(A) Schematic representation of the donor (green, attached to primer strand) and acceptor (magenta, attached to thumb region of Pol I) labeling sites. Pol I is depicted in cartoon form, with the core colored gray and the 5’ nuc domain colored blue. (B) Representative smFRET trajectory (blue) and donor (green) and acceptor (magenta) emission trajectories, for Pol I interacting with mixed flap substrate. The bold line is the idealized state path determined from Hidden Markov modeling. (C) Composite FRET efficiency histograms for states P and N, compiled from n individual FRET trajectories (n value indicated in each plot), for various combinations of DNA substrate and protein. The proteins, from top to bottom, are WT Pol I, Pol I L361A, and KF L361A. The corresponding populations of states P and N are indicated. (D) Transition density plots for Pol I interacting with flap-containing DNA substrates, compiled from a total of n transitions. From left to right: double flap DNA, mixed flap DNA and downstream flap DNA. (E) Rate constants for intramolecular transfer of DNA substrates from pol domain to 5’ nuc domain (P→N) or from 5’ nuc domain to pol domain (N→P).