Fig. 2: Determination of Sloppymerase characteristics.

A Time lapse experiment to assess Sloppymerase processivity. A nicked hairpin (HP) was incubated with either all dNTPs present (4) or with dATP, dGTP, and dTTP present (3), with DNA synthesis followed at different time points (0–120 min). When subjected to denaturing PAGE, the hairpin dissociates into two fragments: a longer 5′ fragment (A) and a shorter 3′ fragment (C). Extension of the longer fragment (B) indicates DNA polymerase activity, while degradation of fragment C indicates 5′–3′ exonuclease activity. A quantification of the bands (A and B) in the gel image are presented, which do not take in account the smear in between that indicate partially extended hairpins. The actual efficiency is hence even higher. B Processivity of Sloppymerase is dependent on the combination of provided dNTPs. A nicked hairpin (HP) was incubated with Sloppymerase and different combinations (either one or two dNTPs were omitted) and concentrations of dNTPs. Bands in position (B) imply full extension of the hairpin, whereas bands in position (A) indicate no elongation. C Structure of Sloppymerase, E. coli DNA polymerase I and DNA polymerase η, as predicted by AlphaFold.