Single-Molecule FRET Measurements of DNA-Pol Interactions

Single-molecule FRET measurements were performed at room temperature using a home-built confocal microscope with 20 kHz alternating-laser excitation between a 532-nm (Samba, Cobolt, operated at 240 μW) and a 638-nm laser (Cube, Coherent, operated at 60 μW), coupled to a 60×, 1.35 numerical aperture (NA), UPLSAPO 60XO objective (Olympus) as previously described (11 (link)). For DNA–DNA measurements, labelled DNA was present at <100 pM and unlabelled Pol (when present) at 3 nM concentration. For Pol-DNA measurements, both Pol and DNA were present at 100 pM concentration. Measurements were taken in ‘Pol buffer’, consisting of 40 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)–NaOH, pH 7.3, 10 mM MgCl₂, 1 mM DTT, 100 μg ml⁻¹ bovine serum albumin, 5% (v/v) glycerol, 1 mM mercaptoethylamine. Photon streams in DD, DA and AA channels were recorded and processed using custom-written software (LabVIEW). Bursts were filtered for the correct labelling stoichiometry (12 (link)), and accurate FRET efficiencies were calculated as described ((13 (link)) and Supplementary Methods). Distances were calculated from the FRET efficiencies, using experimentally determined Förster radii and donor quantum yields (Supplementary Table S4).