Purification of Recombinant Human DNA Polymerase Epsilon

The N-terminal half of POLE that encodes the DNA polymerase and exonuclease domains of human Polϵ (residues 1–1189, 140kDa) was cloned into a pHB-CMV-MCS-EF1-Puro vector as previously described (29 (link)). The plasmids encoding Polϵ-P286R, R375Q, V411L, and P452L were constructed using the primers summarized in Supplementary Table 2. A FLAG tag was added to the N-terminus via PCR. The recombinant FLAG-protein was expressed in 293T cells. Co-immunoprecipitation (Co-IP) was performed to isolate the FLAG-conjugated catalytic subunit using the FLAG-beads (Sigma-Aldrich, St. Louise, MO, USA) following the manufacturer’s instruction. The FLAG-tagged protein band was visualized using Coomassie blue staining and Western blot analysis with an anti-FLAG antibody (#2368T; 1:1000; Cell Signaling Technology, Danvers, MA, USA). The FLAG-tagged wildtype and mutant catalytic fragments were purified from the immunoprecipitated complexes using the 100 kDa Amicon^®Ultra-0.5 filter (MilliporeSigma, Burlington, MA, USA).