(A) Left panel: Electro-mobility shift assay with the E. coli DNA polymerases Pol I (Klenow fragment), Pol II, Pol IIIα, and Pol IV. At 2.5 μM Pol I, Pol II, and Pol IV retain DNA, whereas Pol IIIα does not. The more intense bands for Pol I and Pol II are caused by protein induced fluoresence enhancement (PIFE) (Hwang et al., 2011). Right panel: SDS-page analysis of the same samples used for the electro-mobility shift assay (proteins stained with coomassie blue). Molecular weights: Pol I (Klenow fragment) 70 kDa, Pol II 90 kDa, Pol IIIα 130 kDa, Pol IV 40 kDa. (B) Structural comparison of Pol I (PDB code: 1QTM [Li et al., 1999]), Pol II (PDB code: 3K57 [Wang and Yang, 2009]), PolIIIα (this work), and Pol IV (PDB code: 4IRD [Sharma et al., 2013]). Polymerases were aligned on the 3’ end of the primer, indicated by the solid line. For Pol I, Pol II, and Pol IV, the clamp was modeled based on predicted clamp interacting motifs in the respective polymerases. The distance measured in base pairs between the 3’ end of the primer and the opening of the clamp is indicated on top of the structures, together with the rate of DNA synthesis of each polymerase. (C) Detailed view of the interaction of the polymerase thumb domains and the DNA. All polymerase thumb domains interact with the backbone of the minor groove. Only Pol IIIα inserts a loop into the major groove of the DNA.