Deep enzymology data related to Dukatz et al.: Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase.doi:10.18419/darus-815DaRUS2020-06-183Jeltsch, Albert; Bashtrykov, Pavel; Dukatz, Michael; Adam, Sabrina, 2020, "Deep enzymology data related to Dukatz et al.: Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase.", https://doi.org/10.18419/darus-815, DaRUS, V3Deep enzymology data related to Dukatz et al.: Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase.doi:10.18419/darus-815Jeltsch, AlbertBashtrykov, PavelDukatz, MichaelAdam, SabrinaJE 252/10 - 194537093JE 252/36 - 403074082DaRUSJeltsch, AlbertJeltsch, Albert2020-06-16Medicine, Health and Life SciencesDNA MethylationDNA MethyltransferaseNGS Bisulfite SequencingEnzyme AssayDNMT3BEnzyme ActivityExperimental procedures: Libraries of double stranded DNA substrates with CpG, CpH or CpN sites in randomized sequence context were methlyated by DNMT3B. Reactions were stopped by shock freezing in liquid nitrogen, then treated with proteinase K for 2 hours. Afterwards, the DNA was digested with the BsaI-HFv2 enzyme and a hairpin was ligated using T4 DNA ligase (NEB). The DNA was bisulfite converted using EZ DNA Methylation-Lightning kit (ZYMO RESEARCH) according to the manufacturer protocol, purified and eluted with 10 µL ddH2O. Libraries for Illumina Next Generation Sequencing (NGS) were produced with the two-step PCR approach. In the first PCR, 2 µL of bisulfite-converted DNA were amplified with the HotStartTaq DNA Polymerase (QIAGEN) and primers containing internal barcodes using following conditions: 15 min at 95 °C, 10 cycles of 30 sec at 94 °C, 30 sec at 50 °C, 1 min and 30 sec at 72 °C, and final 5 min at 72 °C; using a mixture containing 1x PCR Buffer, 1x Q-Solution, 0.2 mM dNTPs, 0.05 U/µL HotStartTaq DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. In the second PCR, 1 µL of obtained products were amplified by Phusion Polymerase (Thermo) with another set of primers to introduce adapters and indices needed for NGS (30 sec at 98 °C, 10 cycles - 10 sec at 98 °C, 40 sec at 72 °C, and 5 min at 72 °C). PCRII was carried out in 1x Phusion HF Buffer, 0.2 mM dNTPs, 0.02 U/µL Phusion HF DNA Polymerase, 0.4 µM forward and 0.4 µM reverse primers in a total volume of 20 µL. Obtained libraries were pooled in equimolar amounts and purified using NucleoSpin® Gel and PCR Clean-up kit (Macherey-Nagel), followed by a second purification step of gel extraction and size exclusion with AMPure XP magnetic beads (Beckman Coulter).Raw DNA sequences extracted from Fastq NGS files; Bisulfite-seq DNA methylation analysis<a href="https://pubmed.ncbi.nlm.nih.gov/33105482/">PMID 33105482</a>Michael Dukatz, Sabrina Adam, Mahamaya Biswal, Jikui Song, Pavel Bashtrykov, & Albert Jeltsch: Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase. Nucleic Acids Res. 2020 Nov 18;48(20):11495-1150910.1093/nar/gkaa938Michael Dukatz, Sabrina Adam, Mahamaya Biswal, Jikui Song, Pavel Bashtrykov, & Albert Jeltsch: Complex DNA sequence readout mechanisms of the DNMT3B DNA methyltransferase. Nucleic Acids Res. 2020 Nov 18;48(20):11495-115093B K777A 1 µM CN.txttext/plain3B K777A 2 µM CN.txttext/plain3B N652A 1 µM CN.txttext/plain3B N652A 2 µM CN.txttext/plain3B N656A 0.5 µM CN.txttext/plain3B N656A 1.0 µM CN.txttext/plain3B N656D 10 µM CN.txttext/plain3B N656D 5 µM CN.txttext/plain3B N658A 3 µM CN.txttext/plain3B N658A 6 µM CN.txttext/plain3B N779A 0.5 µM CN.txttext/plain3B N779A 1.0 µM CN.txttext/plain3B N779D 0.5 µM CN.txttext/plain3B N779D 1 µM CN.txttext/plain3B R661A 1.5 µM CN.txttext/plain3B R661A 3.0 µM CN.txttext/plain3B R823A 1.5 µM CN.txttext/plain3B R823A 3.0 µM CN.txttext/plain3B T775A 10 µM CN.txttext/plain3B T775A 5 µM CN.txttext/plain3B V657A 1 µM CN.txttext/plain3B V657A 2 µM CN.txttext/plain3B WT 0.5 µM CN.txttext/plain3B WT 1.0 µM CN.txttext/plainh3B 0.5 µM HM.txttext/plainh3B 1 µM HM.txttext/plainno enzyme CN.txttext/plainR823A 3 µM HM.txttext/plainreadme.pdfapplication/pdf