Cell cultivation and treatment
Mouse embryonic fibroblasts (MEFs) and human breast cancer cell line MCF7 were maintained in Dulbecco's modified Eagle's medium (DMEM; Merck, Darmstadt, Germany) supplemented with 10% fetal bovine serum FCS (Merck), penicillin (1 U/ml), and streptomycin (100 µg/ml) at 37°C in a humidified atmosphere containing 5% CO2. For the experiment of dependent recruitment of ac4C on cell cycles, we used HeLa-Fucci cells expressing RFP-Cdt1 in the G1 phase and GFP-geminin in the S/G2/M phases, as have previously been described in detail by Sakaue-Sawano et al. [21]. HeLa-Fucci cells were cultivated in the same in vitro conditions. 53BP1-deficient and 53BP1 wild type immortalized mouse embryonic fibroblasts (iMEFs) were a gift from Michela Di Virgilio, Laboratory of DNA Repair and Maintenance of Genome Stability, Max Delbrück Center for Molecular Medicine in the Helmholtz Association, Berlin, Germany. Immortalized MEFs were cultured in DMEM medium supplemented with 10% FBS, 2 mM L-Glutamine, and Penicillin-Streptomycin at 37°C and 5% CO2 levels [33]. Rif1-deficient HCT116 cells and wildtype HCT116 cells (a generous gift from prof. David M. Gilbert, San Diego Biomedical Research Institute, USA, [34]) were cultivated in DMEM (Merck) supplemented with 10% FBS, and Penicillin-Streptomycin.
We inhibited RNA polymerase I or poly (ADP ribose) polymerase (PARP). In this case, cells were treated at 50% confluence with actinomycin D (#A9415, Merck; final concentration 0.5 µg/ml, 2 h treatment before microirradiation), or by Olaparib (#S1060, Selleckchem, Germany; final concentration 10 µM, treatment 24 h before microirradiation) [35, 36].
Treatments by enzymes: We used Turbo-DNase (#AM2238, ThermoFisher Scientific, Waltham, MA, USA), RNase A (#R5503, Merck), and RNase H1 (#EN0201, ThermoFisher Scientific). The cells were permeabilized with cold 0.1% Triton X-100 in PBS for 10 sec, washed twice in phosphate-buffered saline (PBS), and incubated in 300 µl RNase A (0,5 mg/ml in PBS) or DNase I (5 U in 1x DNase Reaction buffer) or RNase H1 (2U in 1x RNase Reaction buffer) for 8 min at 37°C before immunostaining [17, 37]. Subsequently, fixation was performed with 4% formaldehyde and permeabilization with 0.3% Triton X. After that, it was followed by further enzymatic treatment for 1 h at 37°C. Relative fluorescence intensity was evaluated in 25 nuclei, and statistical analysis was performed.
Irradiation By Uv-light
The cells seeded on 35 mm glass-bottom dishes (#D35-20-1-N, Cellvis Mountain View, CA, USA) and at 50% confluence were sensitized with 10 µM BrdU (#11296736001, Merck) for 16 h before UVA treatment. The cells were irradiated by the UVA lamp (model GESP-15, 15 W, UVA 330–400 nm wavelength, with maximum efficiency at 365 nm) or UVC lamp (Philips, Amsterdam, The Netherlands, model TUV 30 W T8, UVC 254 nm wavelength). Irradiation was performed for 10 min. After UVC irradiation, the cells were fixed at multiple intervals (5 min, 20 min, 60 min, and 120 min after irradiation). The lamp distance from the sample was 2 cm for the UVA source and 60 cm for the UVC source [18]. The statistical analysis was performed in 40 cells.
Immunofluorescence And Confocal Microscopy
The immunofluorescence protocol was adapted according to Svobodova et al. [38] and modified. The cells were fixed with 2 ml 4% formaldehyde (prepared from paraformaldehyde, PFA; #AAJ19943K2, Thermo Fisher Scientific) for 5 min at room temperature (RT), and then 200 ml 1% SDS was added, and incubation proceeded with an additional 7 min. Afterward, samples were permeabilized with 0.2% Triton X-100 for 15 min and washed twice in PBS for 15 min. As a blocking solution, we used 1% bovine serum albumin (Merck), dissolved in 0.1% 1x PBSTween 20 (BSAT) for 1 hour at RT. Dishes with fixed cells were washed for 15 min in PBS and incubated with primary antibodies at a 1:100 dilution in 1% BSAT at 4°C overnight. For immunofluorescence analysis, the following antibodies were used: anti-N4-acetylcytidine/ac4C in RNA (#A18806 Abclonal, Woburn, MA, USA), anti-phosphorylated histone H2AX (γH2AX; phospho S139) (#05-636, Merck), anti-fibrillarin (#ab4566, Abcam), anti- phospho-ATM; Ser1981 (#MAB3806-C, Merck), anti-α-tubulin (#ab80779 Abcam), and anti-NAT10 (B-4) (#sc-271770, Santa Cruz Biotechnology, Dallas, TX, USA). After incubation with primary antibodies, the samples were washed twice in PBS for 15 min and incubated with the following secondary antibodies, diluted 1:300 in 1% BSAT: Alexa 488-conjugated goat anti-mouse (#ab150077, Abcam), Alexa 594-conjugated goat anti-rabbit (#A11037, Thermo Fisher Scientific), Alexa 488-conjugated goat anti-mouse (#A11029, ThermoFisher Scientific), Alexa Fluor 594-conjugated goat anti-mouse (#A11032, Thermo Fisher Scientific), and Alexa 647-conjugate goat anti-rabbit (#A21245, Thermo Fisher Scientific). The DNA content was visualized using 4′,6-diamidino-2-phenylindole (DAPI; Merck), and Vectashield (Vector Laboratories, USA) was used as the mounting medium. Samples were also incubated without primary antibodies for negative control staining.
Local Laser Microirradiation And Laser Scanning Confocal Microscopy
For the micro irradiation experiments using UVA lasers (wavelength 355 nm), cells were seeded on 35 mm gridded microscope dishes (#81166, Ibidi, Fitchburg, WI, USA), and at 50% confluence, cells were sensitized with 10 µM BrdU for 16h. For microscopy, the cells were maintained under optimal cultivation conditions in an incubation chamber (EMBL) at 37°C, supplemented with 5% CO2. In the selected cell nuclei, we irradiated only the defined region of interest (ROI) using a laser connected to TCS SP5-X confocal microscope system (Leica, Wetzlar, Germany). The microscope settings for induction of local DNA damage were as follows: power laser (355 nm) 25 mW, 512×512 pixel resolution, 400 Hz, bidirectional mode, 48 lines, zoom 4, and 63x oil objective (HCX PL APO, lambda blue) with a numerical aperture (NA) = 1.4 [18]. The maximum exposure of the cells to the laser was 45 minutes, and we monitored approximately 100 cell nuclei. The analysis of 3 biological replicates was performed. After the immunostaining procedure, locally microirradiated cells were localized according to registered coordinates on gridded microscope dishes. We studied the level of the epigenetic marker N4-acetylcytidine in RNA, NAT10 acetyltransferase, and the presence of γH2AX (phospho S139), which was also used for the optimization of microirradiation experiments. For image acquisition and analysis of fluorescence intensity (FI) we used LEICA LAS X software.
Western Blotting
Western blotting was performed using the methods reported in [38]. We used the following primary antibodies: anti-phosphorylated histone H2AX (γH2AX; phospho S139; #ab2893, Abcam), anti-α-tubulin (#ab80779 Abcam), anti-NAT10 (B-4) (#sc-271770, Santa Cruz Biotechnology). As secondary antibodies, we used anti-rabbit IgG (#A-4914, Merck; dilution 1:2000), anti-mouse IgG (#A-9044, Merck; dilution 1:2000) and anti-mouse IgG1 (#ab97240, Abcam; dilution 1:5000).
Gel Analysis Of Rna
RNAs (1–1,5 µg) intended for dot blot were mixed with 2x RNA loading dye (#R0641, Thermo Fisher Scientific) in a ratio of 1:1 and heated to 70°C for 5 min, followed by chilling on ice. These RNA samples were separated in a 10% denaturing polyacrylamide gel containing 7 M urea. As running buffer was used 1x TBE (90 mM Tris, 90 mM borate, and 2 mM EDTA, pH = 8.0) and electrophoresis conditions were 35 mA for 1 hour in cold. After electrophoretic separation, the gel was washed in 1x TBE for 10 min and incubated in GelRed (#41003, Biotium, Fremont, CA, USA) for 15 min. Additional washing was in 1x TBE. The RNA bands were visualized using an Amersham Imager 680 (GE Healthcare, Freiburg, Germany).
Dot Blots
Samples of RNA were diluted to a final concentration of 250 ng/µl or 200 ng/µl. The method was based on Abcam RNA Dot Blot Protocol (https://www.abcam.com/protocols/rna-dot-blot-protocol) and modified according to our conditions. Diluted RNA was denatured at 95°C in a heat block for 3 min, immediately placed on ice for 1 min, and loaded onto Hybond-N + membranes. Membranes were crosslinked by UVC 254 nm lamp for 30 min. The parameters were calculated so that the total energy was 300 mJ/cm2. After that, membranes were washed in 10 ml of TBST (1X TBS, 0.1% Tween-20) for 5 min at RT, blocked with 4% non-fat milk in TBST for 1 h at RT with gentle shaking, and incubated overnight with primary antibodies Anti-N4-acetylcytidine/ac4C (#ab252215, Abcam or #A18806, Abclonal) in blocking buffer at 4°C (dilution 1:2000 or 1:1000). Membranes were washed tree time for 10 min in TBST. As the secondary antibody, we used anti-rabbit IgG (#A-4914, Merck; dilution 1:5000) and visualized spots by Amersham Imager 680 (GE Healthcare).
Isolation Of Total Rna
Total RNA was purified from MEFs (the second day after seeding) using the Quick-RNA Miniprep Kit (#R1054; Zymo Research, Irvine, CA, USA). For isolation of small RNA was used mirVana™ miRNA Isolation Kit (#AM1560, Thermo Fisher Scientific). RNA isolations were done according to the manufacturer’s instructions. Large forms of RNA were separated using both kits in a special step according to the manufacturer's instructions. Following purification, RNA was quantified using a spectrophotometer (NanoDrop™ 2000/2000c Spectrophotometers (#ND-2000, ThermoFisher Scientific). RNA samples for Dot blot analysis were isolated from 3 biological replicates.
Statistical analysis
Fluorescence intensity values were measured by LAS X software and subsequently analyzed in Python 3 software. The obtained data were compared statistically using the ANOVA One-Way test, available in GraphPad Prism software, version 9 for Windows (GraphPad Software, San Diego, CA, USA). All cases labeled in the graphs by asterisks differ significantly from control values.