Transformation of 5-Carboxylcytosine to Cytosine Through C–C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation

Abstract

Open AccessCCS ChemistryRESEARCH ARTICLE1 Apr 2021Transformation of 5-Carboxylcytosine to Cytosine Through C–C Bond Cleavage in Human Cells Constitutes a Novel Pathway for DNA Demethylation Yang Feng, Neng-Bin Xie, Wan-Bing Tao, Jiang-Hui Ding, Xue-Jiao You, Cheng-Jie Ma, Xiaoxue Zhang, Chengqi Yi, Xiang Zhou, Bi-Feng Yuan and Yu-Qi Feng Yang Feng Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072 , Neng-Bin Xie Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072 , Wan-Bing Tao Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072 , Jiang-Hui Ding Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072 , Xue-Jiao You Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072 , Cheng-Jie Ma Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072 , Xiaoxue Zhang State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871. , Chengqi Yi State Key Laboratory of Protein and Plant Gene Research, School of Life Sciences, Peking University, Beijing 100871 Peking-Tsinghua Center for Life Sciences, Academy for Advanced Interdisciplinary Studies, Peking University, Beijing 100871 Department of Chemical Biology and Synthetic and Functional Biomolecules Center, College of Chemistry and Molecular Engineering, Peking University, Beijing 100871. , Xiang Zhou Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072 , Bi-Feng Yuan *Corresponding author: E-mail Address: [email protected] Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072 and Yu-Qi Feng Key Laboratory of Analytical Chemistry for Biology and Medicine (Ministry of Education), Sauvage Center for Molecular Sciences, Department of Chemistry, Wuhan University, Wuhan 430072 https://doi.org/10.31635/ccschem.020.202000286 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Active demethylation of 5-methylcytosine (5mC) can be realized through ten-eleven translocation (TET) dioxygenase-mediated oxidation of 5mC to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC), followed by thymine DNA glycosylase (TDG)-initiated base excision repair (BER). The TDG-BER pathway may lead to the generation of DNA strand breaks, potentially compromising genome integrity. Alternatively, direct decarboxylation of TET-produced 5caC is highly attractive because this mechanism allows for conversion of 5mC to cytosine without the formation of DNA strand breaks. However, cleavage of the C–C bond in 5caC in human cells remains an open question. We examined this reaction in cell extract and live cells using 5caC-carrying hairpin DNA substrate. After incubation with whole-cell protein extract or transfection into human cells, we monitored the transformation of 5caC to cytosine through direct decarboxylation or BER using liquid chromatography–tandem mass spectrometry (LC-MS/MS) analyses at both the mononucleotide and oligodeoxynucleotide levels. Our results clearly showed the direct conversion of 5caC to cytosine in human cells, providing evidence to support a novel pathway for active DNA demethylation. Download figure Download PowerPoint Introduction 5-Methylcytosine (5mC) at CpG sites in DNA plays an important role in epigenetic regulation of gene expression in vertebrates.1,2 5mC is produced and maintained by DNA (cytosine-5)-methyltransferases (DNMTs) via transfer of a methyl group from S-adenosylmethionine (SAM) to the C5 position of cytosine in genomic DNA.3 The levels and distributions of 5mC in genomic DNA are dynamic, and the current model of active demethylation of 5mC in mammals involves stepwise oxidation of 5mC by ten-eleven translocation (TET) dioxygenases to generate 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC).4–11 Thymine DNA glycosylase (TDG) is able to recognize and cleave the N-glycosidic bonds of 5fC and 5caC to produce abasic sites (AP-sites), which are further processed by base excision repair (BER) machinery to restore the unmethylated cytosine.12–16 Although this TET-TDG-BER pathway is the best characterized mechanism of active demethylation of 5mC in DNA of vertebrates,17,18 it may not account for all scenarios. In particular, demethylation of 5mC via this pathway involves the formation of DNA strand breaks as intermediates, which may compromise genome integrity.19 Additionally, it was reported that active demethylation of 5mC in mouse zygotes was not perturbed by genetic deletion of TDG, indicating the presence of a demethylation mechanism initiated by TET-mediated oxidation, but independent of TDG-mediated BER.20 An alternative pathway for active demethylation of 5mC could be possible through direct decarboxylation of TET-produced 5caC. This is a highly attractive mechanism because it realizes the direct conversion of 5mC to cytosine without the generation of DNA strand breaks as intermediates. Along this line, a previous study revealed that both 5fC and 5caC could undergo thiol-mediated and acid-catalyzed C−C bond cleavage to yield cytosine in vitro.21 The elimination of formyl and carboxyl groups in 5fC and 5caC requires transient addition of a nucleophile, for example, thiol, to the C6 position of the modified nucleobases via a 1,6 addition reaction followed by the liberation of formic acid and CO2, respectively.21 The corresponding nucleophile is subsequently detached from cytosine in a similar mechanism as cytosine methylation employed by DNMT proteins.22,23 Some bacterial and mammalian DNA (cytosine-5)-methyltransferases also display potential activity in decarboxylation of 5caC in DNA in vitro.24 Furthermore, biochemical data has demonstrated that recombinant isoorotate decarboxylase, initially identified to be capable of catalyzing decarboxylation of 5-carboxyluracil to uracil in some fungi species, exhibited relatively weak activity in catalyzing the direct decarboxylation of 5caC to cytosine.25 These in vitro studies suggest that deformylation of 5fC and decarboxylation of 5caC may also occur in vivo. Recently, Carell et al.26 reported the conversion of 5fC to cytosine via direct C–C bond cleavage in genomic DNA, providing the first line of evidence to support the direct deformylation of 5fC in cells. However, it remains unclear whether C–C bond cleavage occurs in 5caC in cells. A previous investigation suggested that treatment of 5caC-containing synthetic DNA with nuclear extract of mouse embryonic stem cells led to the conversion of 5caC to cytosine,27 raising the possibility of direct decarboxylation of 5caC in cells. Herein, we have investigated C–C bond cleavage of 5caC in DNA in cell extracts and live cells. Our results provide strong evidence for the direct conversion of 5caC to cytosine in human cells, supporting the idea of a decarboxylation pathway in active demethylation of 5mC in human cells. Experimental Methods Chemicals and reagents 2′-Deoxyadenosine (dA), thymidine (T), 2′-deoxycytidine (dC), 2′-deoxyguanosine (dG), 2′-deoxyadenosine 5′-monophosphate (dAMP), thymidine 5′-monophosphate (TMP), 2′-deoxycytidine 5′-monophosphate (dCMP), and 2′-deoxyguanosine 5'-monophosphate (dGMP) were purchased from Sigma-Aldrich (Beijing, China). 5-Methyl-2′-deoxycytidine (5mdC), 5-hydroxymethyl-2′-deoxycytidine (5hmdC), 5-formyl-2′-deoxycytidine (5fdC), and 5-carboxyl-2′-deoxycytidine (5cadC) were purchased from Berry & Associates (Dexter, MI, USA). Venom phosphodiesterase I was purchased from Sigma-Aldrich (Beijing, China). S1 nuclease, DNase I, calf-intestinal alkaline phosphatase (CIAP), and DNA marker were obtained from Takara Biotechnology Co. Ltd. (Dalian, China). SspI restriction endonuclease was obtained from New England Biolabs (Ipswich, MA, USA). Dulbecco's Modified Eagle medium (DMEM) and fetal bovine serum were purchased from Thermo-Fisher Scientific (Waltham, MA, USA). Chromatographic grade methanol and acetonitrile were purchased from Merck (Darmstadt, Germany). DNA substrates A 40-mer 5caC-containing DNA (5caC-DNA) was synthesized and purchased from Takara Biotechnology Co. Ltd. (Dalian, China). This DNA substrate contains a 5caC site, a biotin tag at the 3′ terminus, with seven oxygen atoms replaced by sulfur atoms in the phosphodiester bonds ( Supporting Information Figure S1 and Table S1). 5caC-DNA can form hairpin structures and carry SspI restriction enzyme sites in the stem region of the hairpin ( Supporting Information Figure S2). A DNA substrate was dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA) to a final concentration of 50 μM. The DNA substrate (10 μM) was annealed to form a hairpin structure in a buffer containing 20 mM Tris-HCl (pH 7.0), 50 mM NaCl, 10 mM MgCl2, and 1 mM DTT by heating at 95 °C for 5 min, followed by gradual cooling to 20 °C. 5caC-DNA hairpin substrate was digested with 4 μL of SspI restriction enzyme (5 U/μL), 6 μL of 10× CutSmart buffer (NEB), and incubated at 37 °C for 2 h. The resulting product was analyzed with 20% polyacrylamide gel electrophoresis. The sequences of 5caC-DNA and other synthesized DNA are listed in Supporting Information Table S1. Cell culture and stable isotope labeling Human embryonic kidney epithelial cell line (HEK293T), human cervical carcinoma cell line (HeLa), and human breast adenocarcinoma cell line (MCF-7) were obtained from the China Center for Type Culture Collection (Wuhan, China). These cells were maintained in DMEM medium containing 10% fetal bovine serum, 100 U/mL penicillin, and 100 μg/mL streptomycin at 37 °C in a 5% CO2 atmosphere. For the stable isotope-labeling tracing monitored by liquid chromatography–tandem mass spectrometry (LC-MS/MS), HEK293T cells were cultured in DMEM medium supplemented with 100 μM of 15N3-dC (Cambridge Isotope Laboratories, Tewksbury, MA, USA) to metabolically label genomic DNA. HEK293T cells were passaged every 2 days in DMEM medium containing 15N3-dC. After 2 weeks, cells were applied for transfection experiments. In vitro decarboxylation assay HEK293T cells were collected by centrifugation at 1500g under 4 °C for 5 min and then washed twice with ice-cold phosphate-buffered saline (PBS) to remove the medium and fetal bovine serum. Approximately 1 × 107 cells were lysed using 200 μL of RIPA lysis buffer (Beyotime Biotech Inc, Shanghai, China) with 1 mM of phenylmethylsulfonyl fluoride (Sigma-Aldrich, Beijing, China). For the in vitro decarboxylation assay, 200 μL of cell extract was added to 50 pmol of annealed 5caC-DNA substrate and incubated at 37 °C for different time. 5caC-DNA was isolated using streptavidin-coated magnetic beads (PMG015, PuriMag Biotech, Xiamen, China) according to the manufacturer's instructions. Briefly, 50 μL of beads (10 mg/mL) were pipetted into a nuclease-free tube and maintained for 30 s using a magnetic separator. The supernatant was removed, and the beads was suspended in 200 μL of binding buffer (20 mM Tris-HCl pH 7.0, 1 M NaCl, 1 mM EDTA, 0.02% Triton X-100). The suspended beads were then added into the reaction solution and incubated for 15 min at 25 °C. The beads were separated using a magnetic separator and washed twice with 200 μL of binding buffer, and then suspended in 20 μL of H2O for subsequent nuclease digestion or SspI restriction enzyme digestion. Nuclease digestion and analysis of nucleotides by LC-MS/MS Nuclease digestion was carried out according to a previous report with slight modification.28 Briefly, to the aforementioned 20 μL of DNA substrate (attached on streptavidin-coated magnetic beads), 2 μL of S1 nuclease (180 U/μL), 2 μL phosphodiesterase I (0.001 U/μL), 3 μL of 10× buffer (500 mM Tris–HCl, 100 mM NaCl, 10 mM MgCl2, 10 mM ZnSO4, pH 7.0), and 3 μL of H2O were added. Enzymatic digestion was continued at 37 °C for 3 h. After adding 170 μL of H2O, the resulting solution was extracted with chloroform twice.29 The aqueous layer, which contains the nucleotides released from digestion, was collected, lyophilized to dryness, and then reconstituted in H2O for LC-MS/MS analysis. LC-MS/MS analysis was conducted on a system consisting of a Shimadzu 8045 mass spectrometer (Kyoto, Japan) with an electrospray ionization (ESI) source coupled with a Shimadzu LC-30AD UPLC system (Tokyo, Japan). The chromatographic separation of nucleotides was performed on a Thermo Accucore C18 column (150 mm × 2.1 mm i.d., 2.6 μm) at 35 °C. Nucleotides were separated by a 20-min gradient with the use of 2 mM NH4HCO3 in H2O (A) and methanol (B) as the mobile phases.30,31 The flow rate was set at 0.3 mL min−1. A gradient of 0–5 min 5% B, 5–10 min 5–80% B, 10–13 min 80% B, 13–16 min 80–5% B, and 16–20 min 5% B was employed. The nucleotides were detected with multiple-reaction monitoring (MRM) in the positive-ion mode. The mass spectrometric parameters for MRM analysis were optimized by direct injection, and the optimized conditions are listed in Supporting Information Table S2. SspI digestion and analysis of oligodeoxynucleotides by high-resolution LC/MS For the analysis of the decarboxylated DNA substrate, to 20 μL of DNA substrate (attached on streptavidin-coated magnetic beads) were added 4 μL of SspI restriction enzyme (5 U/μL), 6 μL of 10× CutSmart buffer (NEB), and 30 μL of H2O. The mixture was incubated at 37 °C for 2 h. Then 2 μL of CIAP (30 U/μL) was added and incubated at 37°C for 1 h. After adding 138 μL of H2O, the resulting solution was extracted with chloroform twice. The aqueous layer was collected, lyophilized to dryness, reconstituted in H2O, and then subjected to high-resolution LC/MS analysis. High-resolution LC/MS analysis was run on an LTQ-Orbitrap Elite high-resolution mass spectrometer (Thermo-Fisher Scientific, Waltham, MA, USA) equipped with an ESI source and Dionex UltiMate 3000 UPLC system (Thermo-Fisher Scientific, Waltham, MA, USA). The chromatographic separation of oligodeoxynucleotides was performed on a Thermo Accucore C18 column (150 mm × 2.1 mm i.d., 2.6 μm) at 25 °C. 50 mM TEAA in H2O (A) and acetonitrile (B) were employed as the mobile phases. The flow rate was set at 0.2 mL min−1. A gradient of 0–10 min 5% B, 10–25 min 5–80% B, 25–27 min 80%–5% B, 27–40 min 5% B was used. Full MS scans were obtained under negative mode with a mass range of m/z 500–2000 at a resolution of 60,000. Data analysis was performed using Xcalibur version 3.0 (Thermo-Fisher Scientific, Waltham, MA, USA). Decarboxylation assay in human cells HEK293T cells were transfected with 300 pmol of annealed 5caC-DNA substrate using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions. Cells were harvested at different time points after transfection (6, 12, 24, and 48 h). The harvested cells were washed with PBS buffer three times and then suspended in 200 μL of PBS. Then 4 μL of DNase I (5 U/μL) was added into the cell solution and incubated at 37 °C for 30 min to remove the potentially adhesive 5caC-DNA substrate on the cell surface or in the residual medium. The resulting cells were washed with 200 μL of PBS three times. Cells were lysed according to a previously described method with slight modification.32 Briefly, the obtained cells were suspended in 500 μL of buffer A (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 0.15% NP40) and kept on ice for 10 min to allow for cell swelling. The cells were then lysed by sonication. Nuclei were collected by centrifugation at 1000g for 10 min. The cytoplasmic fraction was obtained by collecting the supernatant. The nuclear pellet was suspended in 250 μL buffer B (20 mM HEPES pH 7.9, 1.5 mM MgCl2, 0.42 M KCl, 0.2 mM EDTA, 0.5 mM DTT, and 0.1% NP40) and incubated for 30 min on ice. After centrifugation at 14,000 rpm for 15 min at 4 °C, the nuclear extract was obtained by collecting supernatant. The cytoplasmic fraction and nuclear extract were then combined. Proteinase K (0.1 mg/mL) was added into the mixture and incubated at 55 °C for 2 h. The solution was incubated at 95 °C for 10 min to inactivate proteinase K. The resulting solution was extracted with chloroform twice and the aqueous layer was collected. The DNA substrate was isolated using streptavidin-coated magnetic beads and suspended in 20 μL of H2O. The procedures for LC-MS/MS analysis of nucleotides and SspI digested oligodeoxynucleotides were the same as those for in vitro decarboxylation assay. TDG inhibition assay Juglone (Yuanye Bio-Technology, Shanghai, China) and Closantel (Aladdin, Shanghai, China) that were previously reported to be able to inhibit TDG activity33 were utilized in the current study. HEK293T cells were passaged and grown in DMEM medium containing different concentrations of Juglone or Closantel. Genomic DNA of HEK293T cells was extracted using a Tissue DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA). To evaluate the inhibitory effects of Juglone or Closantel, 10 μg of genomic DNA were enzymatically digested for LC-MS/MS analysis. To assess the direct decarboxylation of 5caC in cells with attenuated TDG activity, HEK293T cells were first cultured in DMEM medium with 20 μM of Juglone for 24 h. 5caC-DNA was transfected and then harvested at different time points. DNA substrates were isolated according to aforementioned procedures. Isolated DNA substrates were digested into nucleotides or oligodeoxynucleotides and then analyzed by LC-MS/MS, as described earlier. Decarboxylation assay in TDG knockdown cells Knockdown of TDG was performed using siRNA (Sigma-Aldrich, Beijing, China) targeting human TDG mRNA. Nontargeting siRNA was used as the negative control. HEK293T cells were transfected with siRNA using Lipofectamine 3000 according to manufacturer's instructions. After 24 h, 5caC-DNA substrate was transfected into cells. The DNA substrate was isolated using streptavidin-coated magnetic beads according to the aforementioned procedures. Isolated DNA substrates were digested into nucleotides or oligodeoxynucleotides followed by LC-MS/MS analysis. Total RNA was isolated from HEK293T cells to assess the efficiency of siRNA knockdown. Total RNA was treated with gDNA eraser (Takara, Dalian, China) to degrade potentially contaminated genomic DNA. PrimeScriptTM RT reagent kit (Takara, Dalian, China) was used to carry out the reverse transcription. Quantitative real-time polymerase chain reaction (PCR) was fulfilled on a CFX Connect Real-Time System (Bio-Rad Laboratories, Hercules, USA). The PCR mixture (25 μL) contained 2 μL of reverse transcription solution, 1 μL of TDG forward primer (10 μM), 1 μL of TDG reverse primer (10 μM), 12.5 μL 2 × SYBR Premix Ex Taq II (Takara, Dalian, China), and 8.5 μL of H2O. The PCR conditions included 95 °C for 5 min and 40 cycles at 95 °C for 10 s, 60° C for 30 s, and 72 °C for 20 s. Glyceraldehyde 6-phosphate dehydrogenase gene (GAPDH) was employed as the reference gene for normalization. Sequences of PCR primers are listed in Supporting Information Table S1. siRNA knockdown of TDG was also validated by Western blotting. Briefly, human HEK293T cells were lysed with RIPA lysis buffer. Antibodies that specifically recognize TDG (Abcam, ab154192, Cambridge, MA) and GAPDH (Abcam, ab181602, Cambridge, MA) were used at 1∶1000 and 1∶8000 dilutions, respectively. Horseradish peroxidase-conjugated secondary goat anti-rabbit antibody (Abcam, ab6721, Cambridge, MA) was used at a 1∶10,000 dilution. Decarboxylation assay in TDG knockout cells The TDG knockout HEK293T cell line was generated according to a previous study using CRISPR-Cas9 system.34 TDG protein expression was examined by Western blotting. Decarboxylation assays in TDG knockout cells were carried out in the same way as that in wild-type HEK293T cells. Exploration of endogenous decarboxylase We investigated whether DNMT1 and orotidine 5′-monophosphate decarboxylase (ODCase) were endogenous decarboxylase toward 5caC. Knockdown of DNMT1 or ODCase was performed by using siRNA in 15N3-dC labeled HEK293T cells. siRNA for knockdown of DNMT1 was purchased from Dharmacon (catalog D-004605-01). siRNA for knockdown of ODCase was obtained from Sangon Biotech (Shanghai, China). Nontargeting siRNA was used as the negative control. Sequences of siRNA are listed in Supporting Information Table S1. Quantitative real-time PCR was applied for the relative quantification of mRNA of DNMT1 and ODCase. The levels of gene expression were normalized to GAPDH. The sequences of PCR primers are listed in Supporting Information Table S1. DNMT1 or ODCase siRNA was transfected into 15N3-dC labeled HEK293T cells (HEK293T cells were continuously cultured in the medium containing 15N3-dC) using Lipofectamine 3000 according to manufacturer's instructions. After 24 h, 5caC-DNA substrate was transfected into cells for an additional 48 h. The DNA substrate was then isolated using streptavidin-coated magnetic beads according to aforementioned procedures. Isolated DNA substrates were digested into nucleotides or oligodeoxynucleotides followed by LC-MS/MS analysis. Results and Discussion Direct decarboxylation of 5caC in cell lysate In this study, we aimed to explore the direct decarboxylation of 5caC in vitro and in cells. To this we synthesized a hairpin DNA substrate (5caC-DNA) a a biotin on the terminus, and seven sulfur which replaced the oxygen atoms in seven phosphodiester bonds ( Supporting Information Figure S1). 5caC-DNA was treated by cell extract or transfected into human cells for in After of we analyzed the levels of enzymatically released 5-carboxyl-2′-deoxycytidine and 2′-deoxycytidine as as the oligodeoxynucleotides from 5caC-DNA by In this decarboxylation of nuclease digestion of the DNA substrate released similar digestion of the 5caC-DNA In with SspI digestion, the of the released oligodeoxynucleotide from 5caC-DNA was the corresponding oligodeoxynucleotide released from decarboxylated 5caC-DNA the in and and that the restriction released from 5caC-DNA and decarboxylated 5caC-DNA can be to assess the and of direct decarboxylation of 5caC by LC-MS/MS analysis. Figure 1 LC-MS/MS for monitoring the direct decarboxylation of 5caC in cell of the method in direct decarboxylation of 5caC in cell for monitoring and enzymatically released from a 5caC-DNA substrate. of and to the of from DNA at different treatment time. High-resolution LC/MS for monitoring the of oligodeoxynucleotides released by SspI from 5caC-DNA substrate and decarboxylation product m/z and 1 cell lysate was negative 2 RIPA solution was used of cell DNA containing the same as but with 5caC replaced with a The of m/z the of the decarboxylation product data of the decarboxylation of 5caC-DNA on oligodeoxynucleotide analysis from and Supporting Information Figure in and are the 5caC-DNA substrate and decarboxylation respectively. independent were and the the of the Download figure Download PowerPoint We annealed 5caC-DNA to yield a hairpin structure ( Supporting Information Figure and incubated it with the lysate of HEK293T cells at 37 °C followed by nuclease digestion The and which were released from 5caC-DNA and decarboxylation were monitored by LC-MS/MS and Supporting Information Figure The of DNA from cell lysate on magnetic beads was ( Supporting Information Figure that levels of of DNA from cell lysate can be The LC-MS/MS results revealed a in the of which is with a in the of and Supporting Information Figure indicating the direct decarboxylation of 5caC-DNA in cell was in negative 1 cell lysate was or negative 2 RIPA solution was used of cell treatment for 4 h, of was the of was to the DNA substrate with the same as the 5caC-DNA that the 5caC was replaced with a Supporting Information Table indicating a of decarboxylation of 5caC in the cell lysate 4 h. We examined the decarboxylation of 5caC-DNA at the oligodeoxynucleotide After incubation with cell the 5caC-DNA substrate and processed were digested with SspI restriction and subjected to high-resolution LC/MS analysis The released oligodeoxynucleotide m/z from 5caC-DNA substrate was in the negative was treated with cell but was not detected in the DNA without 5caC The product from the decarboxylation of 5caC m/z the MS is in Supporting Information Figure was clearly at 1 h, and with incubation time Supporting Information and to the analysis performed at the quantification data of oligodeoxynucleotides revealed that a 4 incubation of 5caC-DNA with cell lysate led to decarboxylation of 5caC Supporting Information and Direct decarboxylation of 5caC in human cells We in decarboxylation of 5caC. The 5caC-DNA substrate was transfected into HEK293T cells, and the DNA was subsequently isolated from cells, digested with and subjected to LC-MS/MS analysis In this transfection of 5caC-DNA substrate showed that 5caC-DNA was in both the and with the of 5caC-DNA in the ( Supporting Information Figure We clearly detected both and in the digestion mixture of DNA substrate isolated from cells, and the of with which is with a in that of and and Supporting Information Figure showed that

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