Abstract
Cloning and site-directed mutagenesis: The gene of the catalytic, C-terminal domain of murine DNMT3C (amino acid residues 439-740 of P0DOY1) was obtained in E. coli codon optimized form from IDT Integrated DNA Technologies. The gene fragment was cloned with the StrataClone PCR Cloning Kit (Stratagene) into a StrataClone Vector Mix amp/kan (Stratagene) and plasmid transformation was performed with StrataClone SoloPack competent E. coli cells (Stratagene). Cells were recovered in SOC medium at 37 °C with shaking at 200 rpm for 1 h and cell suspension was plated on 2% LB-agar containing ampicillin at 37 °C. The next day, white single colonies were used for inoculating 2% LB liquid cultures containing ampicillin, which were shake grown overnight at 37 °C with 200 rpm. The plasmid DNA was then isolated using the NucleoSpin® Plasmid Kit (MACHEREY NAGEL). Next, the DNMT3C fragment was cloned into a His-tagged pET28 expression vector (Gowher & Jeltsch, 2002) using Gibson assembly. The entire pET28 vector containing the DNMT3C catalytic domain was amplified by rolling circle PCR using Pfu DNA Polymerase (ThermoFisher SCIENTIFIC) and subsequent digested with DpnI. Plasmid purification was performed using NucleoSpin® Gel and PCR Clean-up Kit (MACHEREY-NAGEL) and finally transformed into One Shot®Stbl3™ Chemically Competent E. coli cells (ThermoFischer Scientific). Cell recovery was done in SOC medium at 37 °C with shaking at 200 rpm for 1 h. The recovered cell liquid was then grown on 2% LB-agar containing kanamycin and incubated overnight at 37 °C. Single colonies were used for inoculating 2% LB liquid cultures containing kanamycin, which were shake grown overnight at 37 °C with 200 rpm. Plasmid DNA was isolated with NucleoSpin® Plasmid Kit (MACHEREY NAGEL). Site-directed mutagenesis of the DNMT3C WT plasmid was performed by using mutagenic primers containing one or two mismatches at the desired position to precisely insert the mutation and PCR amplification of the vector using Pfu DNA polymerase (ThermoFisher SCIENTIFIC) basically as described above. Transformation and purification was done as described above. The DNA sequences in all steps of the procedure as well as the final plasmid DNA sequences were confirmed by Sanger DNA sequencing (Microsynth Seqlab GmbH).Protein overexpression and purification: Plasmids expressing His-tagged DNMT3C catalytic domain and its C543N, V547A, E590K and C543N/V547A (NA) mutants were transformed and overexpressed in E. coli BL21 (DE3) codon plus RIL cells (Stratagene). The overexpression and purification were performed as described for DNMT3A catalytic domain (Emperle et al. 2014). Some precipitation of the protein occurred during dialysis for the DNMT3C WT as well as mutant proteins. The protein concentration and purity were determined using Coomassie-stained 12% SDS polyacrylamide gels revealing a purity of >95% of all purified proteins. Analysis of flanking sequence preference with a randomized substrate and bioinformatic data: For analysis of the flanking sequence preference, substrate with CpG or CpX sites in a 10 bp randomized sequence context were prepared as described (Dukatz, et al. 2020; Dukatz, et al. 2022). Substrate methylation reactions were performed with different enzyme concentrations (0.1875-30 µM) in methylation buffer (20 mM HEPES pH 7.5, 1 mM EDTA, 50 mM KCl, 0.25 mg/mL bovine serum albumin, 1 mM AdoMet (Sigma)) using 1 µM randomized substrate. Methylation reactions were incubated at 37 °C for 30-60 min. The reactions were stopped by freezing in liquid N2, followed by 2 h digestion with proteinase K (NEB) at 42 °C and purification with NucleoSpin® Gel and PCR Clean-up Kit (Macherey-Nagel). Bisulfite conversion was performed as described in the standard protocol EZ DNA Methylation-Lightning™ Kit (Zymo Research). Samples were eluted with RNase free H2O. Library preparation was performed with two PCRs using variable primer pairs to introduce sample specific barcodes and indices for sample distinction and the sequencing reactions. Bioinformatic analysis of the NGS data was conducted as described (Dukatz, et al. 2020; Dukatz, et al. 2022). For determination of the methylation rates of all 256 NNCGNN sequences by one enzyme, methylation reactions of individual substrates were assumed to be independent and reaction velocities are of first order with respect to the substrate concentrations. The results of the individual reactions with different enzyme concentrations and incubation times were fitted to monoexponential reaction progress curves using variable virtual time values. Fitting was conducted with MatLab as described except that convergence was validated by serial fitting (Adam, et al. 2022).
Links and resources
Tags
