Figure 4. PhADE reveals single molecules of Fen1^KikGR at replication forks and measures Fen1^KikGR off rate from DNA.

(a) Imaging sequence for Figure 4b–c. (b) Replication in the presence of 0.25 μM WT Fen1^KikGR. (top) A representative kymograph of Fen1^KikR on λ DNA imaged every 10 s. After the final round of PhADE, replication bubbles and DNA were detected. 132 Fen1^KikR foci were localized to 113 replication bubbles. Scale bar: 1 μm. (bottom) Intensity histogram of Fen1^KikR foci on bubbles (black) versus single molecules of biotin-mKikR (gray). (c) As in Figure 4b but using 0.125 μM WT Fen1^KikGR. 112 Fen1^KikR foci were found on 129 replication bubbles. (d) DNA was replicated in the presence of 0.125 μM wild-type Fen1^KikGR and Fen1^KikR was imaged as in Supplementary Figure 7a before (first frame) and after photoactivation (frames 2–6). Afterwards, replication bubbles and DNA were detected. Scale bar: 1 μm. (e) The duration of wild-type Fen1^KikR binding at replication bubbles is compared to the fluorescence on times of immobilized Biotin-mKikR. (wild-type Fen1^KikR: mean ± s.d. of 3 experiments, n = 40 events; Biotin-mKikGR: mean ± s.d. of 6 experiments, n = 1,165 events, points are fit to a mono-exponential decay). (f) DNA was replicated in the presence of 2 μM WT Fen1^KikGR. Fen1^KikR was photoactivated once (black triangle) and imaged once a minute as depicted in Supplementary Figure 7c. Scale bar: 1 μm. (g) Normalized, integrated intensity of the region in Figure 4f (squares) is compared to Biotin-mKikR photobleaching (solid line) under the same imaging conditions. Biexponential fit (dashed line) reveals a mean life-time of 0.7 ± 0.3 min for Fen1^KikGR on DNA.