Resource s

Four recombinant versions of P. abyssi Q9UZY3 were synthesized and cloned (Genecust) as described in Figure S2. Following the same protocol as for PAN expression, proteins were overexpressed in E. coli BL21(DE3) and cell pellets were resuspended into Buffer 2 (20 mM Tris–HCl, pH 8.0, 150 mM NaCl), complemented with 0.1% Triton X‐100, 25 mM MgSO₄, 0.25 mg ml⁻¹ lysozyme (Euromedex), 0.05 mg ml⁻¹ DNase I grade II (Roche), 0.2 mg ml⁻¹ RNase (Roche) and EDTA‐free protease inhibitor (cOmplete™, Roche). The cells were disrupted by sonication and incubated at 25°C for 30 min. Then the cell extract was treated by heating at 70°C for 15 min and the lysate was clarified by centrifugation at 10,000×g for 1 h. His‐tagged Q9UZY3 was purified by using (i) an affinity column (5‐ml HiTrap Chelating, GE Healthcare) with a linear gradient of 20–250 mM imidazole; (ii) a cation exchange column (Resource S, GE Healthcare) with a linear gradient of 50–500 mM NaCl and (iii) a size exclusion column (Superose 12, GE Healthcare) with elution in Buffer 2. For the untagged Q9UZY3, only the purification steps (ii) and (iii) were performed. The final Q9UZY3 concentration was calculated by measuring the absorbance at 280 nm using the predicted extinction coefficient (ProtParam, ExPASy) listed in Table S1. The primers and cloning strategies for expression in bacteria cells are listed in Table S2.

The chicken MBD2_MBD (amino acids 1–71) and human MBD2_MBD+IDR (amino acids 150–214) were expressed and purified as previously reported (11 (link),13 (link)). Chicken MBD2_MBD+IDR, which likewise includes an additional 46 residues (amino acids 1–117), was cloned into the previously described pET32a based vector (13 (link)), and further modified to replace the thrombin cleave site with a TEV protease cleavage site. The resulting thioredoxin fusion construct was transformed into BL21(DE3) Escherichia coli cells grown at 37°C until an OD₆₀₀ of 0.7 and induced with 1 mM IPTG. Bacteria were pelleted and lysed in 20 mM Tris pH 8, 1 M NaCl with sonication. Proteins were further purified via HisTrapFF (GE Life Sciences) and cleaved using either thrombin (MBD2_MBD) or TEV protease (MBD2_MBD+IDR). Cleaved proteins were further purified over a Resource-S (GE Life Sciences) ion exchange column and Superdex-75 (GE Life Sciences) using an ÄKTA pure FPLC system (GE Life Sciences). Proteins for NMR analysis were either ²H,¹⁵N,¹³C or ²H,¹⁵N labeled. For fluorescence polarization assays the thioredoxin fused protein was purified via HisTrapFF and Superdex-75. For single-molecule experiments MBD2_MBD+IDR and MBD2_MBD were cloned into pET28a with N-terminal His₆ tags and expressed and purified in a similar manner.

Human wild-type DNA pol β was overexpressed from a pET-30 vector in the BL21-CodonPlus(DE3)-RP Escherichia coli strain. Purification of pol β was carried out as described previously and briefly written here (20 (link)). Cell lysate containing pol β was run over GE HiTrap Heparin HP, GE Resource S, and HiPrep 16/60 Sephacryl S-200 HR columns and fractions containing pure pol β were concentrated and stored at –80°C in 20 mM BisTris propane, pH 7.0 for crystallization and 50 mM HEPES, pH 7.4 for kinetics. Pol β was determined to be pure by SDS page and the final concentration was determined by A₂₈₀ using a NanoDrop One UV-Vis Spectrophotometer (ε = 23 380 M⁻¹ cm⁻¹).