Cloning of wild‐type and mutant DNA polymerases

Clones for the wild‐type DNA polymerase ε subunits were a kind gift from the Hurwitz Lab [27]. The original p261 clone was tagged with a Flag epitope. Since we were interested in the properties of intact complexes rather than the isolated p261, the tag was removed from the large subunit and a Flag tag added to the p58 subunit. The exonuclease dead, P286R and V411L variants were obtained by carrying out point mutation to these clones using the QuikChange II Site‐Directed Mutagenesis Kit (Agilent, Santa Clara, CA, USA). The oligonucleotides used to produce these mutations are shown in Table 2. The changes introduced to make the polymerase devoid of 3′‐5′ exonuclease activity (exonuclease dead) were based on work reported from yeast and mice [9, 10].

Nucleotide changes made to produce the human POLE variants used in this study.

a824c

a830c