Fig. 5. Kinetic competition between FEN1 and Polδ for the NP.

A Fluorescence emission spectra of internal labeled NP (Sub#24; Supplementary Fig. 7) in the presence of pre-loaded PCNA at various concentrations of FEN1 D181A. B Quantification of the data presented in panel A. The experimental datapoints were fitted to a dependence proportional to Eq. (1) (“Methods”). C Fluorescence recovery of the Cy3 donor presented in panel A upon addition of a large excess of FEN1 DNA trap (top; 5 µM of unlabeled phosphotiolated nonequilibrating double flap; Sub#31; Supplementary Fig. 7) or Lig1 (bottom; 2 µM). The experimental datapoints were fitted to Eq. (2) (“Methods”). D Fluorescence emission spectra of Cy3-labeled NP (Sub#28; Supplementary Fig. 7) in the presence of pre-loaded PCNA at various concentrations of Polδ. E Quantification of the data presented in panel D and of similar experiments in which the NP was replaced with either a 2-nt gap (Sub#29; Supplementary Fig. 7) or a double flap (Sub#27; Supplementary Fig. 7). F Fluorescence recovery of the Cy3 donor presented in panel D, upon addition of a large excess of Polδ chemical trap (20 ng/µL of heparin) for the 2-nt gap (top; Sub#29) and the NP (bottom; Sub#28) substrates. The experimental datapoints were fitted to a dependence proportional to Eq. (1) (“Methods”). G Cartoon representation of the kinetic competition between FEN1 and Polδ for the NP from association rate perspective. Association rates (k_on) were determined from dissociation constants (K_D) and rates (k_off) based on the indicated equation. The f_{i = 1, 2} coefficients represent the engagement probabilities of the NP by FEN1 and Polδ, respectively, based on their association rates and the indicated equation. Source data are provided as a Source Data file.