Assembly of a pol η holoenzyme was monitored via FRET by slight modifications to a published protocol (Hedglin et al., 2013b). (A) Schematic representation of the experiment depicted in B to monitor FRET under equilibrium conditions. A forked P/T DNA substrate (forked Cy3-P/T DNA) in agreement with the minimal requirements for assembly of human RFC and PCNA onto DNA was labeled with an internal Cy3 dye. This substrate also carries a 3′-biotin label. Together with the flap, the 3′-biotin label in complex with Neutravidin prevents loaded clamp from sliding off the DNA. Forked Cy3-P/T DNA (100 nM) was pre-incubated with Cy5-PCNA (100 nM), RFC (100 nM), and ATP (1 mM). Under these stoichiometric conditions, RFC loads all Cy5-PCNA onto Cy3-DNA and, in the absence of polymerase, dissociates back into solution taking all loaded PCNA with it. After the initial loading event, the clamp loading-unloading pathway reaches equilibrium where the rate constants for PCNA loading and unloading are equal and a net change in the FRET signal is no longer observed. At equilibrium, PCNA loading is highly-favored due to a much faster rate constant for PCNA loading compared to PCNA unloading (Hedglin et al., 2013b). During holoenzyme formation, an incoming polymerase captures loaded PCNA from DNA-bound RFC, stabilizing the sliding clamp on DNA and increasing the fraction of bound forked Cy3-P/T DNA•Cy5-PCNA. (B) The fractional saturation increased with free Pol η concentration and displayed a hyperbolic behavior indicative of an equilibrium binding curve. The dashed lines indicate the concentrations of Cy3-P/T DNA, Cy5-PCNA, and RFC (100 nM each, X = 100 nM) and 100% holoenzyme formation (y = 1.0). The data fit best to a one-site binding model yielding a KD of 28.0 ± 5.38 nM for the forked Cy3-P/T DNA•Cy5-PCNA•Pol η complex. Such behavior is in stark contrast to that observed for pol δ in a recent report from our lab, shown in C. (C) Fractional saturation of the P/T DNA•PCNA complex as a function of the total concentration of DNA polymerase. Shown in grey is the data from B for pol η. Shown in red is the data for pol δ from reference (Hedglin et al., 2013b). In contrast to pol η, pol δ stabilized a stoichiometric amount of PCNA on DNA under the same conditions and, hence, displayed a linear increase. The line plateaued when the concentrations of total pol δ, PCNA, and RFC were equivalent (100 nM of each), and flat-lined thereafter (Hedglin et al., 2013b). This indicates that the total concentration of the DNA•PCNA complex (100 nM) is much greater (>10 fold) than the KD of pol δ for the DNA•PCNA complex (Goodrich and Kugel, 2007). This sets an upper limit of 10 nM for the KD of pol δ for PCNA loaded onto DNA, in excellent agreement with the value reported in an independent study (7.1 ± 1.0 nM) (Zhou et al., 2012).