(A) ϕX174 phage DNA (5.4 kb) primer extension assays, showing the activity of fluorescently labeled and unlabeled proteins. In the labeled reactions, fluorescently labeled clamp, labeled clamp loader, and labeled polymerase (Pol IIIcore, Pol II, or Pol IV) were used. Reaction were quenched at 0, 0.5, 1, 2, and 5 min. The increased background in the ‘Pol IIIcore Fluo label +" lanes is caused by the presence of αAtto488, whose fluorescent signal overlaps with that of the fluorescein label on the DNA primer (B) Coomassie stained SDS-page showing unlabeled and labeled proteins (marked with *). (C) Three-wavelength fluorescent scan of the same gel shown in B (before coomassie staining) revealing the presence of the labeled proteins. (D–F) Lifetimes of fluorescent dyes Atto 488, Atto 565, and Atto 674N under experimental conditions. The dyes were attached to the β-clamp that was loaded onto the DNA substrate used in the experiments. (G–F) Lifetime of Pol IIIcore on clamp-DNA before and after photobleaching of the Atto 488 fluorophore on the DNA substrate. All values represent mean lifetime/lag time ±s.e.m.