All proteins were expressed in E. coli BL21 (DE3).14 (link),18 (link) Enhanced clamp binding mutants of polymerase and exonuclease were used with amino acid changes for the polymerase in residues QADMF to QLDLF from 920–924 and for the exonuclease QTSMAF to QLSLPL from 182–187.14 (link) Pol IIIα, β and ε subunits were expressed and purified as previously described19 (link) and were flash frozen in liquid nitrogen and stored at −80 °C. θ mutant E41C was created to incorporate a Cys residue for maleimide labeling. The Quikchange lightning kit (Agilent) was used for site-directed mutagenesis. θ was purified on a Histrap HP column and a Superdex 75 gel filtration column. The θ subunit containing a E41C mutation was labeled with Cy5 maleimide (GE Healthcare) following manufacturer instructions. Pol III core complexes consisting of α, β and ε were assembled at equistoi-chiometric ratio and then chromatographically separated by gel filtration (Superdex 75 column). Proteins were flash frozen in liquid nitrogen and stored at −80 °C until needed.