Cell culture media were obtained from Lonza (Switzerland) and MP Biomedicals (USA). Fetal bovine serum and penicillin–streptomycin were purchased from Gibco (USA). Radioisotope-labeled nucleotides were purchased from Revvity (USA) and BRIT (India). Antibodies were purchased from Santa Cruz Biotechnology (USA), Sigma (USA), BD Bioscience (USA), Invitrogen (USA), Cell Signaling Technology (USA), and BioLegend (USA). Caffeine was bought from Merck (USA).
Mammalian cell culture
Human cell lines HeLa (human cervical cancer), Molt4 (acute lymphoblastic leukemia), and Jurkat (T-cell leukemia) were purchased from the National Centre for Cell Science, Pune, India. HMF-3S (human mammary fibroblasts) were kindly gifted by Dr Ramray Bhat (IISc, Bangalore). Cells were grown in DMEM or RPMI medium supplemented with 10% FBS and 100 μg/ml penicillin G and streptomycin. Cells were incubated at 37°C in a humidified atmosphere containing 5% CO_2_ [17, 18].
Oligomers
Oligomers used in the study are presented as Supplementary Table S1. Oligomers were gel purified as described before [19].
Plasmids
The in vivo NHEJ, HR, and MMEJ construct pim-EJ5GFP, DR-GFP (pJS65), and EJ2-GFP (pJS248), respectively, were a kind gift from Dr Jeremy Stark, USA [20]. I-SceI overexpression vector (pcDNA3β-myc-NLS-I-SceI) was obtained from Dr Ralph Scully, USA [21]. Plasmid coexpression vector ligase IV and XRCC4 was gifted by Dr M. Modesti (France). XRCC4 was cloned into pET28a vector as described previously (Modesti et al., 1999). T133A mutant XRCC4 was generated by performing site-directed mutagenesis on the wild-type XRCC4 sequence cloned into the pET28a expression vector. The resulting construct was designated as pSK1.
Animals
Wistar rats (Rattus norvegicus, 4–6 weeks) were purchased and maintained in polypropylene cages in the Central Animal Facility, Indian Institute of Science (IISc). Wistar rats were used for dissection of organs for extract preparation. Animals were maintained according to the guidelines of the Animal Ethical Committee, Indian Institute of Science, India, and approved by the Institute Animal Ethics Committee (CAF/Ethics/871/2021). The animals were kept in polypropylene cages and provided with purified water ab libitum and a standard pellet diet (Altromin, gesundheit für Tiere, Germany). Animals were kept in regulated temperature and humidity levels [(100 000 Clean Air facility, temperature (22°C ± 2), humidity (50%–60%)] with a 12 h light/dark cycle.
Ethics Statement
All animal experiments were carried out in accordance with the approval of the Institute Animal Ethical Committee of the Indian Institute of Science (IISc), Bangalore, India (CAF/Ethics/871/2021).
Radiolabeled oligomeric DNA substrate preparation
The 5′ end-labeling of the oligomeric DNA was performed using T4 polynucleotide kinase in a buffer containing 20 mM Tris-acetate (pH 7.9), 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT (Dithiothreitol), and γ^32^P-ATP at 37°C for 1 h [22]. The reaction mixture was then passed through the Sephadex G25 column (Sigma, USA) to obtain the labeled purified product, which was stored at −20°C until further use. Double-stranded oligomeric DNA substrate containing 5′ overhangs (compatible ends) was prepared by annealing γ^32^P-ATP end-labeled 75 nt oligomer, SCR19 with unlabeled 75 nt complementary oligomer, SCR20 in 10 mM NaCl, and 1 mM EDTA in a boiling water bath for 10 min, followed by slow cooling [23, 24]. Double-stranded oligomeric DNA substrate containing 5′-5′ noncompatible overhangs was prepared by annealing γ^32^P-ATP end-labeled oligomers, SCR19, DG11, DG15, and DG13, with unlabeled complementary oligomers, VK11, DG12, DG16, and DG14, respectively. 5′-3′ noncompatible end harboring substrate was prepared by annealing 5′ γ^32^P-ATP end-labeled substrate SCR19 with its complementary sequence VK13.
Preparation of cell-free extracts from rat tissues
Rat tissue extract was prepared from the testes of 4- to 6-week-old male Wistar rats, Rattus norvegicus as described earlier [24–28]. Following dissection, the testes were washed with ice-cold 1× PBS. Further, the tissues were minced into single-cell suspension in 1× PBS, counted under microscope, and approximately 8 × 10^7^ cells were resuspended in a hypotonic solution (Buffer A: 10 mM Tris–HCl [pH 8.0], 1 mM EDTA, 5 mM DTT, and 0.5 mM PMSF [Phenylmethylsulfonyl fluoride]) and homogenized in the presence of protease inhibitors (1 μg/ml each of leupeptin, aprotinin, and pepstatin). Buffer B containing 10 mM Tris–HCl (pH 8.0), 10 mM MgCl_2_, 2 mM DTT, 0.5 mM PMSF, 25% sucrose, and 50% glycerol was added and incubated for 30 min on ice. The neutralized saturated ammonium sulfate solution was then added and incubated at 4°C for 30 min. The lysate was then ultracentrifuged at 42 000 RPM for 3 h at 4°C to separate the supernatant. Neutralized, saturated ammonium sulfate (65% cut-off) was added at 4°C; the precipitated protein pellet was collected by centrifugation at 14 000 RPM. Lastly, the pellet was dissolved and then dialyzed at 4°C overnight using Buffer C, containing 25 mM HEPES-KOH (pH 7.9), 100 mM KCl, 12 mM MgCl_2_, 1 mM EDTA, and 2 mM DTT. Extracts were snap-frozen and stored at −80°C until further use.
Preparation of cell-free extract from cancer cell lines
Cell-free extract (CFE) was prepared from HMF-3S, HeLa, Molt4 and Jurkat cells as described previously [8, 26, 29, 30]). 3 × 10^7^ cells were resuspended in 2 volumes of hypotonic buffer (10 mM Tris–HCl [pH 8.0], 1 mM EDTA, and 5 mM DTT and protease inhibitors) followed by homogenization and incubation at 4°C for 20 min. Following this, half volume of high-salt buffer (50 mM Tris–HCl [pH 7.5], 1 M KCl, 2 mM EDTA, and 2 mM DTT) was added, homogenized, and incubated for 20 min at 4°C. Extracts were ultracentrifuged at 4°C for 3 h at 42 000 RPM in a TLA-100 rotor. Supernatant was collected and dialyzed in dialysis buffer (20 mM Tris–HCl [pH 8.0], 0.1 M potassium acetate, 20% v/v glycerol, 0.5 mM EDTA, 1 mM DTT and 0.1 mM PMSF) overnight. Extracts were aliquoted, snap frozen and stored at -80°C until further use.
DNA end-joining reactions using cell-free extracts
Extracts prepared from primary tissue (testes), human primary cell (HMF-3S) and human cancerous cell lines (Molt4, HeLa and Jurkat) (1 µg) were incubated with radiolabeled double-stranded (ds) DNA oligomers at 25°C in NHEJ buffer [30 mM HEPES-KOH (pH 7.9), 7.5 mM MgCl_2_, 1 mM DTT, 2 mM ATP, 50 μM dNTP,s and 0.1 μg BSA] in the presence of increasing concentration of caffeine [25, 26, 27, 31]. The reaction mixture in which the extract was not added served as the no protein control (NPC). The joining reaction was terminated by adding EDTA (10 mM). The DNA products were purified by phenol:chloroform extraction and precipitated with chilled ethanol and glycogen. The dried pellet was dissolved in 10 μl of TE and resolved on 8% denaturing PAGE, depending on the DNA substrate size. The gel was dried at 80°C for 45 min, exposed, and the signal was detected using phosphorImager FLA9000 (Fuji, Japan). MultiGauge software was used to quantify joined products in Photostimulated Luminescence Units (PSLU). The extent of inhibition in the caffeine-treated samples was quantified in PSLU.
Isolation of plasmid DNA
Plasmid DNA was isolated [pJS296, DR-GFP (pJS65), EJ2-GFP (pJS248), I-SceI, ligase I, ligaseIV/XRCC4, XRCC4, T133A mutant XRCC4, KU70/KU80 expression vector] from bacteria Escherichia coli DH5α as described before [32, 33]. Competent cells were transformed with the plasmids and plated to generate individual colonies. LB media containing appropriate antibiotics were inoculated with the primary bacterial culture and allowed to grow for 14 h at 37°C. Cells were pelleted down at 6000 rpm for 10 min at 4°C, and the pellet was resuspended in Solution I (10 mM EDTA and 25 mM Tris–HCl, pH 8.0). Solution II (0.2 N NaOH and 1% SDS) was added, mixed slowly, and kept at room temperature for 10 min. Solution III (3 M potassium acetate and 2 M glacial acetic acid) was added to the tubes, mixed slowly, and incubated on ice for 20 min. Samples were centrifuged at 14 000 rpm for 20 min, followed by precipitation with equal volume of isopropanol at room temperature for 20–30 min. The pellet was washed with 70% ethanol, dried, resuspended in TE, and treated with RNaseA at 37°C. Following phenol:chloroform extraction, the DNA was dissolved in 1× Tris-EDTA (10 mM:1 mM). The quality of the prepared plasmids was checked on 0.8% agarose gel.
Site-directed mutagenesis of XRCC4 (T133A) in pET-28a vector
The T133A point mutation in XRCC4 was introduced into the pET-28a-XRCC4 construct using a PCR based site-directed mutagenesis approach and was designated as pSK1. Primers carrying the desired nucleotide substitution (ACC→GCC) were designed such that the altered codon was centrally located, flanked by approximately 15 bp of complementary sequence on each side. PCR amplification was performed using a high-fidelity DNA polymerase (Phusion) with pET-28a-XRCC4 as the template under standard cycling conditions recommended by the manufacturer. The parental methylated plasmid was digested with DpnI at 37°C for 30 min to selectively remove template DNA, and the reaction mixture was transformed into E. coli DH5α competent cells. Transformants were selected on LB agar plates containing kanamycin (50 µg/ml), and plasmid DNA was isolated from individual colonies by miniprep. The presence of the T133A mutation was confirmed by sequencing. Verified mutant constructs were subsequently transformed into E. coli Rosetta(DE3)pLysS for recombinant protein expression.
Overexpression and purification of proteins
The plasmid with His-tagged ligase IV and XRCC4 was a kind gift from Dr. Mauro Modesti [34]. Rosetta(DE3)pLysS cells were transformed with this expression vector, grown to an OD_600_ of 0.6, followed by induction with 1 mM IPTG (Isopropyl β-D-1-thiogalactopyranoside) (16 h at 16°C). Proteins were purified as described earlier [28, 34, 35]. Briefly, cells were harvested at 6000 RPM, resuspended in the extraction buffer (20 mM Tris–HCl [pH 8.0], 0.5 M KCl, 20 mM imidazole [pH 7.0], 20 mM β-mercaptoethanol, 10% glycerol, 0.2% Tween 20, and 1 mM PMSF) and loaded on Ni-NTA column as per manufacturer’s instructions (Novagen, USA). Fractions were collected, and the purest fractions were pooled and reloaded onto the UNOsphere Q anion exchange column (BioRad, USA), following which the protein was eluted by a gradient of KCl. Appropriate fractions were pooled and dialyzed (Dialysis buffer: 20 mM Tris–HCl [pH 8.0], 150 mM KCl, 2 mM DTT, and 10% glycerol) overnight. Post dialysis, the protein was snap-chilled and stored at −80°C. The purity and identity of the proteins were confirmed by SDS–PAGE.
For the purification of His-tagged ligase IV and XRCC4 T133A mutant protein, Rosetta(DE3)pLysS cells were transformed with the expression vector, grown to an OD_600_ of 0.6, followed by induction with 1 mM IPTG (16 h at 16°C). Briefly, cells were harvested at 6000 RPM, resuspended in the extraction buffer and the prepared lysate was loaded on Ni-NTA column. The protein was eluted using an increasing gradient of imidazole. Pure fractions were pooled and dialyzed overnight at 4°C. His-tagged ligase I and XRCC4 was purified as described previously [28, 35]). For His-tagged ligase I, wild-type XRCC4 and T133A mutant XRCC4 protein purification, Rosetta(DE3)pLysS cells were transformed with the overexpression constructs. Bacteria were grown to an OD_600_ of 0.6 and then induced with 1 mM IPTG for 16 h at 16°C for ligase I and wild-type XRCC4, whereas at 37°C for 6 h for the mutant XRCC4. The lysate was prepared and then loaded on Ni-NTA column. The protein was eluted using an increasing gradient of imidazole. Pure fractions were pooled and dialyzed (Dialysis buffer: 20 mM Tris–HCl [pH 8.0], 150 mM KCl, 2 mM DTT, and 10% glycerol) overnight at 4°C. His-tagged KU70/80, Artemis, and XLF were purified as described previously [36–39].
Electrophoretic mobility shift assay
To evaluate the effect of caffeine on KU70/80 binding to DNA substrates harboring double-strand breaks, purified KU70/80 protein was incubated in the presence of increasing concentrations of caffeine (10, 20, 40, and 60 mM) for 30 min at 4°C in electrophoretic mobility shift assay (EMSA) buffer containing 25 mM Tris (pH 8.0), 100 mM NaCl, 0.1 mM EDTA, 0.05% Triton X-100, 50 μg/ml of BSA/ml, 10% glycerol, and 2 mM DTT [28, 40]. Purified KU70/80 was a kind gift from Dr J B Charbonnier, France. Following this incubation, the radiolabeled noncompatible oligomeric DNA substrates were added and incubated at 4°C for 30 min. Reaction products were resolved on 6% native polyacrylamide gel at 4°C. The radioactive signals were detected with phosphorImager as described above. MultiGauge software was used to quantify KU-DNA bound products in PSLU.
DNA end-joining reactions using purified protein
To check the effect of caffeine on the joining activity of ligase IV/XRCC4 protein, the purified protein was incubated in the presence of increasing concentrations of caffeine (5, 10, 15, 20, 40, and 60 mM) in the joining buffer [30 mM HEPES-KOH (pH 7.9), 7.5 mM MgCl_2_, 1 mM DTT, 2 mM ATP, 50 μM dNTPs, and 0.1 μg BSA] at 25°C for 30 min [41, 42]. Further, the radiolabeled compatible ends DNA substrate was added to the individual reaction, and the reaction mixture was incubated at 25°C for 1 h. The reaction products were purified by phenol:chloroform extraction. The DNA was precipitated with chilled ethanol and glycogen. Following precipitation, the pellet was dried and dissolved in 10 μl of TE. The joined products were resolved on 8% denaturing PAGE. The gel was dried and exposed, and the signal was detected using phosphorImager FLA9000 (Fuji, Japan). MultiGauge software was used to quantify joined products and measured in PSLU.
In vitro nuclease assay
5′ end labeled substrate (5′-5′ noncompatible double-stranded DNA, SCR19/VK11) was incubated with Artemis in 25 mM Tris (pH 8.0), 10 mM KCl, 10 mM MgCl_2_, 1 mM dithiothreitol, and 50 µg/ml bovine serum albumin in a total volume of 10 µl reaction in the presence of increasing concentration of caffeine (5, 20, and 60 mM). An aliquot of purified Artemis was from Dr JB Charbonnier, France [36]. Samples were incubated for 1 h at 37°C and then denatured for 10 min at 95°C in an equal volume of denaturing gel loading dye. Reaction mixtures were resolved on 12% denaturing PAGE, and the gel image was obtained using phosphorImager as described above. MultiGauge software was used for the quantitation of the cleaved products in PSLU.
Complementation of the inhibition of caffeine using purified ligase IV/XRCC4 or ligase I protein
Briefly, inhibition of end joining with CFE and caffeine (10 mM) was conducted as described in the previous section. Following the inhibition reactions, the purified ligase IV/XRCC4 or ligase I was added back, and the reaction was incubated further for 1 h at 25°C. Following termination of the reaction, DNA was purified by phenol:chloroform extraction and precipitated using glycogen and chilled ethanol. The DNA pellet was dried and dissolved in 10 µl of TE. The joined products were resolved on 8% denaturing PAGE following heat denaturation. MultiGauge software was used to quantify the joined products, with measurements expressed in PSLU and plotted as a bar graph using GraphPad Prism software.
Biolayer interferometry
Biolayer interferometry (BLI) was performed with purified XRCC4, mutant XRCC4 (T133A), or XLF protein and increasing concentrations of caffeine at 4°C, as described previously [42–44]. An aliquot of purified XLF was a kind gift from Dr JB Charbonnier, France [37, 38]. Ni-NTA biosensors were used due to the His-tag on these proteins, which has a strong affinity for these sensors. Initially, the sensors were hydrated in 1× PBS buffer for 10 min in a 200 μl volume within a 96-well plate and then loaded onto the ForteBio Octet RED 96 (ForteBio, USA). The samples were incubated at 37°C with a shaking speed of 1000 RPM. The hydrated sensor was then loaded with the protein and dipped into sample wells containing serially diluted caffeine. Another sensor used as reference was similarly dipped into wells but was not loaded with protein and was used for the subtraction of the background signals. The dissociation constant (Kd) value was calculated using a global fit curve (1:1, association: dissociation model) in the Octet data analysis software (version 8.0).
Docking study using NHEJ proteins
The known crystal and cryo-EM structures of proteins used for the docking studies were downloaded from the RCSB Protein Data Bank: Artemis (PDB ID: 7ABS) [45], ATM (PDB ID: 7NI6) [46], DNA PKcs (PDB ID: 3KGV) [47], KU70/80 (PDB ID: 7AXZ) [48], ligase IV (PDB ID: 3W5O) [49], PAXX (PDB ID: 4WJA) [50], Pol λ (PDB ID: 5CB1) [51], XLF (PDB ID: 2R9A) [52], XRCC4 (PDB ID: 1FU1) [53], and the ligase IV/XRCC4 complex (PDB ID: 3II6) [54]. The structure of caffeine was obtained through PubChem (CID: 2519). Since some PDB structures were bound to ligand, ligand/ion removal was performed with UCSF ChimeraX, developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco, with support from National Institutes of Health R01-GM129325 and the Office of Cyber Infrastructure and Computational Biology, National Institute of Allergy and Infectious Diseases. The docked structures were also visualized using ChimeraX.
Molecular docking was performed in the CB-Dock and CB-Dock2 portals using default parameters [55, 56]. The first step in this process involved cavity detection, which helps in predicting the binding sites, followed by cavity sorting. The docking center and docking size were calculated, which was then used for molecular docking using AutoDock Vina. The top binding scores of the ligand–protein interactions are presented in the table. LigPlot+ was used to generate plots depicting the molecular interactions of caffeine with amino acid residues using default parameters [57].
In silico stability analysis, molecular docking, and visualization of caffeine interaction with XRCC4 and XRCC4 T133A
To assess the impact of mutating T133A in the XRCC4 protein, we performed stability analysis using three different tools: DynaMut [58], mCSM [59], and I-Mutant2.0 [60]. The mutated protein was extracted from DynaMut and molecular docking with caffeine was performed using CB-Dock2 for wild-type and mutant XRCC4. The interactions were visualized using LigPlot+ .
NHEJ reporter assay
Intracellular NHEJ activity was performed as described previously [20, 28, 61, 62]. Briefly, Molt4 and HeLa cells (4 × 10^5^) were seeded in six-well plates and transfected with 20 μg of I-SceI overexpression construct and 15 μg of pimEJ5-GFP reporter construct using branched PEI (polyethylenimine) in the presence of increasing concentrations of caffeine (0.5, 1, 2, and 5 mM). Following transfection, cells were incubated at 37°C. After 48 h, cells were harvested, and the number of GFP-positive cells was analyzed by Flow cytometry (Cytoflex S, Beckman Coulter, USA). A violin plot was plotted for the percentage of GFP-positive cells. SCR7 (20 µM) was used as a positive control for NHEJ inhibition. To normalize transfection efficiency, a plasmid encoding the Green Fluorescent Protein (pmaxGFP) was used as a transfection control.
HR reporter assay
HeLa cells (4 × 10^5^) were seeded in six-well plates and transfected with 5 μg each of I-SceI overexpression construct and DR-GFP (pJS65) reporter construct using branched PEI in the presence of increasing concentrations of caffeine (0.5, 1, 2, and 5 mM). Following transfection, cells were incubated at 37°C. After 48 h, cells were harvested, and the number of GFP-positive cells was analyzed by Flow cytometry (Cytoflex S, Beckman Coulter, USA).
MMEJ reporter assay
HeLa cells (4 × 10^5^) were seeded in six-well plates and co-transfected with 5 µg each of the I-SceI overexpression plasmid and the pJS248 reporter construct using branched PEI in the presence of increasing concentrations of caffeine (0.5, 1, 2, and 5 mM). Following transfection, cells were incubated at 37°C for 48 h, after which they were harvested and analyzed for GFP-positive populations by flow cytometry (CytoFLEX S, Beckman Coulter, USA).
Cell viability assay
Cell viability after treatment with caffeine followed by exposure to irradiation was assayed by trypan blue exclusion assay [63–65]. Briefly, 50 000 cells/ml Jurkat or Molt4 cells were seeded. Cells were treated with caffeine (2 and 5 mM) followed by exposure to IR (5 Gy), and harvested at 48 h time point post IR. The number of viable cells was determined by mixing cells with equal volume of 0.4% Trypan blue stain (Sigma Chemical Co., St Louis, MO, USA). The cells were then counted using a hemocytometer and the % viable cells was plotted as bar graphs using GraphPad Prism 7.
Cell proliferation was measured using Alamar Blue Assay [66]. For this assay, 10 000 cells were seeded and incubated with increasing concentration of caffeine (2 and 5 mM) followed by IR exposure (5 Gy) for 48 h at 37°C. After 48 h of treatment, 1× alamar blue dye was added and incubated at 37°C for 2–3 h followed by measuring the absorbance at 570 and 600 nm wavelength using microplate reader (VICTOR Nivo, PerkinElmer). Bar graphs were plotted indicating the percentage of cell proliferation using GraphPad Prism 7 software.
Immunofluorescence
Cells were cultured on coverslips (50 000 cells/ml for 24 h). The cells were then treated with increasing concentrations of caffeine (1, 2, 5, and 10 mM) for 5 h at 37°C and 5% CO_2_ and subjected to immunofluorescence study as described previously [32, 67]. Cells were washed with phosphate-buffered saline (PBS, 1×) and fixed using 2% paraformaldehyde (10 min at room temperature). Following fixing, cells were permeabilized using 1× PBS + 0.1% Triton X-100 (PBST) for 10 min at room temperature. Cells were then blocked using PBST + 0.1% BSA + 10% FBS for 1 h at 4°C and incubated with appropriate primary antibodies, γ-H2AX (Cell Signaling Technology, USA), 53BP1 (Invitrogen, USA), and XRCC4 (BD Biosciences) at 4°C, overnight. Corresponding Alexa Fluor conjugated secondary antibodies (Life Technologies, USA) were added, and the cells were incubated at room temperature for 2 h. Following antibody staining, the coverslip was mounted using DAPI and diazabicyclo[2.2.2]octane (DAPI:DABCO) mix. Images were captured using a laser confocal microscope (Olympus, FLUOVIEW FV3000, Japan) and processed using Olympus software.
To assess the time-dependent resolution of DSBs in caffeine treated cells upon exposure to the DSB inducer, HeLa and HeLa ligase IV knock out cells (HeLa.Lig4.catDΔ.1; indicated in the study as HeLa LIV KO) were treated with 5 mM caffeine for 5 h followed by exposure to ionizing radiation (IR; 5 Gy). Cells were harvested at 0.5, 4, 12, and 24 h post-irradiation and proceeded for immunofluorescence as described above.
For colocalization analysis of XRCC4 and γ-H2AX, HeLa cells were pretreated with 5 mM caffeine for 5 h and subsequently exposed to IR (5 Gy). Cells were fixed 30 min post-irradiation and subjected to XRCC4 and γ-H2AX co-immunostaining as described above.
TUNEL assay
Apoptotic DNA fragmentation was detected using a terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay (Promega, USA) [68]. HeLa cells were cultured on coverslips (50 000 cells/ml). The cells were then treated with increasing concentrations of caffeine (5 and 10 mM) for 24 h at 37°C and 5% CO_2._ Following treatment cells were fixed in 4% paraformaldehyde for 10 min at room temperature. After two PBS washes, cells were permeabilized with 0.2% Triton X-100 at room temperature for 10 min, rinsed with 1× PBS, and equilibrated in the manufacturer’s equilibration buffer. Labeling was performed using the TUNEL reaction mix according to the kit instructions for 2 h at 37°C in a humidified chamber. Reactions were stopped by adding 2× SSC buffer. Nuclei were counterstained with DAPI and coverslips were mounted with anti-fade mounting medium, DABCO. Slides were imaged by fluorescence microscopy (Nikon, Shinagawa, Tokyo, Japan).
Comet assay
The generation of DSBs upon caffeine treatment following irradiation exposure was determined by performing a neutral comet assay as described previously [35, 69]. Cells were treated with caffeine (5 mM for 5 h) followed by exposure to gamma rays (5 Gy). Cells were harvested at 0.5, 4, 18, and 24 h post-irradiation and washed using 1× PBS. Approximately 10 000 cells were mixed with low-melting agarose and spread on an agarose-coated glass slide. The slides were subjected to lysis overnight at 37°C using neutral lysis buffer (0.5 M EDTA, 2% Sarkosyl, and 0.5 mg/ml Proteinase K). Following lysis, slides were thoroughly rinsed with neutral electrophoresis buffer [90 mM Tris–HCl (pH 8.0), 90 mM boric acid, and 2 mM EDTA] and electrophoresed at 12 V for 25 min. After electrophoresis, slides were washed with double-distilled water and stained with propidium iodide (1 µg/ml) for 20 min at room temperature. Excess stain was removed by washing with double-distilled water for 15 min, and slides were stored at 4°C in a moist chamber until imaging. Images were acquired at 10× magnification using a Nikon epifluorescence microscope and processed with ImageJ software. Quantitative analysis of DNA damage (Olive tail moment) was performed using CometScore software, and data were represented as line plot.
Clonogenic assay
Clonogenic survival assays were performed to assess the long-term proliferative capacity of HeLa and HeLa LIV KO cells following caffeine treatment [70]. Cells were trypsinized, counted, and seeded in six-well plates at densities optimized to yield approximately 50–200 colonies per well under control conditions (1000 cells). Cells were allowed to adhere overnight followed by treatment with increasing concentration of caffeine (1, 2, 5, 10, and 20 mM) for 24 h. Following treatment, medium was changed, washed with 1× PBS and cells were incubated in drug free medium at 37°C in a humidified incubator with 5% CO_2_ for 14 days to allow colony formation. Colonies were then fixed with methanol:acetic acid (3:1) for 30 min, stained with 0.5% (w/v) crystal violet prepared in 50% methanol for 20 min, gently rinsed with distilled water, and air-dried. Colonies consisting of ≥50 cells were counted manually.
Immunoprecipitation
For immunoprecipitation (IP), equal amounts of Molt4 CFE (30 µg) were incubated with 2 µg anti-ligase IV or normal IgG (negative control) overnight at 4°C with gentle rotation [71]. The immune complexes were captured by adding 15 µl of pre-washed Protein A/G agarose beads (Santa Cruz Biotechnology) and incubated for an additional 2 h at 4°C with rotation. Beads were spun down to collect the supernatant (immunodepleted fraction), which was then used for NHEJ assay. Beads were then washed three times with ice-cold IP buffer to remove nonspecific proteins, ensuring minimal bead loss during each wash. For western blotting, whole cell and immunodepleted extracts were electrophoresed on SDS–PAGE, transferred onto a PVDF membrane, and probed with anti-ligase IV.
Immunoblotting
For the immunoblotting assay, 30 µg protein from Molt4, Jurkat, HeLa and HMF-3S extract was resolved on an 8% SDS–PAGE [8, 17, 72–74]. Following electrophoresis, proteins were transferred to an activated PVDF membrane (Millipore, USA) at 120 mA and blocked with 5% skimmed milk powder for 1 h at room temperature. Proteins of interest were probed with appropriate primary antibodies KU70, ligase IV, Polymerase λ, Polymerase µ, XLF, XRCC4, PCNA ( Proliferating Cell Nuclear Antigen), APLF, and Artemis for overnight at 4°C. The proteins were then probed with the appropriate secondary antibodies as per standard protocol. The blots were developed using a chemiluminescent substrate (Immobilon^™^ Western, Millipore), and images were acquired using a chemiluminescence documentation system (Bio-Rad).
Statistical analysis
Statistical analysis was carried out using GraphPad Prism software (Version 6 and 7). Data are presented as mean ± standard error of the mean (SEM) from three independent biological replicates, unless otherwise specified. Comparisons between two groups were analyzed using the unpaired two-tailed Student’s t-test, while comparisons among more than two groups were evaluated using one-way analysis of variance (ANOVA). Differences were considered statistically significant at P < 0.05 and *, **, ***, **** denotes *P *< 0.05, *P *< 0.005, *P *< 0.001, and *P *< 0.0001, respectively.
Results
Caffeine inhibits the joining of DSBs irrespective of the sequence, DNA end configuration, and the cell types
The configuration of DSBs, and sequence of DNA terminus can vary when DNA breaks occur within cells. The DSB configuration can be a blunt, a 5′ overhang, or a 3′ overhang, depending on the context. The majority of the DSBs generated inside the cells are generally noncompatible in nature (i.e. without base complementarity and cannot be repaired by direct ligation), and majority of such broken DNA ends are efficiently repaired by NHEJ. To investigate whether caffeine can inhibit NHEJ, firstly, a biochemical assay system was used in which oligomeric DNA substrates possessing different noncompatible ends were incubated with CFE from two cancer cell lines, Jurkat and Molt4 (Supplementary Fig. S1A). For this, we used a 5′–5′ noncompatible end dsDNA substrate (SCR19/VK11) and a 5′–3′ noncompatible end dsDNA substrate (SCR19/VK13) (Fig. 1A). The CFE was prepared from leukemic cell lines, Jurkat and Molt4, in which the efficiency of NHEJ was known to be high [25]. An increasing concentration (0.5, 1, 2, 5, 10, and 20 mM) of caffeine was incubated with cell extracts and appropriate DNA substrate. The joined product formation was evaluated by resolving the purified DNA products on a denaturing polyacrylamide gel (Supplementary Fig. S1A). Results showed a concentration-dependent decrease in the joining efficiency when oligomeric DNA with a 5′–5′ noncompatible end was incubated with Jurkat CFE in the presence of caffeine (Fig. 1B and C). Compared to the untreated control, where the joining efficiency was maximum, even the addition of lowest concentration of caffeine resulted in a significant reduction in the joining efficiency (Fig. 1B and C). A comparable inhibitory effect was observed when Molt4 CFE was incubated with the 5–5′ noncompatible end (SCR19/VK11) (Fig. 1D and E). When 5′–3′ noncompatible ends were used, a caffeine concentration-dependent inhibition in the joining was observed in the case of Jurkat and Molt4 cell extracts (Fig. 1F–I). >50% of the inhibition of NHEJ was observed when incubated with 10 and 20 mM caffeine in both the cell lines. The effect was more pronounced when the 5′–5′ noncompatible ends DNA substrate (SCR19/VK11) and the 5′–3′ noncompatible ends DNA substrate (SCR19/VK13), were incubated with a higher concentration of caffeine in the presence of Jurkat as well as Molt4 CFE (Supplementary Fig. S1B–I).
*Evaluation of the effect of caffeine on the joining of DSBs with the noncompatible end when incubated with CFEs.(A) Pictorial representation of the sequence of oligomers with DSBs having 5′–5′ or 5′–3′ noncompatible ends (SCR19/VK11 and SCR19/VK13, respectively) used for the end-joining assay. (B) Representative denaturing PAGE profile showing the effect of increasing concentration of caffeine (0.5, 1, 2, 5, 10, and 20 mM) on the joining of noncompatible end substrate (SCR19/VK11) in the presence of CFE from Jurkat cells. (C) The bar diagram showing the quantitation of the experiment shown in panel (B) (n = 3). The joined product was quantified using Multi Gauge V3.0 software; the data represents mean ± SEM. (D) The PAGE profile shows the impact of caffeine on the end joining of the substrate shown in panel (A) in the presence of CFE from Molt4 cells. (E) The bar diagram showing the quantitation of the experiment shown in panel (D) (n = 3); the data show mean ± SEM. (F) Representative denaturing PAGE gel showing the effect of caffeine (0.5, 1, 2, 5, 10, and 20 mM) on the joining of 5′–3′ noncompatible end substrate (SCR19/VK13) in the presence of Jurkat CFE. (G) The bar diagram showing the quantitation of the experiment shown in panel (F) (n = 3); the data shown are mean ± SEM. H. Denaturing PAGE gel image depicting the impact of caffeine on the end joining of SCR19/VK13 in the presence of Molt4 extract. (I) The bar diagram showing the quantitation of the experiment shown in panel (H) (n = 3); the data represent mean ± SEM. (J and K) The bar diagram showing the quantitation of the effect of caffeine on the joining of 5′–5′ noncompatible substrate DG11/12 (AT rich DNA) when incubated with CFE prepared from Jurkat (J) and Molt4 cells (K); the data represent mean ± SEM (n = 3). (L and M) The bar graph showing the effect of caffeine on the joining of 5′–5′ noncompatible substrate DG13/14 (GC-rich DNA) when incubated with CFE from Jurkat(L) and Molt4 (M) cells; the data represent mean ± SEM. In all panels, ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.001, ***P < 0.0001.
The above results suggest that caffeine may interact with/inhibit the activity of the key protein/proteins involved in the NHEJ-mediated DSB repair. When the joining assay was performed using 5′–5′ noncompatible substrates with different flank sequences (AT- and GC-rich sequences) by incubating with CFE from Jurkat or Molt4, a dose-dependent inhibition of joined product formation was observed in the presence of caffeine with the maximum inhibition at the highest concentrations (Fig. 1J–M and Supplementary S2A–E). Overall, the above results demonstrated that exposure to caffeine leads to the inhibition of the joining of noncompatible end DNA substrate, irrespective of the nature of the DNA end sequence or the polarity.
For the above studies highly proliferative cancer cells were used as they exhibit elevated replication stress and a greater reliance on DSB repair pathways to maintain genomic integrity and sustain rapid cell division. Therefore, they provide a sensitive and biologically relevant system to investigate how caffeine influences NHEJ activity. To determine whether the caffeine-induced inhibition of end joining was not specific to cancer cells, we next assessed NHEJ efficiency of normal, but transformed fibroblast cells, HMF-3S. Results showed a concentration-dependent decrease in the joining efficiency when oligomeric DNA with a 5′–5′ or 5′–3′ noncompatible end were incubated with HMF-3S CFE in the presence of caffeine (Fig. 2A–D), with a marked reduction observed at ≥5 mM.
*Assessment of effect of caffeine on joining of DSBs with noncompatible end using CFE from cell lines derived from primary cells, RTE and efficacy of joining when cells were irradiated to activate NHEJ proteins.(A) Representative denaturing PAGE profile showing the effect of increasing concentrations of caffeine (0.5, 1, 2, 5, 10, and 20 mM) on the joining of a 5′–5′ noncompatible end substrate (SCR19/VK11) in the presence of CFE derived from HMF-3S cells. (B) The experiment shown in panel (A) was performed three independent times, and the joined products were quantified using Multi Gauge V3.0 software. The resulting data are presented as a bar graph representing mean ± SEM. (C) Representative denaturing PAGE image showing the effect of increasing concentration of caffeine (0.5, 1, 2, 5, 10, and 20 mM) on the joining of 5′–3′ noncompatible end substrate (SCR19/VK13) in the presence of CFE from HMF-3S (human mammary fibroblasts) cells. (D) The bar graph representing the quantitation of the experiment shown in panel (C) (n = 3). The joined products were quantified using Multi Gauge V3.0 software; the data represents mean ± SEM. (E) Western blot analysis to determine the levels of various NHEJ repair proteins (KU70, ligase IV, Polymerase λ, Polymerase µ, XLF, XRCC4, APLF, and Artemis) in Molt4, Jurkat, HeLa, and HMF-3S extracts. (F) Bar graph showing the relative protein expression levels of key NHEJ proteins in cancer cell lines (Molt4, Jurkat, HeLa) and normal human fibroblasts (HMF-3S). Each of the protein levels were quantified and normalized to total protein content using ponceau. Data represent mean ± SEM from two independent experiments. Statistical significance was calculated with respect to Jurkat cell line. (G) SDS–PAGE showing the protein profile of extract prepared from unirradiated and irradiated (5 Gy) Jurkat cells. (H and J) Representative denaturing PAGE profile showing the impact of increasing concentration of caffeine (0.5, 1, 2, 5, 10, 20, 40, and 60 mM) on the end joining of 5′–5′ noncompatible end substrate (SCR19/VK11) derived from unirradiated Jurkat cells (H) and Jurkat cells exposed to 5 Gy IR (J) to promote NHEJ complex assembly. (I and K) The bar graph represents the quantitation of the experiment represented in panel (H) (n = 3) and (J) (n = 3). The joined products were quantified using Multi Gauge V3.0 software; the data represent mean ± SEM. (L) Representative denaturing PAGE image showing the impact of increasing concentration of caffeine (0.5, 1, 2, 5, 10, and 20 mM) on the end joining of 5′–5′ noncompatible end substrate (SCR19/VK11) in the presence of RTE. (M) The bar diagram showing the quantitation of the experiment showed in panel (L) and Supplementary Fig. S3A (n = 3); the data shown are mean ± SEM. (N) Representative denaturing PAGE image showing the impact of caffeine (0.5, 1, 2, 5, 10, and 20 mM) on the end joining of 5′–3′ noncompatible end substrate (SCR19/VK13) in the presence of RTE. (O) The bar diagram showing the quantitation of the experiment shown in panel (N) and Supplementary Fig. S3B (n = 3); the data represent mean ± SEM. In all panels, ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.001, ***P < 0.0001.
To test whether these cell lines indeed possessed the core NHEJ machinery, western blotting analysis was performed for the presence of key NHEJ proteins. HeLa cells were also included in this analysis, and in subsequent studies as it is an adherent cell line. All cell lines expressed the major NHEJ proteins, including KU70, DNA ligase IV, XRCC4, XLF, and the polymerases Pol μ and Pol λ, confirming the presence of major NHEJ machinery (Fig. 2E). However, notable differences in expression levels were observed among the cell types. KU70 and ligase IV were highly abundant in the lymphoid-derived Molt4 and Jurkat cells compared to HMF-3S. XRCC4 and XLF levels were comparable in all the four cell lines tested. Among the polymerases, Pol μ was more prominently expressed in the lymphoid lines, whereas Pol λ was present at comparable levels across all cell types (Fig. 2E and F). Accessory NHEJ factors such as APLF and Artemis were detectable in all cell lines, though APLF showed lower abundance in Jurkat cells. The PCNA was used as the loading control to confirm equal protein loading across the samples. Overall, these results indicate that while all tested cell lines harbor the core NHEJ components, Molt4 and Jurkat cells exhibit relatively higher basal expression of NHEJ proteins compared to HeLa and HMF-3S (Fig. 2E and F).
Since there was a difference in the basal expression levels of NHEJ proteins, we explored strategies to enhance the assembly of active NHEJ repair complexes in cultured cells. To facilitate recruitment and stabilization of NHEJ factors at DNA damage sites, Jurkat cells were exposed to DSB inducing agent such as IR. Following IR treatment (5 Gy), cells were harvested at 0.5 h time point, the CFE was prepared and joining assay was performed using 5′–5′ noncompatible end substrate (Fig. 2G). CFE prepared from unirradiated cells served as the control (Fig. 2G). Results showed that extracts derived from irradiated Jurkat cells displayed enhanced joining activity compared to unirradiated control (lanes 2 and 3, Fig. 2H and J), consistent with an increased abundance or activation of repair complexes following DSB induction. Importantly, even in the DSB-induced extract, caffeine treatment led to a dose dependent inhibition of joining, though the extent of inhibition was slightly reduced at lower concentrations as compared to unirradiated control (Fig. 2H–K) suggesting partial functional compensation by the damage-induced upregulation of NHEJ components. Together, these findings demonstrate that caffeine suppresses the NHEJ activity in a concentration-dependent manner, and that DSB induction enhances the assembly and catalytic efficiency of NHEJ machinery in cell extracts, thereby partially mitigating the inhibitory impact of caffeine.
To test whether the above results of caffeine-mediated inhibition of NHEJ of human cell line extracts are also seen in a normal cellular context, we performed a joining assay using extract prepared from primary rat tissue, the testes. The rat testicular extract (RTE) was used for the study, as it possesses the highest efficiency of end joining among different organs [26, 27]. To investigate the effect of caffeine on NHEJ-mediated repair catalysed by the testes extract, we performed the joining assay with oligomeric DNA with different noncompatible ends in the presence of an increasing concentration of caffeine (0.5, 1, 2, 5, 10, 20, 40, and 60 mM). Results showed a significant inhibition of end joining with the increasing concentration of caffeine starting from the lowest concentration (0.5 mM). Overall, a notable decrease in the joined product formation was observed in the case of both 5′–5′ noncompatible end substrate (SCR19/VK11) (Fig. 2L and M; Supplementary Fig. S3A) as well as 5′–3′ noncompatible end substrate (SCR19/VK13) (Fig. 2N and O, and Supplementary Fig. S3B). Moreover, when another 5′–5′ noncompatible end DNA substrate (DG15/16) harboring different end sequences was used for the study in the presence of RTE, we observed similar inhibition in the joined product formation (Supplementary Fig. S3C and D). Thus, results suggest that caffeine inhibited end joining irrespective of the configuration of DSBs, even when the extract from primary cells was used.
Caffeine inhibits the NHEJ within human cells
An extrachromosomal assay system was employed to evaluate the effect of caffeine on NHEJ within the human cells (Fig. 3A and B) [20, 28, 61]. To do this, we used the NHEJ reporter construct, pimEJ5-GFP, having the disrupted GFP gene and I-SceI sites as described before [20]. When the DSBs induced by I-SceI in pimEJ5-GFP episome are repaired by NHEJ, GFP expression is restored (Fig. 3B). Molt4 cells were transiently transfected with pimEJ5-GFP reporter and I-SceI overexpression constructs in the presence of increasing concentrations of caffeine (0.5, 1, 2, and 5 mM; Fig. 3C). Cells were harvested after 48 h and subjected to flow cytometry analysis to determine the number of GFP-positive cells upon expressing I-SceI, which reflects repair through NHEJ (Fig. 3C and D). Interestingly, upon the addition of caffeine, a decrease in the GFP-positive cells was observed, which suggests the inhibition of NHEJ in the presence of caffeine at the intracellular level (Fig. 3C and D). SCR7 (20 µM) was used as the positive control and exhibited a comparable reduction in GFP-positive cells (Fig. 3C and D). Plasmid encoding GFP (pmaxGFP) served as an internal control to verify successful transfection and to normalize for variations in transfection efficiency between samples (Fig. 3C and D). The study was also performed in an independent cell line, HeLa (Fig. 3E). In HeLa cells, caffeine concentrations of above 1 mM significantly reduced GFP-positive populations, with a marked inhibition (∼70%–80%) observed at 5 mM compared to untreated controls (Fig. 3E and Supplementary Fig. S4). In summary, the treatment of caffeine resulted in the inhibition of NHEJ within cells.
*Evaluation of impact of caffeine on NHEJ-mediated DSB repair within human cells.(A) Representative cartoon showing the steps followed to evaluate the impact of caffeine on NHEJ in human cells. (B) Schematic representation of intracellular NHEJ assay. PimEJ5-GFP is the reporter construct that harbors the disrupted GFP sequence flanked by I-SceI sites. Upon transfection with the I-SceI overexpression construct, a double-strand break is induced on PimEJ5-GFP, which, upon repair by NHEJ, can restore the GFP expression. (C) Histograms representing the comparison of intracellular NHEJ activity in caffeine-treated Molt4 cells as compared to untreated cells. SCR7 (20 µM) was used positive control. GFP expression plasmid (pmaxGFP) was used to normalize transfection efficiency. (D) GFP positive cells, post-transfection of Molt4 with PimEJ5-GFP and I-SceI overexpression constructs in the presence of caffeine were analyzed using flow cytometry and plotted as a violin plot representing mean ± SEM. (E) GFP-positive cells obtained after co-transfection in HeLa cells with the PimEJ5-GFP reporter and I-SceI expression constructs in the presence of caffeine were analyzed by flow cytometry, and the results are presented as a violin plot showing mean ± SEM. (F) Schematic representation of the HR reporter assay (DR-GFP system). The reporter plasmid (pJS65) contains two nonfunctional GFP fragments, an I-SceI recognition site within the sceGFP sequence and a downstream internal GFP fragment (iGFP). Expression of the I-SceI endonuclease introduces a site-specific DSB within sceGFP. Successful HR-mediated repair using the iGFP sequence as the template restores the functional GFP gene, resulting in GFP expression. (G) Graph representing the GFP positive cells as a measure of intracellular HR activity upon transfection in HeLa cells with pJS65 and I-SceI overexpression constructs in the presence of caffeine. (H) Schematic representation of the MMEJ reporter assay (pJS248). The reporter plasmid contains a disrupted GFP gene with an I-SceI recognition site flanked by 8 nt microhomology sequence. Expression of I-SceI introduces a site-specific DSB. Repair through the MMEJ pathway aligns the microhomology regions, resulting in a 35 nt deletion and restoration of a functional GFP gene. GFP expression thus serves as a readout for MMEJ-mediated DSB repair efficiency. (I) Graph representing the percentage of GFP positive cells as a measure of intracellular MMEJ activity upon transfection in HeLa cells with pJS248 and I-SceI overexpression constructs in the presence of caffeine. A total of 10 000 events were acquired per sample during flow cytometry analysis. (In panles D, E, G and I; ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.001, ***P < 0.0001).
To determine whether the inhibitory effect of caffeine on DNA repair was specific to the NHEJ pathway, we performed parallel experiments using the HR reporter system (DR-GFP) in HeLa cells. In this assay, cells were treated with increasing concentrations of caffeine followed by transfection with the DR-GFP construct (Fig. 3F). Results showed that caffeine treatment led to a dose-dependent reduction in the percentage of GFP-positive cells, indicating a diminished HR repair efficiency (Fig. 3F and G), which was consistent with previous studies [11, 12]. These findings suggest that caffeine not only interferes with the NHEJ pathway but also compromises the HR-mediated repair of DSBs, thereby exerting a broad inhibitory effect on the major DSB repair mechanisms in human cells.
Further, to assess whether caffeine also influences the alternative end joining pathway, MMEJ activity was evaluated using the EJ2-GFP reporter system (Fig. 3H). Results showed that caffeine treatment led to a reduction in MMEJ-mediated repair at 1 mM caffeine concentration; however, the effect was not dose-dependent (Fig. 3I). Furthermore, the inhibition was more pronounced in the case of NHEJ, suggesting that the classical end joining pathway is more sensitive to caffeine exposure than MMEJ. Nonetheless, the precise mechanism by which caffeine impacts MMEJ remains unclear and warrants further investigation. Collectively, these findings demonstrate that caffeine inhibits NHEJ- and HR-mediated DSB repair in a dose-dependent manner across multiple cell types.
Inhibition of NHEJ by caffeine inside the human cells leads to the accumulation of unrepaired DSBs
Based on the above observations, we were curious to test whether the inhibition of NHEJ inside the cells could result in the accumulation of unrepaired endogenous chromosomal DSBs at the genome level. To test this, we treated HeLa (cancer cell line) and HMF-3S (primary cell line) cells with an increasing concentration of caffeine (1, 2, 5, and 10 mM) followed by immunofluorescence using the γ-H2AX antibody (Fig. 4A–E). Results showed an increase in the levels of γ-H2AX foci in HeLa cells with the increasing concentration of caffeine, which is indicative of the accumulation of unrepaired DSBs within cells (Fig. 4B and C, and Supplementary Fig. S5A), suggesting that caffeine blocked the NHEJ-mediated repair even in chromosomal DNA. SCR7 (50 µM) was used as the positive control. Comparable results were observed in HMF-3S cells (Fig. 4D and E). 53BP1 (p53-binding protein 1) was also used to detect DSBs in DNA because 53BP1 helps to select the repair pathway choice for DSBs [9]. Following 53BP1 staining in HeLa cells post-caffeine treatment, we observed a remarkable increase in the induction of DSBs, demonstrating accumulation of DNA breaks upon caffeine treatment (Fig. 4F and Supplementary Fig. S5B). These results suggest that caffeine blocks the NHEJ-mediated repair inside the mammalian cells, leading to the accumulation of unrepaired DSBs.
*Evaluation of the effect of caffeine on chromosomal DSB repair within the human cells.(A) Schematic presentation showing the steps associated with evaluating the impact of caffeine on the repair of DSBs within HeLa cells. (B) Representative immunofluorescence images showing γ-H2AX foci as a marker for DSBs inside the nucleus. HeLa cells were treated with caffeine (1, 2, 5, and 10 mM) for 5 h and were subjected to immunofluorescence analysis using anti-γ-H2AX antibody. The nucleus was stained using DAPI. In each case, a merged image for both signals has been shown as a panel on the right. SCR7 (50 µM) was used as a positive control for NHEJ inhibition. Scale bar represents 10 µm. (C) The experiment was repeated a minimum of three times, and approximately 100 cells were evaluated for each sample. The number of foci is represented as a violin plot showing mean ± SEM. (D) Representative immunofluorescence images show γ-H2AX foci as a marker for DSBs in HMF-3S cells following caffeine treatment. HMF-3S cells were treated with caffeine (1, 2, 5, and 10 mM) for 5 h and were subjected to immunofluorescence using anti-γ-H2AX antibody. SCR7 (50 µM) was used as positive control. The nucleus was stained using DAPI. In each case, a merged image for both signals has been shown as a panel on the right. Scale bar represents 10 µm. (E) The experiment was repeated three independent times, and approximately 100 cells were evaluated for each sample. The foci are represented as a violin plot showing mean ± SEM. (F) Violin plot representing the number of 53BP1 foci in HeLa cells following caffeine (1, 2, 5, and 10 mM) treatment. The experiment was repeated twice, and approximately 100 cells were evaluated for each sample and the average foci are represented as a violin graph showing mean ± SEM. (G) Representative neutral comet assay images showing DNA strand breaks in HeLa cells treated with caffeine (5 mM), exposed to IR (5 Gy), or both in combination, harvested at indicated time points post IR exposure. Scale bar represents 100 µm (In panels C, E and F; ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.001, ***P < 0.0001).
To examine whether caffeine affects the repair kinetics of IR-induced DNA DSBs, a neutral comet assay was performed at different time points post-irradiation (0.5, 4, 18, and 24 h) (Fig. 4G). HeLa cells were treated with caffeine (5 mM, 5 h) followed by exposure to IR (5 Gy), and the extent of DNA damage was assessed based on comet tail formation. Control cells displayed intact nuclei with minimal or no comet tails, indicating negligible DNA breaks. Cells exposed to IR alone showed pronounced comet tails immediately after irradiation (0.5 h), reflecting substantial DNA damage, followed by a gradual reduction at later time points (18–24 h), consistent with efficient DSB repair (Fig. 4G and Supplementary Fig. S5C). Treatment with caffeine also resulted in detectable comet tails, indicating accumulation of DNA breaks likely due to inhibition of the NHEJ repair pathway, leading to unresolved DSBs. Notably, cells treated with both caffeine and IR exhibited extensive and persistent comet tails even at 18 and 24 h post-irradiation, indicating a marked delay in DNA repair. The persistence of DNA damage in the caffeine + IR group (Fig. 4G and Supplementary Fig. S5C) suggests that caffeine interferes with the efficient resolution of IR-induced DSBs, likely through inhibition of the NHEJ pathway. We observed a significant increase in DSBs at the higher concentrations of caffeine, particularly at 5 and 10 mM concentrations, and these DBSs, if left unrepaired, can be harmful as this could lead to cell death. To test for the presence of DSBs, we performed a TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay in HeLa cells following treatment with caffeine. Cells were exposed to increasing concentrations of caffeine (5 and 10 mM) for 24 h, and DNA fragmentation (a hallmark of persistent or unrepaired DSBs) was visualized using fluorescently labeled dUTP incorporation at free DNA ends. Cells treated with the NHEJ inhibitor SCR7 served as a positive control for DSBs accumulation. While untreated control cells exhibited minimal FITC fluorescence, indicating low basal levels of DNA breaks under normal conditions, cells treated with caffeine displayed a marked increase in green FITC signal intensity, indicative of enhanced DNA fragmentation and accumulation of DNA ends accessible to TUNEL labeling (Fig. 5A). The extent of fluorescence increased with caffeine concentration, with 10 mM caffeine showing a pronounced signal compared to 5 mM, suggesting dose-dependent increase in cell death. The SCR7-treated cells also exhibited strong FITC staining, similar to the 10 mM caffeine group, further validating that the observed effect results from inhibition of the NHEJ repair pathway.
*Impact of different concentration of caffeine treatment on cell survival in human cells. (A) Representative fluorescence microscopy images showing TUNEL staining in HeLa cells following treatment with increasing concentrations of caffeine (5 and 10 mM) for 24 h. DAPI (blue) marks the nuclei, and FITC (green) indicates TUNEL positive cells representing DNA fragmentation. In each case, a merged image for both signals has been represented as a panel on the right. Scale bar represents 100 µm. SCR7 (50 µM) was used as a positive control. (B and D) Bar graph representing the percentage of viable cells in caffeine (2 and 5 mM) treated Jurkat (B) and Molt4 (D) cells following IR (5 Gy) exposure as determined by trypan blue assay. The bar graph represents mean ± SEM (n = 3). (C and E) Alamar blue assay representing the percentage of cell proliferation in caffeine (2 and 5 mM) treated Jurkat (C) and Molt4 (E) cells upon exposure to IR (5 Gy). The experiment was repeated three independent times, and the bar graph represents mean ± SEM (In panels B-E; ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.001, ***P < 0.0001).
To evaluate the effect of caffeine on cell viability and proliferation following DSBs induction, Jurkat and Molt4 cells were treated with increasing concentrations of caffeine (2 and 5 mM) followed by exposure to IR (5 Gy). Cell viability was determined using the trypan blue exclusion assay, and proliferation was assessed by Alamar blue assay at 48 h post-treatment. In Jurkat cells, IR exposure alone resulted in a reduction in viability compared to untreated controls (Fig. 5B and C). However, co-treatment with caffeine led to a dose-dependent decrease in the percentage of viable cells, with a significant reduction observed even at 2 mM caffeine. At 5 mM caffeine, cell viability dropped drastically, indicating a strong cytotoxic effect when combined with IR (Fig. 5B and C). Similarly, caffeine markedly suppressed proliferation of Molt4 cells following DSBs induction (Fig. 5D and E). These findings demonstrate that caffeine suppresses cellular DSB repair capacity, leading to the persistence of unrepaired DNA lesions that can ultimately contribute to cell death in a concentration-dependent manner.
In silico screening studies identify potential NHEJ protein targets for caffeine
Since the above results suggest that caffeine can inhibit NHEJ-mediated repair of double-strand breaks, we were interested in identifying the key protein/proteins associated with NHEJ that is/are targeted by caffeine. Molecular docking of caffeine with various NHEJ proteins was performed using the mathematical algorithm-based web portal CB-Dock and CB-Dock2 for the determination of the docking score (Fig. 6A). CB-Dock is a protein-ligand docking method that automates the identification of binding sites, computes their centers and sizes, customizes docking box dimensions based on the query ligands, and conducts molecular docking using AutoDock Vina [75].
Evaluation of target protein for caffeine when it inhibits NHEJ.(A) Table showing binding scores for NHEJ proteins following docking analysis of caffeine with various NHEJ proteins using CB Dock and CBDock2. (B) Representative image showing the docked pose of caffeine with KU70 heterodimer. The red color blot corresponds to caffeine molecule. (C) A representative image showing the docked position of Artemis upon interaction with caffeine (red blot). (D) Cartoon representation showing the structure of XRCC4 with ligand (caffeine) bound conformation. The orange colour blot representation corresponds to caffeine molecule. (E) SDS–PAGE profile showing the purification of XRCC4 using Ni-NTA column. (F)BLI sensorgrams depicting the real-time binding of caffeine with XRCC4. (G) Table representing Kd values for caffeine interaction with different NHEJ proteins. Binding study of caffeine with XRCC4, XRCC4 (T133A) mutant and XLF protein showed a Kd value of 3.8, 26.8, and 11.1 µM, respectively. (H) 3D structure of XRCC4 protein showing the docked position with caffeine. (I) BLI sensograms depicting the binding of caffeine with XLF protein. (J) BLI sensorgrams illustrating the real-time interaction between caffeine and XRCC4 mutant, T133A.
The known crystal and cryo-EM structures of major NHEJ proteins such as Artemis (PDB ID: 7ABS) [45], ATM (PDB ID: 7NI6) [46], DNA PKcs (PDB ID: 3KGV) [47], KU70/80 (PDB ID: 7AXZ) [48], ligase IV (PDB ID: 3W5O) [49], PAXX (PDB ID: 4WJA) [50], Pol λ (PDB ID: 5CB1) [51], XLF(PDB ID: 2R9A) [52], and XRCC4 (PDB ID: 1FU1) [53] as well as the ligase IV/XRCC4 complex (PDB ID: 3II6) [54] were downloaded from the Protein Data Bank (PDB). ChimeraX was used to remove the ligand or ion/molecule from the protein, which was then used for blind docking. The docking score was used to screen the affinity of the ligand of interest (caffeine) to the NHEJ protein/s. Among various NHEJ proteins, KU70/80, Artemis, and XRCC4 showed the highest docking score, indicating their potential as targets for caffeine binding, thereby inhibiting the NHEJ-mediated joining of DSBs (Fig. 6A–D; Supplementary Figs S6A–I and S7A–C). ATM was used as the positive control for the study [14, 76]. It is important to emphasize that most XRCC4 amino acid residues interacting with caffeine were located within the XRCC4 interaction domain with ligase IV (Supplementary Fig. S6I). This suggests that caffeine may disrupt the XRCC4–ligase IV interaction during the NHEJ-mediated joining.
Biophysical and biochemical studies identify XRCC4/ligase IV as the target when caffeine inhibits NHEJ
Based on the docking studies, we were interested in determining whether caffeine can inhibit the activity of KU70/80 protein. To investigate this, His-tagged KU70/KU80 was purified as described previously using the Ni-NTA column [39, 48]. The protein was eluted using imidazole and the purity of the protein was checked on SDS–PAGE (Supplementary Fig. S7D). To check the effect of caffeine on KU70/80 protein complex, 5′–5′ noncompatible DNA substrate, SCR19/VK11 was incubated with purified protein in the presence of increasing concentrations of caffeine (10, 20, 40, and 60 mM) (Supplementary Fig. S7E and F). However, the results revealed no inhibition on the binding of KU70/80 protein to the DNA substrate harboring double-strand break, even at the highest caffeine concentration (Supplementary Fig. S7E and F) suggesting that caffeine may not inhibit the binding of KU70/80 protein to the DNA. Since Artemis was also identified as one of the potential target proteins of caffeine, we investigated impact of caffeine on the nuclease activity of purified Artemis protein [36]. For this, we incubated DNA substrate with 5′ overhang (SCR19/VK11) with purified Artemis protein in the presence of increasing concentration of caffeine (5, 20, and 60 mM) for 1 h at 37°C. Following this, the cleaved products were resolved on 12% denaturing PAGE (Supplementary Fig. S7G–I). However, we did not find significant difference in the cleavage efficacy (Supplementar Fig. S7G–I) in the presence of caffeine suggesting that caffeine may not inhibit Artemis activity. Therefore, preliminary studies suggest that KU70/80 protein and Artemis may not act as a target for caffeine.
XRCC4 was also among one of the targets of caffeine obtained from docking studies. To evaluate whether caffeine can bind with the XRCC4 protein, BLI studies were performed as described earlier [35, 42, 44]. For this, we purified XRCC4 protein (55 kDa) using the Ni-NTA column. The protein was eluted using imidazole gradient and resolved on SDS–PAGE (Fig. 6E and Supplementary Fig. S7J). The purity of the purified protein was checked on SDS–PAGE (Fig. 6E and Supplementary Fig. S7J). The XRCC4 protein was bound to Ni-NTA sensors using His-tag and probed against an increasing concentration of caffeine (0.001, 0.01, 0.1, 1, 20, and 40 mM) to determine the time of association and dissociation. Kd value was estimated to be 3.8 μM (Fig. 6F, and G). However, no binding was observed when the purified KU70/80 protein was used in the presence of caffeine (Fig. 6G and Supplementary Fig. S7K), which further validated the observation that caffeine can bind to XRCC4 protein (Fig. 6F, G, and H), but not to KU70/80 protein.
In these experiments, XLF was included as a negative control, as it interacts with XRCC4 but does not form a stable complex with ligase IV. Unlike XRCC4, which showed strong and specific binding to caffeine, XLF exhibited only weak and transient interactions (Supplementary Fig. S7L, and 6G and I). These results indicate that caffeine preferentially binds to XRCC4, confirming its specificity toward the ligase IV/XRCC4 complex rather than other NHEJ proteins.
To further delineate the molecular basis of caffeine–XRCC4 interaction, we performed BLI binding assays using purified recombinant mutant XRCC4 T133A. The objective was to determine whether alteration at threonine 133, a residue implicated in mediating XRCC4’s interaction with caffeine (Supplementary Fig. S6I), affects its binding affinity. Interestingly, the mutant XRCC4 T133A displayed markedly reduced binding to caffeine, as reflected by a substantially lower response amplitude and higher Kd value compared to wild-type XRCC4 (Fig. 6F, G, and J, and Supplementary Fig. S7M). This loss of affinity suggests that the Threonine 133 residue contributes directly to the caffeine-binding interface. Further, it is possible that the mutation could lead to a difference in protein folding affecting its interaction with ligase IV. Together, these results demonstrate that caffeine specifically interacts with the XRCC4, and that this interaction is disrupted in the T133A mutant, implicating Threonine 133 as a key determinant of caffeine recognition. These findings reinforce the conclusion that caffeine targets the XRCC4, thereby impairing efficient DNA end joining during NHEJ.
XRCC4 T133A mutation alters the stability and weakens predicted caffeine interactions
In the wild-type XRCC4 structure, T133 is positioned within an α-helical region that contributes to the structural integrity of the XRCC4 homodimer and lies near the predicted caffeine interaction interface (Supplementary Fig. S10A and B). Through docking analysis, we observed a shift in the pocket where the caffeine docked in wild-type versus mutant XRCC4 and visualized using LigPlot+ (Supplementary Fig. S10C–E). Total number of interactions varied between wild-type (8 residues) and mutant XRCC4 (7 residues) (Supplementary Figs S6I and S10E). Six of these interactions in wild-type were with chain A. In mutant, 2 chain B interactions were gained: Ile 527 and Leu 531. Additionally, 3 chain A interactions were lost: Leu 126, Tyr129, and Ile127 (Fig. S10C–E). Rather than occupying a well-defined pocket, caffeine displayed an increased positional variability in the mutant model, indicative of weakened or transient association (Supplementary Fig. S10C and D). These observations suggest that T133 contributes to maintaining stability of XRCC4 that is permissive for caffeine association. Together, these results support a model in which T133 plays a key role in stabilizing the XRCC4 helical interface targeted by caffeine, and its mutation weakens caffeine association without globally compromising XRCC4 structure. To understand how mutation of XRCC4 residue T133 influences caffeine-mediated inhibition, we examined the structural consequences of this substitution using in silico tools, DynaMut, mCSM and I-Mutant2.0. Results showed a mild to moderate destabilizing effect on the protein (Supplementary Fig. S10F).
Caffeine impairs XRCC4 recruitment to DNA damage sites following ionizing radiation
To investigate whether caffeine interferes with the recruitment of XRCC4 to DSB sites in cells exposed to genotoxic stress, we performed immunofluorescence analysis of XRCC4 and γ-H2AX foci formation in HeLa cells following exposure to IR in the presence or absence of caffeine. γ-H2AX was used as a marker of DSBs, while XRCC4 localization reflected NHEJ complex assembly at the damage sites. Results showed minimal γ-H2AX signal, consistent with low basal DNA damage under normal conditions in control (untreated) cells and thus only a low level of XRCC4 staining was noted (Fig. 7A). Following IR exposure (5 Gy), a distinct increase in γ-H2AX foci was observed, confirming the induction of DSBs. Notably, XRCC4 exhibited enhanced punctate nuclear localization that partially overlapped with γ-H2AX foci, indicating active recruitment of XRCC4 to damage sites and efficient initiation of the NHEJ repair process (Fig. 7A and B). In contrast, cells treated with caffeine alone (5 mM) showed γ-H2AX accumulation but displayed only reduced XRCC4 intensity, suggesting potential destabilization or altered nuclear distribution of XRCC4 in the absence of exogenous DNA damage. Strikingly, co-treatment with caffeine and IR resulted in a marked decrease in XRCC4 foci formation, despite the presence of prominent γ-H2AX signals. The reduced colocalization between XRCC4 and γ-H2AX indicates that caffeine impairs the efficient recruitment of XRCC4 to DSBs generated by irradiation (Fig. 7A and B). Magnified views (right panels) further highlight that in the caffeine + IR condition, XRCC4 foci colocalizing with γ-H2AX foci were less compared to IR alone, suggesting disrupted assembly of the XRCC4/ligase IV repair complex at DNA damage sites in the presence of caffeine. Together, these observations demonstrate that caffeine interferes with the recruitment and stabilization of XRCC4 at DSBs following IR, thereby compromising NHEJ-mediated repair. This disruption likely contributes to the persistence of DNA damage and the accumulation of γ-H2AX foci observed in caffeine-treated cells.
*Caffeine inhibits XRCC4 localization at DSBs following exposure to IR.(A) Representative immunofluorescence images showing colocalization of XRCC4 (green) and γ-H2AX (red) in cells treated with caffeine with/without IR exposure. Nuclei were counterstained with DAPI (blue). Control cells show basal XRCC4 distribution with minimal γ-H2AX foci, whereas IR alone induces prominent γ-H2AX foci and XRCC4 accumulation at DNA damage sites. Caffeine alone and Caffeine + IR treatment results in altered XRCC4 localization at the damaged DNA sites (indicated by γ-H2AX), suggesting impaired DSB repair. Magnified images (right panels) highlight colocalization of XRCC4 and γ-H2AX signals; scale bar: 10 µm. (B) Box-and-whisker plot depicting Mander’s colocalization coefficient for green (XRCC4) and red (γ-H2AX) signals as evaluated by JACoP plug-in of ImageJ software. (C) Bar graphs representing the GFP positive cells as a measure of intracellular NHEJ activity following transfection with PimEJ5-GFP and I-SceI overexpression constructs in HeLa WT and HeLa LIV KO cells upon caffeine (1, 2, and 5 mM) treatment. HeLa wild-type cells were used as a control to compare NHEJ efficiency between the wild-type and ligase IV knockout backgrounds. A total of 10 000 events were acquired per sample during flow cytometry analysis (In panels B-C; ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.001, ***P < 0.0001).
To further validate that the inhibitory effect of caffeine on NHEJ is primarily mediated through XRCC4 and its interaction with ligase IV, we next examined the impact of caffeine on NHEJ efficiency in a ligase IV-deficient HeLa cell line (HeLa.Lig4.catDΔ.1.NK, hereafter referred to as HeLa L4 KO) [67]. In the wild-type HeLa cells, caffeine treatment led to a significant reduction in NHEJ activity; however, in HeLa L4 KO cells, no further decrease in NHEJ efficiency was observed upon caffeine treatment (Fig. 7C). The comparable levels of NHEJ between untreated and caffeine treated HeLa L4 KO cells suggest that the inhibitory effect of caffeine is specifically dependent on the presence of functional ligase IV. These findings reinforce that caffeine exerts its inhibitory action on NHEJ by interfering with the ligase IV/XRCC4 complex, rather than through general suppression of DNA repair processes.
Caffeine inhibits DNA end-joining of DSBs with sticky ends
In order to assess the effect of caffeine on end joining of DNA substrate with compatible ends, 75 nt long double-stranded oligomeric DNA, SCR19/20, harboring sticky end (Fig. 8A) was incubated with increasing concentrations of caffeine (10, 20, 40, and 60 mM) in the presence of Jurkat (Fig. 8B and C) or Molt4 CFE (Fig. 8D and E). Compatible sticky ends were used for the study, as DNA can be quickly joined by a ligase. We observed a concentration-dependent decrease in the efficiency of joined product formation when incubated with the increasing concentration of caffeine in the case of both Jurkat as well as Molt4 cells (Fig. 8B–E). This revealed that treatment with caffeine led to inhibition of the end joining of DSBs with compatible DNA ends. Similar results were also observed when RTE was used for the joining (Fig. 8F and G). However, it was also noted that the efficiency of inhibition of joining by caffeine in case of RTE was less as compared to that of the cell line extracts (Fig. 8F and G).
*Evaluation of the effect of caffeine joining of sticky DNA ends and impact of mutation of XRCC4 on the inhibition.(A) The figure shows the sequence of compatible (sticky) end DNA substrate used for the joining assay. (B) Denaturing PAGE profile showing caffeine’s impact on joining of compatible DSBs when incubated with Jurkat CFE. (C) Quantitation of experiment shown in panel B (n = 3) using Multi Gauge V3.0 software and presented as a bar graph in the panel below, showing mean ± SEM. (D) Denaturing PAGE profile depicting the impact of caffeine on the end joining of sticky end substrate when incubated with Molt4 CFE. (E) The experiment was repeated a minimum of three times. The joined product was quantified and presented as a bar graph in the panel below, showing mean ± SEM. (F) Representative denaturing PAGE image showing the impact of caffeine on the end joining of compatible end substrate (SCR19/20) in the presence of RTE. (G) The bar diagram showing the quantitation of the experiment showed in panel F (n = 3); the data shown is mean ± SEM. (H) Representative denaturing PAGE gel profile showing the effect of caffeine on the ligation of compatible end substrate by purified ligase IV/XRCC4 protein. (I) The experiment was repeated three times, the joined product was quantified and presented as a bar graph, showing mean ± SEM. (J) Representative denaturing PAGE profile showing impact of caffeine on joining of compatible DSBs when incubated with purified ligase IV/XRCC4 and ligase IV/XRCC4 T133A mutant protein. (K) The experiment shown in panel (J) was repeated three independent times and the joined product was quantified and presented as a bar graph, showing mean ± SEM (In all panels; ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.001, ***P < 0.0001). (L) A representative 3D image showing the docked position of interphase of ligase IV/XRCC4 protein complex upon interaction with caffeine. (M) LigPlot diagram depicting the interacting residues of ligase IV/XRCC4 with caffeine.
Since the XRCC4 protein was also among the targets of caffeine identified from docking studies, we investigated the effect of caffeine on the joining mediated by the ligase IV/XRCC4 complex. Ligase IV/XRCC4 complex was purified using Ni-NTA column as described previously [28], eluted using imidazole gradient, and resolved on SDS–PAGE (Supplementary Fig. S8A). The identity of the protein was confirmed by western blotting (Supplementary Fig. S8B). Different fractions were dialyzed, and the joining assay using compatible end substrates (SCR19/20) was performed to test the activity of the purified protein (Supplementary Fig. S8C). Further, to evaluate the effect of caffeine on the ligase IV/XRCC4 complex, the joining assay was performed using compatible end DNA substrate (SCR19/20) with the purified ligase IV/XRCC4 protein in the presence of increasing concentrations of caffeine (5, 10, 15, 20, 40, and 60 mM) followed by resolving the joined products on a denaturing PAGE (Fig. 8H). The result showed a dose-dependent inhibition of joined product formation in the presence of caffeine (Fig. 8H and I), suggesting that caffeine interferes with the activity of ligase IV/XRCC4 complex and inhibits NHEJ.
To further substantiate that caffeine specifically interferes with the functional interaction between ligase IV and XRCC4, we next examined whether disrupting the caffeine-binding interface on XRCC4 could reduce the inhibitory effect. To examine this, an in vitro end-joining assay was performed using the ligase IV/XRCC4 T133A mutant protein. Consistent with previous observations, caffeine treatment significantly inhibited end joining in the wild-type ligase IV/XRCC4 reaction (Fig. 8J and K). However, when the assay was performed in the presence of the ligase IV/XRCC4 T133A mutant, the inhibition by caffeine was partially reduced, as evidenced by increased ligation product compared to the wild-type control (Fig. 8J and K, Supplementary Fig. S8B). Importantly, the addition of the ligase IV/XRCC4 T133A did not completely restore end joining activity to untreated levels, instead resulted in a partial recovery, suggesting that the mutation selectively weakens, rather than abolishing, caffeine-mediated inhibition. Together, these data suggest that substitution of T133 with Alanine diminishes the inhibitory effect of caffeine, likely by altering the local structural environment required for effective caffeine association. Moreover, the partial recovery of ligation activity in the presence of ligase IV/XRCC4 T133A further supports that caffeine directly targets the ligase IV/XRCC4, impairing its stability and function during the terminal ligation step of NHEJ rather than general suppression of enzymatic activity (Fig. 8J and K).
Further, in silico analysis revealed that caffeine interacts with specific amino acid residues of ligase IV (Fig. 8L and M), which are involved in forming a complex with XRCC4 during the final ligation step of the NHEJ pathway and thereby inhibiting the joining mediated by this complex. These results further provide evidence that caffeine specifically targets the ligase IV/XRCC4 complex, rather than broadly affecting other components of the DNA repair machinery, thereby confirming the selectivity of caffeine’s inhibitory action on the terminal ligation step of NHEJ.
Exposure to caffeine leads to reduced cell survival and delays in DSB repair in a ligase IV/XRCC4-dependent manner
To determine whether the inhibitory effect of caffeine on DSB repair correlates with reduced cell survival and whether this effect depends on ligase IV/XRCC4, we performed clonogenic survival and γ-H2AX foci assays in HeLa and HeLa LIV KO cells. Results showed that the exposure to increasing concentrations of caffeine (1, 2, 5, 10, and 20 mM) resulted in a pronounced, dose-dependent decrease in colony-forming ability in WT HeLa cells (Fig. 9A). The treatment with ≥5 mM caffeine led to a significant reduction in colony numbers, with near complete inhibition of growth observed at 10–20 mM. Interestingly, caffeine treatment decreased the viability of HeLa LIV KO cells (Fig. 9B); however, the extent of reduction in colony formation was less pronounced than that observed in wild-type cells, suggesting that the cytotoxic effect of caffeine is at least partly mediated through inhibition of ligase IV-dependent NHEJ activity.
Effect of caffeine on cell survival and DNA damage response in HeLa and HeLa ligase IV knock out cells.(A and B) Clonogenic survival assay showing the effect of increasing concentrations of caffeine on the colony-forming ability of HeLa (A) and HeLa ligase IV knock out cells (B). Cells were treated with the indicated concentrations of caffeine for 24 h, followed by incubation in drug free medium for 14 days to allow colony formation. Colonies were fixed, stained with crystal violet, and imaged. (C) Representative confocal images showing γ-H2AX foci (red) and nuclear DNA stained with DAPI (blue) in HeLa treated with IR (5 Gy) with or without caffeine treatment (5 mM) at various time points (0.5, 4, 12, and 24 h) post-irradiation; scale bar: 10 μm. (D) Quantification of γ-H2AX foci in HeLa and HeLa ligase IV knock out cells exposed to IR (5 Gy) in the presence or absence of caffeine (5 mM). Experiments were performed three independent times and cells were fixed at the indicated time points post-IR and immunostained for γ-H2AX. The average number of γ-H2AX foci per nucleus was quantified and presented as line plot showing mean ±SEM. (E) Representative confocal images of γ-H2AX foci and DAPI-stained nuclei in HeLa Ligase IV knockout (LIV KO) cells following exposure to 5 Gy ionizing radiation (IR), in the presence or absence of caffeine (5 mM), at indicated time points (0.5, 4, 12, and 24 h) post-IR.
To assess whether caffeine influences DSB repair kinetics following IR, we examined γ-H2AX foci formation and resolution in both HeLa and HeLa LIV KO cells treated with IR (5 Gy) in the presence or absence of caffeine (5 mM). Representative confocal images revealed rapid induction of γ-H2AX foci in both cell types shortly after irradiation (0.5 h), confirming efficient DSB formation (Fig. 9C, D and E). In control cells, γ-H2AX foci gradually diminished over time (12–24 h), indicating active DNA repair (Fig. 9C, and D). However, in caffeine-treated HeLa cells, γ-H2AX foci persisted for longer durations, suggesting delayed DSB resolution. Notably, HeLa LIV KO cells exhibited sustained γ-H2AX signal even without caffeine, consistent with their defective NHEJ background, and caffeine treatment did not further exacerbate the foci persistence to the same extent observed in wild-type cells (Fig. 9D and E). In wild-type HeLa cells, caffeine significantly delayed the decline in γ-H2AX foci post-IR compared to irradiated controls, indicating compromised DSB repair kinetics. On the contrary, HeLa LIV KO cells showed inherently high levels of persistent γ-H2AX foci after IR, and caffeine co-treatment did not markedly alter this profile (Fig. 9C, D and E). Together, these results demonstrate that caffeine impairs DSB repair efficiency and reduces clonogenic survival in a ligase IV-dependent manner. The differential response between wild-type and ligase IV-deficient cells strongly supports the conclusion that caffeine targets the ligase IV/XRCC4-mediated NHEJ pathway, thereby contributing to the accumulation of unrepaired DSBs and reduced cell survival.
Reconstitution of NHEJ by complementation of purified ligase IV/XRCC4 could reverse the inhibition of end joining by caffeine
To confirm the specificity of caffeine-mediated inhibition of DNA end joining, ligation of DSBs with noncompatible DNA ends was performed using ligase IV immunodepleted Molt4 CFE. Ligase IV was depleted from the extracts by performing IP, and the efficiency of end joining was compared to the IgG control Molt4 CFE (Fig. 10A and B). Results showed that while control Molt4 CFE efficiently catalyzed DNA end joining which was inhibited by caffeine in a concentration-dependent manner, ligase IV-depleted extracts displayed reduced basal ligation activity, consistent with the essential role of ligase IV in the NHEJ pathway. Notably, the extent of caffeine-mediated inhibition was reduced in the absence of ligase IV, indicating that caffeine’s suppressive effect primarily depends on the presence of the ligase IV/XRCC4 complex (Fig. 10B and C).
*Reconstitution studies following caffeine-mediated inhibition of NHEJ catalyzed by human CFEs.(A) Western blot showing IP of ligase IV from Molt4 CFE. PCNA was used as a loading control. Ponceau staining confirms equal protein loading. (B) Representative denaturing PAGE showing the effect of increasing concentrations of caffeine (1, 5, 10, and 20 mM) on the joining of 5′–5′ noncompatible DNA substrate (SCR19/VK11) in the presence of immunodepleted ligase IV CFE (lanes 8–13) and control CFE (lanes 2–7). (C) The experiment was repeated three independent times, and the bar graph represents mean ± SEM. (D) A representative denaturing PAGE showing reconstitution of the end joining of compatible end substrate (SCR19/20) catalyzed by Molt4 CFE after inhibition due to caffeine treatment (10 mM). Following caffeine treatment, ligase IV/XRCC4 protein (0.125, 0.25, and 0.5 µg) was added to determine, if the joining could be reconstituted. Initially, inhibition of end joining with CFE in the presence of caffeine (10 mM) was conducted. Following the inhibition reaction, the increasing concentration of overexpressed and purified ligase IV/XRCC4 was added back to the samples incubated with caffeine to evaluate the effect of caffeine on ligase IV/XRCC4 mediated joining. (E) The joined product was quantified and presented as a bar graph representing mean ±SEM (n = 4). (F–I) Representative denaturing PAGE image for end joining of 5′–5′ (SCR19/VK11, F) and 5′–3′ noncompatible end substrate (SCR19/VK13, H), respectively post ligase IV/XRCC4 complementation upon treatment with caffeine (10 mM) in the presence of Molt4 CFE. The joined products for the substrates SCR19/VK11 (G) and SCR19/VK13 (I) were quantified and presented as a bar graph. The experiments were conducted four times, and the error bar represents mean ±SEM. (J) Representative denaturing PAGE image for end joining of 5′–5′ noncompatible end substrate following ligase I complementation upon treatment with caffeine (10 mM) in the presence of Molt4 CFE. The joined products were quantified and presented as a bar graph. (K) The experiments were conducted thrice, and the error bar represents mean ±SEM. (In all panels; ns: not significant, *P < 0.05, **P < 0.005, ***P < 0.001, ***P < 0.0001). (L) Cartoon depicting the molecular mechanism by which caffeine inhibits NHEJ. Caffeine inhibits NHEJ by selectively interfering with the binding of XRCC4 to the ligase IV complex, thereby reducing their functional activity while leaving the KU70/80 function unaffected.
A reconstitution assay was performed to confirm the specificity of caffeine as an inhibitor of NHEJ in human CFEs (Supplementary Fig. S9A). The Molt4 CFEs were treated with caffeine (10 mM) for 1 h to inhibit end-joining activity. Purified ligase IV/XRCC4 complex protein was then added, and the reaction products were resolved on denaturing PAGE to assess the repair outcome (Supplementary Fig. S9A). Results showed that the addition of caffeine (10 mM) to the mammalian CFEs abrogated the end-joining of compatible end substrate (SCR19/20), as evidenced by a significant reduction in product formation (Fig. 10D and E). Interestingly, addition of purified ligase IV/XRCC4 to the Molt4 CFE resulted in a partial recovery of end-joining activity that was reduced upon caffeine treatment (Fig. 10D and E). Comparable results were observed when this assay was performed using a compatible end substrate in the presence of HeLa CFE (Supplementary Fig. S9B and C). These findings demonstrate that caffeine-mediated inhibition of NHEJ is ligase IV/XRCC4 dependent, highlighting it as a critical molecular target for caffeine in suppressing the NHEJ repair pathway.
Since most physiological DSBs generated inside the cells are noncompatible ends, we were interested in investigating whether ligase IV/XRCC4 can rescue the end joining of noncompatible end substrates. Two distinct noncompatible end substrates (SCR19/VK11 and SCR19/VK13) were used for the investigation. Remarkably, the addition of purified ligase IV/XRCC4 restored the joining of noncompatible ends substrate, as evidenced by a robust increase in the joined product formation (Fig. 10F–I). These results are significant and underscore the specificity of caffeine in targeting ligase IV/XRCC4. Further, to assess the specificity, we performed the assay using 5′–3′ noncompatible (SCR19/VK13) DNA substrates in the presence of DNA ligase I. In contrast to ligase IV/XRCC4, ligase I failed to restore end joining of DNA substrates in caffeine treated samples, showing no detectable increase in ligation product formation (Fig. 10J and K, and Supplementary Fig. S9D). This lack of rescue confirms that the observed joining activity is specific to ligase IV/XRCC4 and not a general property of DNA ligases (Fig. 10J and K). Collectively, the result from this study provides evidence that caffeine acts as an inhibitor of NHEJ by directly interfering with ligase IV/XRCC4 activity (Fig. 10L).
Discussion
Caffeine has been widely used to study DNA damage and cell cycle checkpoint responses due to its ability to inhibit ATM and ATR kinases [14]. Building on previous studies that highlighted caffeine’s inhibitory effects on HR-mediated repair, our study demonstrates that caffeine also suppresses the NHEJ-mediated DSB repair pathway both in vitro and in vivo. The inhibition of NHEJ resulted in the accumulation of DSBs within the cells.
We investigated the effect of caffeine on NHEJ using a cell-free repair assay with double-stranded oligomeric DNA substrates harboring compatible or noncompatible DSBs. Results showed a dose-dependent decrease in joined product formation with increasing caffeine concentration, indicating inhibition of NHEJ. This inhibition was consistent across multiple human cancer cell lines (Jurkat, Molt4, HeLa), normal fibroblasts (HMF-3S), and rat primary tissue extracts, as well as different DNA end configurations and sequences used for the study, suggesting that caffeine targets key protein(s) involved in the NHEJ pathway. Using an extrachromosomal NHEJ reporter assay, we observed reduced GFP-positive cells upon caffeine treatment, indicating NHEJ inhibition in mammalian cells. Additionally, immunofluorescence analysis revealed an increase in γ-H2AX and 53BP1 foci, leading to the accumulation of unrepaired endogenous DSBs following caffeine treatment. We recognize that the endogenous DSBs observed following caffeine treatment may not be exclusively attributed to the inhibition of NHEJ but could also result from the suppression of the HDR pathway.
Molecular docking studies identified KU70/80, Artemis, and XRCC4 as potential targets of caffeine during NHEJ inhibition. However, EMSA showed that caffeine does not inhibit the binding of KU70/80 to DNA. Further, results from the nuclease assay using purified Artemis protein also ruled out the possibility of Artemis as the target for caffeine during the observed NHEJ inhibition. However, joining assays with purified ligase IV/XRCC4 complex indicated that caffeine inhibited the activity of ligase IV/XRCC4 in a dose-dependent manner. BLI further confirmed caffeine’s binding to XRCC4 with a Kd value of 3.8 μM, but not to KU70/80, validating XRCC4 as the primary target. On the other hand, the high dissociation constant in the mutant XRCC4 T133A protein provides evidence that Thr133 is a critical residue for caffeine recognition and that this interaction interferes with the structural integrity or catalytic efficiency of the ligase IV/XRCC4 complex. The inability of caffeine to inhibit KU70/80 DNA binding or Artemis nuclease activity, despite its predicted docking potential, strongly supports that XRCC4/ligase IV constitutes the primary functional target during its inhibition on NHEJ. The lack of inhibition in ligase IV-depleted extracts and the successful rescue by reconstitution with purified ligase IV/XRCC4 complex further confirms this specificity. Moreover, caffeine failed to affect ligation catalyzed by DNA ligase I, reinforcing that the compound selectively interferes with the NHEJ-specific ligation step by targeting XRCC4. Immunofluorescence analysis revealed that caffeine prevents XRCC4 from efficiently localizing to γ-H2AX-marked DSBs following IR. This impaired recruitment suggests that caffeine binding inhibits the localization of XRCC4 at the DSB sites or disrupts its interaction with ligase IV, thereby hindering repair complex assembly at damage sites. Such interference at the structural level may underlie the persistent γ-H2AX and 53BP1 foci observed in caffeine-treated cells, reflecting defective end-joining and accumulation of unrepaired DSBs. The increased γ-H2AX foci and TUNEL positivity following caffeine treatment demonstrate persistent unrepaired DSBs, validating functional inhibition of NHEJ inside cells. Overall, the results suggest that caffeine inhibited NHEJ-mediated DSB repair by targeting the XRCC4 protein in the ligase IV/XRCC4 complex.
Previous studies demonstrated that caffeine inhibits the HR repair pathway by impairing Rad51 recombinase filament formation on ssDNA, a process essential for gene conversion, and is independent of its inhibition of the Mec1^ATR^/Tel1^ATM^ DNA damage response (Tsabar et al., 2015b). Apart from this, caffeine’s inhibitory effects on HR-mediated repair extend through multiple mechanisms, including the rapid loss of Sae2 and Dna2, nucleases essential for 5′ to 3′ end resection (Tsabar et al., 2015a), and increased interactions between Rad51 and nonhomologous DNA, which hinder strand invasion during joint molecule formation (Zelensky et al., 2013). Additionally, caffeine inhibited DNA-PKcs activity at concentrations up to 10 mM. However, 10-fold higher concentration of caffeine was required to inhibit the autophosphorylation of DNA-PK (a crucial step required for NHEJ-mediated repair) in vitro but it did not impair DNA-PKcs-dependent DSB repair in vivo (Block et al., 2004). Interestingly, caffeine abolishes radiation-induced checkpoint responses and sensitizes NHEJ-deficient cells to radiation, suggesting that its radiosensitization effects are related to checkpoint inhibition rather than NHEJ disruption (Wang et al., 2003).
Caffeine has been previously reported to modulate HR, particularly under conditions of replication stress [11, 13]. However, our present study specifically highlights the direct effect of caffeine on the NHEJ pathway in response to IR-induced DSBs, thereby distinguishing its influence on NHEJ from its previously known effects on HR. In summary, we report that in addition to HR, caffeine can inhibit NHEJ-mediated repair by abolishing the activity of XRCC4 in the ligase IV/XRCC4 complex (Fig. 10L). Besides, understanding caffeine’s effects on NHEJ could provide novel insights into its broader role in modulating DNA repair pathways and radiosensitization, which could have implications for therapeutic applications during cancer treatment. The differential inhibitory effects of caffeine observed on DNA repair pathways and kinase activities at varying concentrations, as reported in previous studies (from 1 mM to as high as 60 mM), guided our selection of higher concentrations of caffeine for investigating its effect on NHEJ [10, 11, 13, 14].
Previous studies have reported that caffeine can exert radioprotective effects under specific conditions. At low to moderate concentrations, caffeine has been shown to scavenge ROS generated by IR, thereby reducing oxidative DNA damage and lipid peroxidation. Its antioxidant properties help preserve cellular redox balance and protect DNA, proteins, and membranes from radiation-induced injury. In some models, caffeine pretreatment decreased chromosomal aberrations and micronuclei formation, suggesting protection against genotoxic stress [77–79]. In a recent study, we demonstrated that caffeine could protect DNA from IR [80]. At low concentrations (0.1–1 mM) achievable through moderate consumption, caffeine exerts a radioprotective effect by effectively scavenging reactive oxygen and nitrogen species (ROS/RNS), thereby reducing IR-induced DNA strand breaks, and promoting normal cell cycle progression (Kumari et al., MCB, 2025, in press). Under these conditions, caffeine does not interfere with the core DNA repair pathways, including NHEJ and HR, allowing cells to maintain genomic stability. In contrast, at higher concentrations (>2 mM), caffeine displays a dual inhibitory behaviour, where it not only suppresses ATM/ATR kinase activity and impairs checkpoint signaling as demonstrated in previous reports but also directly interferes with the assembly and function of NHEJ repair complexes. The present study reveals that high-dose caffeine disrupts the ligase IV/XRCC4 interaction, leading to reduced XRCC4 recruitment at DNA damage sites and subsequent inhibition of the terminal ligation step of NHEJ. Collectively, these findings define a biphasic model in which caffeine acts as a radioprotectant at low doses through its antioxidant capacity, while at higher doses it compromises DNA repair fidelity by targeting key components of the NHEJ and HR machinery.
Following oral administration in humans, caffeine is instantly and almost completely (99%) absorbed from the small intestine (Fredholm & Arnaud, 2011; Samah & Heard, 2013) and generally reaches peak plasma concentrations within 30–120 min after administration (May et al., 1982; White Jr et al., 2016). It has been reported that the peak plasma concentration in humans after ingestion of one cup of coffee (one cup of coffee contains 80–100 mg caffeine) ranges between 1.5 and 1.8 μg/ml (Perera et al., 2010; White Jr et al., 2016). This peak plasma concentration is estimated to be approximately 10 μM. In our study, we demonstrated that lower concentration of caffeine does not have any effect on NHEJ; on the other hand, a higher concentration of caffeine (≥2 mM) showed an inhibitory effect on NHEJ-mediated repair in an in vitro condition. In in vivo condition, this inhibitory effect of caffeine on NHEJ was observed from 2 mM.
Given that caffeine inhibits NHEJ-mediated repair, our findings provide an opportunity to optimize its concentration for therapeutic applications. At appropriately low doses, caffeine could be strategically employed in combination with conventional anti-cancer drugs or radiotherapy to enhance treatment efficacy. Elucidating the precise molecular targets of caffeine within the NHEJ pathway will be critical for developing targeted interventions, particularly in cancers with elevated NHEJ activity or defective HR repair, where cells rely heavily on NHEJ for survival. By preventing DSB repair through NHEJ inhibition, caffeine may selectively induce apoptosis in such cancer cells. Thus, caffeine-mediated radiosensitization likely arises from its combined inhibitory effects on both HR and NHEJ, positioning caffeine as a potential DNA repair inhibitor for improving cancer therapy outcomes.
Supplementary Material
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The reference list from the paper itself. Each links out to its DOI / PubMed record.
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