Analysis of flanking sequence preference with a randomized substrate and bioinformatic data
For analysis of the flanking sequence preference, substrate with different target sites sites in a 9 or 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 in methylation buffers as indicated in the main manuscript . 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).
References
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Dukatz M, Dittrich M, Stahl E, Adam S, de Mendoza A, Bashtrykov P, Jeltsch A. DNA methyltransferase DNMT3A forms interaction networks with the CpG site and flanking sequence elements for efficient methylation. J. Biol. Chem. 298(10), 102462 (2022)
Dukatz M, Adam S, Biswal M, Song J, Bashtrykov P, Jeltsch A. Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase. Nucleic Acids Res 48, 11495-11509 (2020)
